Condenser lens choice for a given objective

[Kyle Michael Douglass]

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Hello listers,


I have a couple of questions about condensers for you. I'd like to do some transmitted light imaging in an inverted microscope using high magnification, oil-immersion objectives. For the moment, I don't need to do anything other than brightfield with a high power LED light source. It might be nice to do phase contrast or DIC in the future, but I don't need it now.


My questions are:


1) What are the rules of thumb for matching a brightfield condenser to an objective? I won't be using anything but oil immersion objectives with NA's greater than 1.4.


2) If I do want to do phase contrast or DIC in the future, should I put special consideration into the condenser lens selection now? I imagine the condenser NA will determine what phase contrast rings I can use, but does it impact DIC?


Thanks!

Kyle


Dr. Kyle M. Douglass
Post-doctoral Researcher
EPFL - The Laboratory of Experimental Biophysics
http://leb.epfl.ch/
http://kmdouglass.github.io

[Barbara Foster]

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Hi, Kyle

The general rule of thumb is that the NA on the condenser should meet
or exceed that of the objective.

If you are using oil immersion objectives, ideally, to achieve that
goal, you should use an oil immersion condenser, otherwise you are
limited to an NA of 0.9.

Also, remember that the aperture iris in the condenser adjusts the
condenser's WORKING  numerical aperture.  Just because the condenser
is marked 1.4 NA doesn't mean that, in a practical experiment, it
will be operating at 1.4.  I follow the guidelines set down by Frits
Zernicke (inventor of Phase contrast):  gently close the aperture
iris to the "Oomph" position: that delicate balance between
sufficient edge definition and optimum resolution (Yes, the condenser
does contribute to resolution).

As for planning for growth:
You might want to invest in a turret condenser early on.  That will
give you the option to add those other contrast techniques as you
grow into them.

And just one more reminder, specifically regarding DIC:
If you are going to use plastic vessels (petri dishes, multi-well
plates, growth flasks), use Hoffman Modulation Contrast instead of
DIC.  DIC uses polarized light.  The plastic will affectt the shear
and cause effects that will be hard to interpret.  Some HMC set-ups
do use pol to control the width of the slit in the condenser, but all
of that is on the incoming side of the sample and will not be
affected by plastic containers.

Good hunting!
Barbara Foster, President & Chief Consultant
Microscopy/Microscopy Education  ... "Education, not Training"
7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P: 972-924-5310
www.MicroscopyEducation.com

Microscopy/Microscopy Education is a division of The Microscopy &
Imaging Place, Inc.


NEW!   Getting involved in Raman or FTIR?
MME is now offering courses in these areas specifically for microscopists!
Now scheduling courses through the mid 2016.  We can customize a
course on nearly any topic, from fluorescence to confocal to image
analysis to SEM/TEM.
Call today for a free training evaluation.




At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Hello listers,
>
>
>I have a couple of questions about condensers for you. I'd like to
>do some transmitted light imaging in an inverted microscope using
>high magnification, oil-immersion objectives. For the moment, I
>don't need to do anything other than brightfield with a high power
>LED light source. It might be nice to do phase contrast or DIC in
>the future, but I don't need it now.
>
>
>My questions are:
>
>
>1) What are the rules of thumb for matching a brightfield condenser
>to an objective? I won't be using anything but oil immersion
>objectives with NA's greater than 1.4.
>
>
>2) If I do want to do phase contrast or DIC in the future, should I
>put special consideration into the condenser lens selection now? I
>imagine the condenser NA will determine what phase contrast rings I
>can use, but does it impact DIC?
>
>
>Thanks!
>
>Kyle
>
>
>Dr. Kyle M. Douglass
>Post-doctoral Researcher
>EPFL - The Laboratory of Experimental Biophysics
>http://leb.epfl.ch/
>http://kmdouglass.github.io

[Kyle Michael Douglass]

*****
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*****

Thanks for the feedback, Barbara. It is very helpful.

I have heard the advice before about the condenser NA needing to be
greater than or equal to the objective NA. Can you offer some physical
explanation or intuition for why this is?

One admittedly incomplete explanation I can think of for the
recommendation goes like this: the light collected by the objective
consists of two parts. One part is the transmitted light that is not
scattered by the sample. The other part is the light scattered by the
sample. If the condenser's working NA is smaller than the objective's,
then the unscattered, transmitted light fills only a portion of the
objective's back focal plane. However, the light scattered by the sample
will probably be dispersed across the entire back focal plane because it
will encode all the spatial frequencies of the sample.

I wonder if it's the inhomogeneous distribution of light from the two
components in the objective's back focal plane that leads to the matched
NA requirements of the condenser and objective. Does this make sense?

Thanks!
Kyle

On 03/17/2016 08:22 PM, Barbara Foster wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi, Kyle
>
> The general rule of thumb is that the NA on the condenser should meet
> or exceed that of the objective.
>
> If you are using oil immersion objectives, ideally, to achieve that
> goal, you should use an oil immersion condenser, otherwise you are
> limited to an NA of 0.9.
>
> Also, remember that the aperture iris in the condenser adjusts the
> condenser's WORKING  numerical aperture.  Just because the condenser
> is marked 1.4 NA doesn't mean that, in a practical experiment, it will
> be operating at 1.4.  I follow the guidelines set down by Frits
> Zernicke (inventor of Phase contrast):  gently close the aperture iris
> to the "Oomph" position: that delicate balance between sufficient edge
> definition and optimum resolution (Yes, the condenser does contribute
> to resolution).
>
> As for planning for growth:
> You might want to invest in a turret condenser early on.  That will
> give you the option to add those other contrast techniques as you grow
> into them.
>
> And just one more reminder, specifically regarding DIC:
> If you are going to use plastic vessels (petri dishes, multi-well
> plates, growth flasks), use Hoffman Modulation Contrast instead of
> DIC.  DIC uses polarized light.  The plastic will affectt the shear
> and cause effects that will be hard to interpret.  Some HMC set-ups do
> use pol to control the width of the slit in the condenser, but all of
> that is on the incoming side of the sample and will not be affected by
> plastic containers.
>
> Good hunting!
> Barbara Foster, President & Chief Consultant
> Microscopy/Microscopy Education  ... "Education, not Training"
> 7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P: 972-924-5310
> www.MicroscopyEducation.com
>
> Microscopy/Microscopy Education is a division of The Microscopy &
> Imaging Place, Inc.
>
>
> NEW!   Getting involved in Raman or FTIR?
> MME is now offering courses in these areas specifically for
> microscopists!
> Now scheduling courses through the mid 2016.  We can customize a
> course on nearly any topic, from fluorescence to confocal to image
> analysis to SEM/TEM.
> Call today for a free training evaluation.
>
>
>
>
> At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your
>> posting.
>> *****
>>
>> Hello listers,
>>
>>
>> I have a couple of questions about condensers for you. I'd like to do
>> some transmitted light imaging in an inverted microscope using high
>> magnification, oil-immersion objectives. For the moment, I don't need
>> to do anything other than brightfield with a high power LED light
>> source. It might be nice to do phase contrast or DIC in the future,
>> but I don't need it now.
>>
>>
>> My questions are:
>>
>>
>> 1) What are the rules of thumb for matching a brightfield condenser
>> to an objective? I won't be using anything but oil immersion
>> objectives with NA's greater than 1.4.
>>
>>
>> 2) If I do want to do phase contrast or DIC in the future, should I
>> put special consideration into the condenser lens selection now? I
>> imagine the condenser NA will determine what phase contrast rings I
>> can use, but does it impact DIC?
>>
>>
>> Thanks!
>>
>> Kyle
>>
>>
>> Dr. Kyle M. Douglass
>> Post-doctoral Researcher
>> EPFL - The Laboratory of Experimental Biophysics
>> http://leb.epfl.ch/
>> http://kmdouglass.github.io

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch

[Aryeh Weiss]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

In a transmitted-light brightfield image, the Rayleigh criterion
includes both the objective NA and the condenser NA
(1.22 lambda/(NA_obj +NA_cond)) . The makes sense because even a very
small NA objective can receive light scattered at a large angle if the
NA of the condenser is large. (This is how dark-field works).
So it would appear that in principle, you benefit from having a
condenser with as large as NA as possible  (although you may not have
much contrast on that brightfield image).

BTW, you can have a "poor" man's dark field scope by using a low-NA
objective with a phase ring made for a higher NA objective.
For example, in my teaching lab, the students get very nice darkfield
images using our 4x/NA=0.1 objective with the ph2 phase ring.

--aryeh



On 18/03/2016 10:26 AM, Kyle Douglass wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Thanks for the feedback, Barbara. It is very helpful.
>
> I have heard the advice before about the condenser NA needing to be
> greater than or equal to the objective NA. Can you offer some physical
> explanation or intuition for why this is?
>
> One admittedly incomplete explanation I can think of for the
> recommendation goes like this: the light collected by the objective
> consists of two parts. One part is the transmitted light that is not
> scattered by the sample. The other part is the light scattered by the
> sample. If the condenser's working NA is smaller than the objective's,
> then the unscattered, transmitted light fills only a portion of the
> objective's back focal plane. However, the light scattered by the
> sample will probably be dispersed across the entire back focal plane
> because it will encode all the spatial frequencies of the sample.
>
> I wonder if it's the inhomogeneous distribution of light from the two
> components in the objective's back focal plane that leads to the
> matched NA requirements of the condenser and objective. Does this make
> sense?
>
> Thanks!
> Kyle
>
> On 03/17/2016 08:22 PM, Barbara Foster wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your
>> posting.
>> *****
>>
>> Hi, Kyle
>>
>> The general rule of thumb is that the NA on the condenser should meet
>> or exceed that of the objective.
>>
>> If you are using oil immersion objectives, ideally, to achieve that
>> goal, you should use an oil immersion condenser, otherwise you are
>> limited to an NA of 0.9.
>>
>> Also, remember that the aperture iris in the condenser adjusts the
>> condenser's WORKING  numerical aperture.  Just because the condenser
>> is marked 1.4 NA doesn't mean that, in a practical experiment, it
>> will be operating at 1.4.  I follow the guidelines set down by Frits
>> Zernicke (inventor of Phase contrast):  gently close the aperture
>> iris to the "Oomph" position: that delicate balance between
>> sufficient edge definition and optimum resolution (Yes, the condenser
>> does contribute to resolution).
>>
>> As for planning for growth:
>> You might want to invest in a turret condenser early on.  That will
>> give you the option to add those other contrast techniques as you
>> grow into them.
>>
>> And just one more reminder, specifically regarding DIC:
>> If you are going to use plastic vessels (petri dishes, multi-well
>> plates, growth flasks), use Hoffman Modulation Contrast instead of
>> DIC.  DIC uses polarized light.  The plastic will affectt the shear
>> and cause effects that will be hard to interpret.  Some HMC set-ups
>> do use pol to control the width of the slit in the condenser, but all
>> of that is on the incoming side of the sample and will not be
>> affected by plastic containers.
>>
>> Good hunting!
>> Barbara Foster, President & Chief Consultant
>> Microscopy/Microscopy Education  ... "Education, not Training"
>> 7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P: 972-924-5310
>> www.MicroscopyEducation.com
>>
>> Microscopy/Microscopy Education is a division of The Microscopy &
>> Imaging Place, Inc.
>>
>>
>> NEW!   Getting involved in Raman or FTIR?
>> MME is now offering courses in these areas specifically for
>> microscopists!
>> Now scheduling courses through the mid 2016.  We can customize a
>> course on nearly any topic, from fluorescence to confocal to image
>> analysis to SEM/TEM.
>> Call today for a free training evaluation.
>>
>>
>>
>>
>> At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>>> posting.
>>> *****
>>>
>>> Hello listers,
>>>
>>>
>>> I have a couple of questions about condensers for you. I'd like to
>>> do some transmitted light imaging in an inverted microscope using
>>> high magnification, oil-immersion objectives. For the moment, I
>>> don't need to do anything other than brightfield with a high power
>>> LED light source. It might be nice to do phase contrast or DIC in
>>> the future, but I don't need it now.
>>>
>>>
>>> My questions are:
>>>
>>>
>>> 1) What are the rules of thumb for matching a brightfield condenser
>>> to an objective? I won't be using anything but oil immersion
>>> objectives with NA's greater than 1.4.
>>>
>>>
>>> 2) If I do want to do phase contrast or DIC in the future, should I
>>> put special consideration into the condenser lens selection now? I
>>> imagine the condenser NA will determine what phase contrast rings I
>>> can use, but does it impact DIC?
>>>
>>>
>>> Thanks!
>>>
>>> Kyle
>>>
>>>
>>> Dr. Kyle M. Douglass
>>> Post-doctoral Researcher
>>> EPFL - The Laboratory of Experimental Biophysics
>>> http://leb.epfl.ch/
>>> http://kmdouglass.github.io
>

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

[Guy Cox-2]

*****
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*****

This is a little bit oversimplified.   The Rayleigh criterion does not apply to a widefield image, but does apply in fluorescence.  The condenser NA normally IS the objective NA, since they are usually one and the same thing, but condenser NA only affects brightness, not resolution.

The Abbe criterion r = 0.5 lambda / NA applies in transmitted light, but ONLY if the condenser aperture equals or exceeds the objective NA.  Reducing the condenser NA does not have the same effect as reducing the objective NA.  Reducing the condenser NA to 0 (parallel illumination)   worsens  the resolution to r = lambda/NA - ie 50% of what the objective should give.  

                                              Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Aryeh Weiss
Sent: Friday, 18 March 2016 11:38 PM
To: [hidden email]
Subject: Re: Condenser lens choice for a given objective

*****
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*****

In a transmitted-light brightfield image, the Rayleigh criterion includes both the objective NA and the condenser NA
(1.22 lambda/(NA_obj +NA_cond)) . The makes sense because even a very small NA objective can receive light scattered at a large angle if the NA of the condenser is large. (This is how dark-field works).
So it would appear that in principle, you benefit from having a condenser with as large as NA as possible  (although you may not have much contrast on that brightfield image).

BTW, you can have a "poor" man's dark field scope by using a low-NA objective with a phase ring made for a higher NA objective.
For example, in my teaching lab, the students get very nice darkfield images using our 4x/NA=0.1 objective with the ph2 phase ring.

--aryeh



On 18/03/2016 10:26 AM, Kyle Douglass wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Thanks for the feedback, Barbara. It is very helpful.
>
> I have heard the advice before about the condenser NA needing to be
> greater than or equal to the objective NA. Can you offer some physical
> explanation or intuition for why this is?
>
> One admittedly incomplete explanation I can think of for the
> recommendation goes like this: the light collected by the objective
> consists of two parts. One part is the transmitted light that is not
> scattered by the sample. The other part is the light scattered by the
> sample. If the condenser's working NA is smaller than the objective's,
> then the unscattered, transmitted light fills only a portion of the
> objective's back focal plane. However, the light scattered by the
> sample will probably be dispersed across the entire back focal plane
> because it will encode all the spatial frequencies of the sample.
>
> I wonder if it's the inhomogeneous distribution of light from the two
> components in the objective's back focal plane that leads to the
> matched NA requirements of the condenser and objective. Does this make
> sense?
>
> Thanks!
> Kyle
>
> On 03/17/2016 08:22 PM, Barbara Foster wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your
>> posting.
>> *****
>>
>> Hi, Kyle
>>
>> The general rule of thumb is that the NA on the condenser should meet
>> or exceed that of the objective.
>>
>> If you are using oil immersion objectives, ideally, to achieve that
>> goal, you should use an oil immersion condenser, otherwise you are
>> limited to an NA of 0.9.
>>
>> Also, remember that the aperture iris in the condenser adjusts the
>> condenser's WORKING  numerical aperture.  Just because the condenser
>> is marked 1.4 NA doesn't mean that, in a practical experiment, it
>> will be operating at 1.4.  I follow the guidelines set down by Frits
>> Zernicke (inventor of Phase contrast):  gently close the aperture
>> iris to the "Oomph" position: that delicate balance between
>> sufficient edge definition and optimum resolution (Yes, the condenser
>> does contribute to resolution).
>>
>> As for planning for growth:
>> You might want to invest in a turret condenser early on.  That will
>> give you the option to add those other contrast techniques as you
>> grow into them.
>>
>> And just one more reminder, specifically regarding DIC:
>> If you are going to use plastic vessels (petri dishes, multi-well
>> plates, growth flasks), use Hoffman Modulation Contrast instead of
>> DIC.  DIC uses polarized light.  The plastic will affectt the shear
>> and cause effects that will be hard to interpret.  Some HMC set-ups
>> do use pol to control the width of the slit in the condenser, but all
>> of that is on the incoming side of the sample and will not be
>> affected by plastic containers.
>>
>> Good hunting!
>> Barbara Foster, President & Chief Consultant Microscopy/Microscopy
>> Education  ... "Education, not Training"
>> 7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P:
>> 972-924-5310 www.MicroscopyEducation.com
>>
>> Microscopy/Microscopy Education is a division of The Microscopy &
>> Imaging Place, Inc.
>>
>>
>> NEW!   Getting involved in Raman or FTIR?
>> MME is now offering courses in these areas specifically for
>> microscopists!
>> Now scheduling courses through the mid 2016.  We can customize a
>> course on nearly any topic, from fluorescence to confocal to image
>> analysis to SEM/TEM.
>> Call today for a free training evaluation.
>>
>>
>>
>>
>> At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>>> posting.
>>> *****
>>>
>>> Hello listers,
>>>
>>>
>>> I have a couple of questions about condensers for you. I'd like to
>>> do some transmitted light imaging in an inverted microscope using
>>> high magnification, oil-immersion objectives. For the moment, I
>>> don't need to do anything other than brightfield with a high power
>>> LED light source. It might be nice to do phase contrast or DIC in
>>> the future, but I don't need it now.
>>>
>>>
>>> My questions are:
>>>
>>>
>>> 1) What are the rules of thumb for matching a brightfield condenser
>>> to an objective? I won't be using anything but oil immersion
>>> objectives with NA's greater than 1.4.
>>>
>>>
>>> 2) If I do want to do phase contrast or DIC in the future, should I
>>> put special consideration into the condenser lens selection now? I
>>> imagine the condenser NA will determine what phase contrast rings I
>>> can use, but does it impact DIC?
>>>
>>>
>>> Thanks!
>>>
>>> Kyle
>>>
>>>
>>> Dr. Kyle M. Douglass
>>> Post-doctoral Researcher
>>> EPFL - The Laboratory of Experimental Biophysics http://leb.epfl.ch/ 
>>> http://kmdouglass.github.io
>

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Hi Kyle,

Your post indicates that for now you just want to get a brightfield
image through your 100x objective lens. The purpose of an objective lens
is two fold:
1. resolution ... emphasized in the replies below ... high NA condenser
results in higher spatial resolution.
2. brightness ... get enough photon flux so you can do your experiment.

NA: at Guy's limit of zero NA on the illumination side, you will few
photons reaching the objective lens and detector(s). You will also have
(practically) infinite depth of focus with respect to imaging dust and
other things on every optical surface. Many of these could be cleaned up
by background subtraction (plus constant).

Brightness is proportional to:

         NA^4
B ~ ----------
          M^2

The numerator is assuming equal NA (ex. epi-illumination with a single
objective lens). I assume this could be re-written as

(NAcond^2)*(NAobj^2)

which is ok until Guy's limit of NAcond = 0. I'll also mention that you
do not even need a transmitted light LED, condenser, or condenser arm if
you have a sensitive enough detector(s): room lights, desk lamps,
computer monitors can provide enough light (I first encountered this
problem/feature working with James Sabry in Jim Spudich's lab using a
back illuminated CCD on an inverted microscope, no recalling what
objective lens, but was 19 years ago and available in their published
papers).

More importantly, M^2 means that your 100x lens is putting 1% as much
photon flux onto a pixel as a 10x lens would.

My advice: go find a long working distance objective lens that gets
enough light onto your specimen to get you a useful brightfield image.
You could later figure out if you need a phase contrast/DIC turret, what
NA and working distance you need etc.
I also suggest instead of hardware contrast (DIC requires polarizers and
prisms ... can avoid illumination side polarizer if using a laser as in
confocal microscope stand DIC using the transmitted light pathway in
reverse; phase contrast requires a phase ring in the objective lens -
usually with a lower NA for a given price point), you start looking into
synthetic contrast options. The simplest is to just go with digital
contrast by background subtraction.
Software to get quantitative phase microscopy data of wet mass (leading
to dry mass) - a couple of links and comments:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3555125/  ... McCarty ...
uses 0.1 NA on condenser side.
http://www.jove.com/video/50988/quantitative-optical-microscopy-measurement-cellular-biophysical 
... McCarty, see downloads (.M files).

Nugent / IATIA (now Ultima Capital) developed the first software only
solution
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624349/.
http://www.ncbi.nlm.nih.gov/pubmed/15800856   ... Nugent / IATIA on confocal
http://onlinelibrary.wiley.com/doi/10.1111/j.1440-1681.2004.04100.x/abstract 

http://aups.org.au/Proceedings/34/121-127/121-127.pdf   ... fig 3
illustrates synthetic phase and DIC; fig 4 shows improves segmentation.
http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging/applicationNotes/comparisonWithOpticalPhaseContrastModalities.pdf
http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging/applicationNotes/measurementOfAreaChanges.pdf 
(I think you can ignore the "Confidence-Publication Pending" at top -
this appnote was posted by the manufacturer and has been online for years).


Hardware assisted (not a complete list - some use holography, others
interferometry):
Ovizio
Gabriel Popescu
Graham Dunn

PubMed has more - a simple search is:   "quantitative phase microscopy"

McCarty's JoVE article now has downloadable .M (MatLab) files. If Anne
Carpenter or anyone on the Cellprofiler team is reading this (or someone
send is it their way), I encourage Anne to work with McCarty and their
University to get "MaCarty QPm" into Cellprofiler.

not QPm, this may still be of interest to listservites:
Direct imaging of phase objects enables conventional deconvolution in
bright field light microscopy
http://www.ncbi.nlm.nih.gov/pubmed/24558478

//

Getting more out of high NA objective lens ... confocal or widefield
interference reflection microscopy (IRM) provides data on
cell-substratum adhesion ... including contact area. In reflection
confocal (ok, for Jim Pawley and Guy Cox: scattered confocal), you can
get optical sections of the cell, "label free". Some IRM data I posted
online:

http://works.bepress.com/gmcnamara/10/
http://works.bepress.com/gmcnamara/7/

Widefield IRM is very simple if your filter cube does not have an
exciter filter (best to do this with a wavelength and intensity
selectable LED illuminator than a broad spectrum arc lamp): just turn on
a wavelength(s) that enable some light to bounce from the
cells/coverglass through the dichroic and emission filter (could do even
better with a dichroic only, and even better with a 50/50 beamsplitter
only). My thanks to Tom DiMatteo, Epi Technology, for telling me about
single LED control on my early gen SOLA. I had a long chat with Tom at
his ABRF booth a couple of years ago.
IRM can be quantitative
http://www.ncbi.nlm.nih.gov/pubmed/23024911
http://www.ncbi.nlm.nih.gov/pubmed/20013754
http://www.ncbi.nlm.nih.gov/pubmed/3900106 ... Verschueren



George



On 3/18/2016 9:00 PM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> This is a little bit oversimplified.   The Rayleigh criterion does not apply to a widefield image, but does apply in fluorescence.  The condenser NA normally IS the objective NA, since they are usually one and the same thing, but condenser NA only affects brightness, not resolution.
>
> The Abbe criterion r = 0.5 lambda / NA applies in transmitted light, but ONLY if the condenser aperture equals or exceeds the objective NA.  Reducing the condenser NA does not have the same effect as reducing the objective NA.  Reducing the condenser NA to 0 (parallel illumination)   worsens  the resolution to r = lambda/NA - ie 50% of what the objective should give.
>
>                                                Guy
>
> Guy Cox, Honorary Associate Professor
> School of Medical Sciences
>
> Australian Centre for Microscopy and Microanalysis,
> Madsen, F09, University of Sydney, NSW 2006
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Aryeh Weiss
> Sent: Friday, 18 March 2016 11:38 PM
> To: [hidden email]
> Subject: Re: Condenser lens choice for a given objective
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> In a transmitted-light brightfield image, the Rayleigh criterion includes both the objective NA and the condenser NA
> (1.22 lambda/(NA_obj +NA_cond)) . The makes sense because even a very small NA objective can receive light scattered at a large angle if the NA of the condenser is large. (This is how dark-field works).
> So it would appear that in principle, you benefit from having a condenser with as large as NA as possible  (although you may not have much contrast on that brightfield image).
>
> BTW, you can have a "poor" man's dark field scope by using a low-NA objective with a phase ring made for a higher NA objective.
> For example, in my teaching lab, the students get very nice darkfield images using our 4x/NA=0.1 objective with the ph2 phase ring.
>
> --aryeh
>
>
>
> On 18/03/2016 10:26 AM, Kyle Douglass wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Thanks for the feedback, Barbara. It is very helpful.
>>
>> I have heard the advice before about the condenser NA needing to be
>> greater than or equal to the objective NA. Can you offer some physical
>> explanation or intuition for why this is?
>>
>> One admittedly incomplete explanation I can think of for the
>> recommendation goes like this: the light collected by the objective
>> consists of two parts. One part is the transmitted light that is not
>> scattered by the sample. The other part is the light scattered by the
>> sample. If the condenser's working NA is smaller than the objective's,
>> then the unscattered, transmitted light fills only a portion of the
>> objective's back focal plane. However, the light scattered by the
>> sample will probably be dispersed across the entire back focal plane
>> because it will encode all the spatial frequencies of the sample.
>>
>> I wonder if it's the inhomogeneous distribution of light from the two
>> components in the objective's back focal plane that leads to the
>> matched NA requirements of the condenser and objective. Does this make
>> sense?
>>
>> Thanks!
>> Kyle
>>
>> On 03/17/2016 08:22 PM, Barbara Foster wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>>> posting.
>>> *****
>>>
>>> Hi, Kyle
>>>
>>> The general rule of thumb is that the NA on the condenser should meet
>>> or exceed that of the objective.
>>>
>>> If you are using oil immersion objectives, ideally, to achieve that
>>> goal, you should use an oil immersion condenser, otherwise you are
>>> limited to an NA of 0.9.
>>>
>>> Also, remember that the aperture iris in the condenser adjusts the
>>> condenser's WORKING  numerical aperture.  Just because the condenser
>>> is marked 1.4 NA doesn't mean that, in a practical experiment, it
>>> will be operating at 1.4.  I follow the guidelines set down by Frits
>>> Zernicke (inventor of Phase contrast):  gently close the aperture
>>> iris to the "Oomph" position: that delicate balance between
>>> sufficient edge definition and optimum resolution (Yes, the condenser
>>> does contribute to resolution).
>>>
>>> As for planning for growth:
>>> You might want to invest in a turret condenser early on.  That will
>>> give you the option to add those other contrast techniques as you
>>> grow into them.
>>>
>>> And just one more reminder, specifically regarding DIC:
>>> If you are going to use plastic vessels (petri dishes, multi-well
>>> plates, growth flasks), use Hoffman Modulation Contrast instead of
>>> DIC.  DIC uses polarized light.  The plastic will affectt the shear
>>> and cause effects that will be hard to interpret.  Some HMC set-ups
>>> do use pol to control the width of the slit in the condenser, but all
>>> of that is on the incoming side of the sample and will not be
>>> affected by plastic containers.
>>>
>>> Good hunting!
>>> Barbara Foster, President & Chief Consultant Microscopy/Microscopy
>>> Education  ... "Education, not Training"
>>> 7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P:
>>> 972-924-5310 www.MicroscopyEducation.com
>>>
>>> Microscopy/Microscopy Education is a division of The Microscopy &
>>> Imaging Place, Inc.
>>>
>>>
>>> NEW!   Getting involved in Raman or FTIR?
>>> MME is now offering courses in these areas specifically for
>>> microscopists!
>>> Now scheduling courses through the mid 2016.  We can customize a
>>> course on nearly any topic, from fluorescence to confocal to image
>>> analysis to SEM/TEM.
>>> Call today for a free training evaluation.
>>>
>>>
>>>
>>>
>>> At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> Post images on http://www.imgur.com and include the link in your
>>>> posting.
>>>> *****
>>>>
>>>> Hello listers,
>>>>
>>>>
>>>> I have a couple of questions about condensers for you. I'd like to
>>>> do some transmitted light imaging in an inverted microscope using
>>>> high magnification, oil-immersion objectives. For the moment, I
>>>> don't need to do anything other than brightfield with a high power
>>>> LED light source. It might be nice to do phase contrast or DIC in
>>>> the future, but I don't need it now.
>>>>
>>>>
>>>> My questions are:
>>>>
>>>>
>>>> 1) What are the rules of thumb for matching a brightfield condenser
>>>> to an objective? I won't be using anything but oil immersion
>>>> objectives with NA's greater than 1.4.
>>>>
>>>>
>>>> 2) If I do want to do phase contrast or DIC in the future, should I
>>>> put special consideration into the condenser lens selection now? I
>>>> imagine the condenser NA will determine what phase contrast rings I
>>>> can use, but does it impact DIC?
>>>>
>>>>
>>>> Thanks!
>>>>
>>>> Kyle
>>>>
>>>>
>>>> Dr. Kyle M. Douglass
>>>> Post-doctoral Researcher
>>>> EPFL - The Laboratory of Experimental Biophysics http://leb.epfl.ch/
>>>> http://kmdouglass.github.io
>
> --
> Aryeh Weiss
> Faculty of Engineering
> Bar Ilan University
> Ramat Gan 52900 Israel
>
> Ph:  972-3-5317638
> FAX: [phone]
>

--



George McNamara, Ph.D.
Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab)
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42
http://works.bepress.com/gmcnamara/75
https://www.linkedin.com/in/georgemcnamara

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

George,

            If you get 0 NA by closing down your condenser diaphragm you will indeed get very little light and will image any dust in your illumination system.  But if you remove your condenser and illuminate the sample with parallel light you won't have either of these problems and you will still have an illumination NA of 0 and a resolution of r = lambda / NA.

  All the tricks in the Abbe diffraction kit depend on parallel illumination.  You probably know this but, for the benefit of others in the list, Jim Pawley, Dennis Dwarte, my son Cassian and I put together  a computerized version of some of the items in the Abbe diffraction demonstration kit.  Zeiss made CDs of it so you might get one from your local Zeiss rep, but otherwise it is available on my website http://www.guycox.com  (free).

                                        Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Sunday, 20 March 2016 4:07 AM
To: [hidden email]
Subject: Re: Condenser lens choice for a given objective

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Kyle,

Your post indicates that for now you just want to get a brightfield image through your 100x objective lens. The purpose of an objective lens is two fold:
1. resolution ... emphasized in the replies below ... high NA condenser results in higher spatial resolution.
2. brightness ... get enough photon flux so you can do your experiment.

NA: at Guy's limit of zero NA on the illumination side, you will few photons reaching the objective lens and detector(s). You will also have
(practically) infinite depth of focus with respect to imaging dust and other things on every optical surface. Many of these could be cleaned up by background subtraction (plus constant).

Brightness is proportional to:

         NA^4
B ~ ----------
          M^2

The numerator is assuming equal NA (ex. epi-illumination with a single objective lens). I assume this could be re-written as

(NAcond^2)*(NAobj^2)

which is ok until Guy's limit of NAcond = 0. I'll also mention that you do not even need a transmitted light LED, condenser, or condenser arm if you have a sensitive enough detector(s): room lights, desk lamps, computer monitors can provide enough light (I first encountered this problem/feature working with James Sabry in Jim Spudich's lab using a back illuminated CCD on an inverted microscope, no recalling what objective lens, but was 19 years ago and available in their published papers).

More importantly, M^2 means that your 100x lens is putting 1% as much photon flux onto a pixel as a 10x lens would.

My advice: go find a long working distance objective lens that gets enough light onto your specimen to get you a useful brightfield image.
You could later figure out if you need a phase contrast/DIC turret, what NA and working distance you need etc.
I also suggest instead of hardware contrast (DIC requires polarizers and prisms ... can avoid illumination side polarizer if using a laser as in confocal microscope stand DIC using the transmitted light pathway in reverse; phase contrast requires a phase ring in the objective lens - usually with a lower NA for a given price point), you start looking into synthetic contrast options. The simplest is to just go with digital contrast by background subtraction.
Software to get quantitative phase microscopy data of wet mass (leading to dry mass) - a couple of links and comments:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3555125/  ... McCarty ...
uses 0.1 NA on condenser side.
http://www.jove.com/video/50988/quantitative-optical-microscopy-measurement-cellular-biophysical
... McCarty, see downloads (.M files).

Nugent / IATIA (now Ultima Capital) developed the first software only solution http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624349/.
http://www.ncbi.nlm.nih.gov/pubmed/15800856   ... Nugent / IATIA on confocal
http://onlinelibrary.wiley.com/doi/10.1111/j.1440-1681.2004.04100.x/abstract 

http://aups.org.au/Proceedings/34/121-127/121-127.pdf   ... fig 3
illustrates synthetic phase and DIC; fig 4 shows improves segmentation.
http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging/applicationNotes/comparisonWithOpticalPhaseContrastModalities.pdf
http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging/applicationNotes/measurementOfAreaChanges.pdf
(I think you can ignore the "Confidence-Publication Pending" at top - this appnote was posted by the manufacturer and has been online for years).


Hardware assisted (not a complete list - some use holography, others
interferometry):
Ovizio
Gabriel Popescu
Graham Dunn

PubMed has more - a simple search is:   "quantitative phase microscopy"

McCarty's JoVE article now has downloadable .M (MatLab) files. If Anne Carpenter or anyone on the Cellprofiler team is reading this (or someone send is it their way), I encourage Anne to work with McCarty and their University to get "MaCarty QPm" into Cellprofiler.

not QPm, this may still be of interest to listservites:
Direct imaging of phase objects enables conventional deconvolution in bright field light microscopy
http://www.ncbi.nlm.nih.gov/pubmed/24558478

//

Getting more out of high NA objective lens ... confocal or widefield interference reflection microscopy (IRM) provides data on cell-substratum adhesion ... including contact area. In reflection confocal (ok, for Jim Pawley and Guy Cox: scattered confocal), you can get optical sections of the cell, "label free". Some IRM data I posted
online:

http://works.bepress.com/gmcnamara/10/
http://works.bepress.com/gmcnamara/7/

Widefield IRM is very simple if your filter cube does not have an exciter filter (best to do this with a wavelength and intensity selectable LED illuminator than a broad spectrum arc lamp): just turn on a wavelength(s) that enable some light to bounce from the cells/coverglass through the dichroic and emission filter (could do even better with a dichroic only, and even better with a 50/50 beamsplitter only). My thanks to Tom DiMatteo, Epi Technology, for telling me about single LED control on my early gen SOLA. I had a long chat with Tom at his ABRF booth a couple of years ago.
IRM can be quantitative
http://www.ncbi.nlm.nih.gov/pubmed/23024911
http://www.ncbi.nlm.nih.gov/pubmed/20013754
http://www.ncbi.nlm.nih.gov/pubmed/3900106 ... Verschueren



George



On 3/18/2016 9:00 PM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> This is a little bit oversimplified.   The Rayleigh criterion does not apply to a widefield image, but does apply in fluorescence.  The condenser NA normally IS the objective NA, since they are usually one and the same thing, but condenser NA only affects brightness, not resolution.
>
> The Abbe criterion r = 0.5 lambda / NA applies in transmitted light, but ONLY if the condenser aperture equals or exceeds the objective NA.  Reducing the condenser NA does not have the same effect as reducing the objective NA.  Reducing the condenser NA to 0 (parallel illumination)   worsens  the resolution to r = lambda/NA - ie 50% of what the objective should give.
>
>                                                Guy
>
> Guy Cox, Honorary Associate Professor
> School of Medical Sciences
>
> Australian Centre for Microscopy and Microanalysis, Madsen, F09,
> University of Sydney, NSW 2006
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Aryeh Weiss
> Sent: Friday, 18 March 2016 11:38 PM
> To: [hidden email]
> Subject: Re: Condenser lens choice for a given objective
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> In a transmitted-light brightfield image, the Rayleigh criterion
> includes both the objective NA and the condenser NA
> (1.22 lambda/(NA_obj +NA_cond)) . The makes sense because even a very small NA objective can receive light scattered at a large angle if the NA of the condenser is large. (This is how dark-field works).
> So it would appear that in principle, you benefit from having a condenser with as large as NA as possible  (although you may not have much contrast on that brightfield image).
>
> BTW, you can have a "poor" man's dark field scope by using a low-NA objective with a phase ring made for a higher NA objective.
> For example, in my teaching lab, the students get very nice darkfield images using our 4x/NA=0.1 objective with the ph2 phase ring.
>
> --aryeh
>
>
>
> On 18/03/2016 10:26 AM, Kyle Douglass wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Thanks for the feedback, Barbara. It is very helpful.
>>
>> I have heard the advice before about the condenser NA needing to be
>> greater than or equal to the objective NA. Can you offer some
>> physical explanation or intuition for why this is?
>>
>> One admittedly incomplete explanation I can think of for the
>> recommendation goes like this: the light collected by the objective
>> consists of two parts. One part is the transmitted light that is not
>> scattered by the sample. The other part is the light scattered by the
>> sample. If the condenser's working NA is smaller than the
>> objective's, then the unscattered, transmitted light fills only a
>> portion of the objective's back focal plane. However, the light
>> scattered by the sample will probably be dispersed across the entire
>> back focal plane because it will encode all the spatial frequencies of the sample.
>>
>> I wonder if it's the inhomogeneous distribution of light from the two
>> components in the objective's back focal plane that leads to the
>> matched NA requirements of the condenser and objective. Does this
>> make sense?
>>
>> Thanks!
>> Kyle
>>
>> On 03/17/2016 08:22 PM, Barbara Foster wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>>> posting.
>>> *****
>>>
>>> Hi, Kyle
>>>
>>> The general rule of thumb is that the NA on the condenser should
>>> meet or exceed that of the objective.
>>>
>>> If you are using oil immersion objectives, ideally, to achieve that
>>> goal, you should use an oil immersion condenser, otherwise you are
>>> limited to an NA of 0.9.
>>>
>>> Also, remember that the aperture iris in the condenser adjusts the
>>> condenser's WORKING  numerical aperture.  Just because the condenser
>>> is marked 1.4 NA doesn't mean that, in a practical experiment, it
>>> will be operating at 1.4.  I follow the guidelines set down by Frits
>>> Zernicke (inventor of Phase contrast):  gently close the aperture
>>> iris to the "Oomph" position: that delicate balance between
>>> sufficient edge definition and optimum resolution (Yes, the
>>> condenser does contribute to resolution).
>>>
>>> As for planning for growth:
>>> You might want to invest in a turret condenser early on.  That will
>>> give you the option to add those other contrast techniques as you
>>> grow into them.
>>>
>>> And just one more reminder, specifically regarding DIC:
>>> If you are going to use plastic vessels (petri dishes, multi-well
>>> plates, growth flasks), use Hoffman Modulation Contrast instead of
>>> DIC.  DIC uses polarized light.  The plastic will affectt the shear
>>> and cause effects that will be hard to interpret.  Some HMC set-ups
>>> do use pol to control the width of the slit in the condenser, but
>>> all of that is on the incoming side of the sample and will not be
>>> affected by plastic containers.
>>>
>>> Good hunting!
>>> Barbara Foster, President & Chief Consultant Microscopy/Microscopy
>>> Education  ... "Education, not Training"
>>> 7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P:
>>> 972-924-5310 www.MicroscopyEducation.com
>>>
>>> Microscopy/Microscopy Education is a division of The Microscopy &
>>> Imaging Place, Inc.
>>>
>>>
>>> NEW!   Getting involved in Raman or FTIR?
>>> MME is now offering courses in these areas specifically for
>>> microscopists!
>>> Now scheduling courses through the mid 2016.  We can customize a
>>> course on nearly any topic, from fluorescence to confocal to image
>>> analysis to SEM/TEM.
>>> Call today for a free training evaluation.
>>>
>>>
>>>
>>>
>>> At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> Post images on http://www.imgur.com and include the link in your
>>>> posting.
>>>> *****
>>>>
>>>> Hello listers,
>>>>
>>>>
>>>> I have a couple of questions about condensers for you. I'd like to
>>>> do some transmitted light imaging in an inverted microscope using
>>>> high magnification, oil-immersion objectives. For the moment, I
>>>> don't need to do anything other than brightfield with a high power
>>>> LED light source. It might be nice to do phase contrast or DIC in
>>>> the future, but I don't need it now.
>>>>
>>>>
>>>> My questions are:
>>>>
>>>>
>>>> 1) What are the rules of thumb for matching a brightfield condenser
>>>> to an objective? I won't be using anything but oil immersion
>>>> objectives with NA's greater than 1.4.
>>>>
>>>>
>>>> 2) If I do want to do phase contrast or DIC in the future, should I
>>>> put special consideration into the condenser lens selection now? I
>>>> imagine the condenser NA will determine what phase contrast rings I
>>>> can use, but does it impact DIC?
>>>>
>>>>
>>>> Thanks!
>>>>
>>>> Kyle
>>>>
>>>>
>>>> Dr. Kyle M. Douglass
>>>> Post-doctoral Researcher
>>>> EPFL - The Laboratory of Experimental Biophysics
>>>> http://leb.epfl.ch/ http://kmdouglass.github.io
>
> --
> Aryeh Weiss
> Faculty of Engineering
> Bar Ilan University
> Ramat Gan 52900 Israel
>
> Ph:  972-3-5317638
> FAX: [phone]
>

--



George McNamara, Ph.D.
Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab) University of Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales http://works.bepress.com/gmcnamara/42
http://works.bepress.com/gmcnamara/75
https://www.linkedin.com/in/georgemcnamara

[Michael Model]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

In my opinion, visibility in bright field is more limited by contrast than
by theoretical resolution...

As for brightness, "in the absence of strong scattering, brightness in
transmitted illumination depends mostly on direct, and not on diffracted,
light (as the popular formula (NAob/M)2 assumes) and thus on the smallest
NA between the objective and condenser; this can be easily verified by
using an objective with a variable numerical aperture. "

Mike Model

On Sat, Mar 19, 2016 at 1:07 PM, George McNamara <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Kyle,
>
> Your post indicates that for now you just want to get a brightfield image
> through your 100x objective lens. The purpose of an objective lens is two
> fold:
> 1. resolution ... emphasized in the replies below ... high NA condenser
> results in higher spatial resolution.
> 2. brightness ... get enough photon flux so you can do your experiment.
>
> NA: at Guy's limit of zero NA on the illumination side, you will few
> photons reaching the objective lens and detector(s). You will also have
> (practically) infinite depth of focus with respect to imaging dust and
> other things on every optical surface. Many of these could be cleaned up by
> background subtraction (plus constant).
>
> Brightness is proportional to:
>
>         NA^4
> B ~ ----------
>          M^2
>
> The numerator is assuming equal NA (ex. epi-illumination with a single
> objective lens). I assume this could be re-written as
>
> (NAcond^2)*(NAobj^2)
>
> which is ok until Guy's limit of NAcond = 0. I'll also mention that you do
> not even need a transmitted light LED, condenser, or condenser arm if you
> have a sensitive enough detector(s): room lights, desk lamps, computer
> monitors can provide enough light (I first encountered this problem/feature
> working with James Sabry in Jim Spudich's lab using a back illuminated CCD
> on an inverted microscope, no recalling what objective lens, but was 19
> years ago and available in their published papers).
>
> More importantly, M^2 means that your 100x lens is putting 1% as much
> photon flux onto a pixel as a 10x lens would.
>
> My advice: go find a long working distance objective lens that gets enough
> light onto your specimen to get you a useful brightfield image. You could
> later figure out if you need a phase contrast/DIC turret, what NA and
> working distance you need etc.
> I also suggest instead of hardware contrast (DIC requires polarizers and
> prisms ... can avoid illumination side polarizer if using a laser as in
> confocal microscope stand DIC using the transmitted light pathway in
> reverse; phase contrast requires a phase ring in the objective lens -
> usually with a lower NA for a given price point), you start looking into
> synthetic contrast options. The simplest is to just go with digital
> contrast by background subtraction.
> Software to get quantitative phase microscopy data of wet mass (leading to
> dry mass) - a couple of links and comments:
>
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3555125/  ... McCarty ...
> uses 0.1 NA on condenser side.
>
> http://www.jove.com/video/50988/quantitative-optical-microscopy-measurement-cellular-biophysical
> ... McCarty, see downloads (.M files).
>
> Nugent / IATIA (now Ultima Capital) developed the first software only
> solution
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624349/.
> http://www.ncbi.nlm.nih.gov/pubmed/15800856   ... Nugent / IATIA on
> confocal
>
> http://onlinelibrary.wiley.com/doi/10.1111/j.1440-1681.2004.04100.x/abstract
> http://aups.org.au/Proceedings/34/121-127/121-127.pdf   ... fig 3
> illustrates synthetic phase and DIC; fig 4 shows improves segmentation.
>
> http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging/applicationNotes/comparisonWithOpticalPhaseContrastModalities.pdf
>
> http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging/applicationNotes/measurementOfAreaChanges.pdf
> (I think you can ignore the "Confidence-Publication Pending" at top - this
> appnote was posted by the manufacturer and has been online for years).
>
>
> Hardware assisted (not a complete list - some use holography, others
> interferometry):
> Ovizio
> Gabriel Popescu
> Graham Dunn
>
> PubMed has more - a simple search is:   "quantitative phase microscopy"
>
> McCarty's JoVE article now has downloadable .M (MatLab) files. If Anne
> Carpenter or anyone on the Cellprofiler team is reading this (or someone
> send is it their way), I encourage Anne to work with McCarty and their
> University to get "MaCarty QPm" into Cellprofiler.
>
> not QPm, this may still be of interest to listservites:
> Direct imaging of phase objects enables conventional deconvolution in
> bright field light microscopy
> http://www.ncbi.nlm.nih.gov/pubmed/24558478
>
> //
>
> Getting more out of high NA objective lens ... confocal or widefield
> interference reflection microscopy (IRM) provides data on cell-substratum
> adhesion ... including contact area. In reflection confocal (ok, for Jim
> Pawley and Guy Cox: scattered confocal), you can get optical sections of
> the cell, "label free". Some IRM data I posted online:
>
> http://works.bepress.com/gmcnamara/10/
> http://works.bepress.com/gmcnamara/7/
>
> Widefield IRM is very simple if your filter cube does not have an exciter
> filter (best to do this with a wavelength and intensity selectable LED
> illuminator than a broad spectrum arc lamp): just turn on a wavelength(s)
> that enable some light to bounce from the cells/coverglass through the
> dichroic and emission filter (could do even better with a dichroic only,
> and even better with a 50/50 beamsplitter only). My thanks to Tom DiMatteo,
> Epi Technology, for telling me about single LED control on my early gen
> SOLA. I had a long chat with Tom at his ABRF booth a couple of years ago.
> IRM can be quantitative
> http://www.ncbi.nlm.nih.gov/pubmed/23024911
> http://www.ncbi.nlm.nih.gov/pubmed/20013754
> http://www.ncbi.nlm.nih.gov/pubmed/3900106 ... Verschueren
>
>
>
> George
>
>
>
> On 3/18/2016 9:00 PM, Guy Cox wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> This is a little bit oversimplified.   The Rayleigh criterion does not
>> apply to a widefield image, but does apply in fluorescence.  The condenser
>> NA normally IS the objective NA, since they are usually one and the same
>> thing, but condenser NA only affects brightness, not resolution.
>>
>> The Abbe criterion r = 0.5 lambda / NA applies in transmitted light, but
>> ONLY if the condenser aperture equals or exceeds the objective NA.
>> Reducing the condenser NA does not have the same effect as reducing the
>> objective NA.  Reducing the condenser NA to 0 (parallel illumination)
>>  worsens  the resolution to r = lambda/NA - ie 50% of what the objective
>> should give.
>>
>>                                                Guy
>>
>> Guy Cox, Honorary Associate Professor
>> School of Medical Sciences
>>
>> Australian Centre for Microscopy and Microanalysis,
>> Madsen, F09, University of Sydney, NSW 2006
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Aryeh Weiss
>> Sent: Friday, 18 March 2016 11:38 PM
>> To: [hidden email]
>> Subject: Re: Condenser lens choice for a given objective
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> In a transmitted-light brightfield image, the Rayleigh criterion includes
>> both the objective NA and the condenser NA
>> (1.22 lambda/(NA_obj +NA_cond)) . The makes sense because even a very
>> small NA objective can receive light scattered at a large angle if the NA
>> of the condenser is large. (This is how dark-field works).
>> So it would appear that in principle, you benefit from having a condenser
>> with as large as NA as possible  (although you may not have much contrast
>> on that brightfield image).
>>
>> BTW, you can have a "poor" man's dark field scope by using a low-NA
>> objective with a phase ring made for a higher NA objective.
>> For example, in my teaching lab, the students get very nice darkfield
>> images using our 4x/NA=0.1 objective with the ph2 phase ring.
>>
>> --aryeh
>>
>>
>>
>> On 18/03/2016 10:26 AM, Kyle Douglass wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>>> posting.
>>> *****
>>>
>>> Thanks for the feedback, Barbara. It is very helpful.
>>>
>>> I have heard the advice before about the condenser NA needing to be
>>> greater than or equal to the objective NA. Can you offer some physical
>>> explanation or intuition for why this is?
>>>
>>> One admittedly incomplete explanation I can think of for the
>>> recommendation goes like this: the light collected by the objective
>>> consists of two parts. One part is the transmitted light that is not
>>> scattered by the sample. The other part is the light scattered by the
>>> sample. If the condenser's working NA is smaller than the objective's,
>>> then the unscattered, transmitted light fills only a portion of the
>>> objective's back focal plane. However, the light scattered by the
>>> sample will probably be dispersed across the entire back focal plane
>>> because it will encode all the spatial frequencies of the sample.
>>>
>>> I wonder if it's the inhomogeneous distribution of light from the two
>>> components in the objective's back focal plane that leads to the
>>> matched NA requirements of the condenser and objective. Does this make
>>> sense?
>>>
>>> Thanks!
>>> Kyle
>>>
>>> On 03/17/2016 08:22 PM, Barbara Foster wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> Post images on http://www.imgur.com and include the link in your
>>>> posting.
>>>> *****
>>>>
>>>> Hi, Kyle
>>>>
>>>> The general rule of thumb is that the NA on the condenser should meet
>>>> or exceed that of the objective.
>>>>
>>>> If you are using oil immersion objectives, ideally, to achieve that
>>>> goal, you should use an oil immersion condenser, otherwise you are
>>>> limited to an NA of 0.9.
>>>>
>>>> Also, remember that the aperture iris in the condenser adjusts the
>>>> condenser's WORKING  numerical aperture.  Just because the condenser
>>>> is marked 1.4 NA doesn't mean that, in a practical experiment, it
>>>> will be operating at 1.4.  I follow the guidelines set down by Frits
>>>> Zernicke (inventor of Phase contrast):  gently close the aperture
>>>> iris to the "Oomph" position: that delicate balance between
>>>> sufficient edge definition and optimum resolution (Yes, the condenser
>>>> does contribute to resolution).
>>>>
>>>> As for planning for growth:
>>>> You might want to invest in a turret condenser early on.  That will
>>>> give you the option to add those other contrast techniques as you
>>>> grow into them.
>>>>
>>>> And just one more reminder, specifically regarding DIC:
>>>> If you are going to use plastic vessels (petri dishes, multi-well
>>>> plates, growth flasks), use Hoffman Modulation Contrast instead of
>>>> DIC.  DIC uses polarized light.  The plastic will affectt the shear
>>>> and cause effects that will be hard to interpret.  Some HMC set-ups
>>>> do use pol to control the width of the slit in the condenser, but all
>>>> of that is on the incoming side of the sample and will not be
>>>> affected by plastic containers.
>>>>
>>>> Good hunting!
>>>> Barbara Foster, President & Chief Consultant Microscopy/Microscopy
>>>> Education  ... "Education, not Training"
>>>> 7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P:
>>>> 972-924-5310 www.MicroscopyEducation.com
>>>>
>>>> Microscopy/Microscopy Education is a division of The Microscopy &
>>>> Imaging Place, Inc.
>>>>
>>>>
>>>> NEW!   Getting involved in Raman or FTIR?
>>>> MME is now offering courses in these areas specifically for
>>>> microscopists!
>>>> Now scheduling courses through the mid 2016.  We can customize a
>>>> course on nearly any topic, from fluorescence to confocal to image
>>>> analysis to SEM/TEM.
>>>> Call today for a free training evaluation.
>>>>
>>>>
>>>>
>>>>
>>>> At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> Post images on http://www.imgur.com and include the link in your
>>>>> posting.
>>>>> *****
>>>>>
>>>>> Hello listers,
>>>>>
>>>>>
>>>>> I have a couple of questions about condensers for you. I'd like to
>>>>> do some transmitted light imaging in an inverted microscope using
>>>>> high magnification, oil-immersion objectives. For the moment, I
>>>>> don't need to do anything other than brightfield with a high power
>>>>> LED light source. It might be nice to do phase contrast or DIC in
>>>>> the future, but I don't need it now.
>>>>>
>>>>>
>>>>> My questions are:
>>>>>
>>>>>
>>>>> 1) What are the rules of thumb for matching a brightfield condenser
>>>>> to an objective? I won't be using anything but oil immersion
>>>>> objectives with NA's greater than 1.4.
>>>>>
>>>>>
>>>>> 2) If I do want to do phase contrast or DIC in the future, should I
>>>>> put special consideration into the condenser lens selection now? I
>>>>> imagine the condenser NA will determine what phase contrast rings I
>>>>> can use, but does it impact DIC?
>>>>>
>>>>>
>>>>> Thanks!
>>>>>
>>>>> Kyle
>>>>>
>>>>>
>>>>> Dr. Kyle M. Douglass
>>>>> Post-doctoral Researcher
>>>>> EPFL - The Laboratory of Experimental Biophysics http://leb.epfl.ch/
>>>>> http://kmdouglass.github.io
>>>>>
>>>>
>> --
>> Aryeh Weiss
>> Faculty of Engineering
>> Bar Ilan University
>> Ramat Gan 52900 Israel
>>
>> Ph:  972-3-5317638
>> FAX: [phone]
>>
>>
>
> --
>
>
>
> George McNamara, Ph.D.
> Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab)
> University of Texas M.D. Anderson Cancer Center
> Houston, TX 77054
> Tattletales http://works.bepress.com/gmcnamara/42
> http://works.bepress.com/gmcnamara/75
> https://www.linkedin.com/in/georgemcnamara
>

[Guy Cox-2]

Look, it all depends on what you are looking for.  I can get an entire class to resolve 500nm with an NA 0.65 objective but it isn’t easy!  These days we are rarely looking for ultimate resolution in brightfield but I think there are still times when we need it.   However, I do understand that usually when we are working at the limit we are in fluorescence where Rayleigh applies.  

                                               Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michael Model
Sent: Sunday, 20 March 2016 11:33 PM
To: [hidden email]
Subject: Re: Condenser lens choice for a given objective

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

In my opinion, visibility in bright field is more limited by contrast than by theoretical resolution...

As for brightness, "in the absence of strong scattering, brightness in transmitted illumination depends mostly on direct, and not on diffracted, light (as the popular formula (NAob/M)2 assumes) and thus on the smallest NA between the objective and condenser; this can be easily verified by using an objective with a variable numerical aperture. "

Mike Model

On Sat, Mar 19, 2016 at 1:07 PM, George McNamara <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Kyle,
>
> Your post indicates that for now you just want to get a brightfield
> image through your 100x objective lens. The purpose of an objective
> lens is two
> fold:
> 1. resolution ... emphasized in the replies below ... high NA
> condenser results in higher spatial resolution.
> 2. brightness ... get enough photon flux so you can do your experiment.
>
> NA: at Guy's limit of zero NA on the illumination side, you will few
> photons reaching the objective lens and detector(s). You will also
> have
> (practically) infinite depth of focus with respect to imaging dust and
> other things on every optical surface. Many of these could be cleaned
> up by background subtraction (plus constant).
>
> Brightness is proportional to:
>
>         NA^4
> B ~ ----------
>          M^2
>
> The numerator is assuming equal NA (ex. epi-illumination with a single
> objective lens). I assume this could be re-written as
>
> (NAcond^2)*(NAobj^2)
>
> which is ok until Guy's limit of NAcond = 0. I'll also mention that
> you do not even need a transmitted light LED, condenser, or condenser
> arm if you have a sensitive enough detector(s): room lights, desk
> lamps, computer monitors can provide enough light (I first encountered
> this problem/feature working with James Sabry in Jim Spudich's lab
> using a back illuminated CCD on an inverted microscope, no recalling
> what objective lens, but was 19 years ago and available in their published papers).
>
> More importantly, M^2 means that your 100x lens is putting 1% as much
> photon flux onto a pixel as a 10x lens would.
>
> My advice: go find a long working distance objective lens that gets
> enough light onto your specimen to get you a useful brightfield image.
> You could later figure out if you need a phase contrast/DIC turret,
> what NA and working distance you need etc.
> I also suggest instead of hardware contrast (DIC requires polarizers
> and prisms ... can avoid illumination side polarizer if using a laser
> as in confocal microscope stand DIC using the transmitted light
> pathway in reverse; phase contrast requires a phase ring in the
> objective lens - usually with a lower NA for a given price point), you
> start looking into synthetic contrast options. The simplest is to just
> go with digital contrast by background subtraction.
> Software to get quantitative phase microscopy data of wet mass
> (leading to dry mass) - a couple of links and comments:
>
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3555125/  ... McCarty ...
> uses 0.1 NA on condenser side.
>
> http://www.jove.com/video/50988/quantitative-optical-microscopy-measur
> ement-cellular-biophysical ... McCarty, see downloads (.M files).
>
> Nugent / IATIA (now Ultima Capital) developed the first software only
> solution http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624349/.
> http://www.ncbi.nlm.nih.gov/pubmed/15800856   ... Nugent / IATIA on
> confocal
>
> http://onlinelibrary.wiley.com/doi/10.1111/j.1440-1681.2004.04100.x/abstract
> http://aups.org.au/Proceedings/34/121-127/121-127.pdf   ... fig 3
> illustrates synthetic phase and DIC; fig 4 shows improves segmentation.
>
> http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging
> /applicationNotes/comparisonWithOpticalPhaseContrastModalities.pdf
>
> http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging
> /applicationNotes/measurementOfAreaChanges.pdf
> (I think you can ignore the "Confidence-Publication Pending" at top -
> this appnote was posted by the manufacturer and has been online for years).
>
>
> Hardware assisted (not a complete list - some use holography, others
> interferometry):
> Ovizio
> Gabriel Popescu
> Graham Dunn
>
> PubMed has more - a simple search is:   "quantitative phase microscopy"
>
> McCarty's JoVE article now has downloadable .M (MatLab) files. If Anne
> Carpenter or anyone on the Cellprofiler team is reading this (or
> someone send is it their way), I encourage Anne to work with McCarty
> and their University to get "MaCarty QPm" into Cellprofiler.
>
> not QPm, this may still be of interest to listservites:
> Direct imaging of phase objects enables conventional deconvolution in
> bright field light microscopy
> http://www.ncbi.nlm.nih.gov/pubmed/24558478
>
> //
>
> Getting more out of high NA objective lens ... confocal or widefield
> interference reflection microscopy (IRM) provides data on
> cell-substratum adhesion ... including contact area. In reflection
> confocal (ok, for Jim Pawley and Guy Cox: scattered confocal), you can
> get optical sections of the cell, "label free". Some IRM data I posted online:
>
> http://works.bepress.com/gmcnamara/10/
> http://works.bepress.com/gmcnamara/7/
>
> Widefield IRM is very simple if your filter cube does not have an
> exciter filter (best to do this with a wavelength and intensity
> selectable LED illuminator than a broad spectrum arc lamp): just turn
> on a wavelength(s) that enable some light to bounce from the
> cells/coverglass through the dichroic and emission filter (could do
> even better with a dichroic only, and even better with a 50/50
> beamsplitter only). My thanks to Tom DiMatteo, Epi Technology, for
> telling me about single LED control on my early gen SOLA. I had a long chat with Tom at his ABRF booth a couple of years ago.
> IRM can be quantitative
> http://www.ncbi.nlm.nih.gov/pubmed/23024911
> http://www.ncbi.nlm.nih.gov/pubmed/20013754
> http://www.ncbi.nlm.nih.gov/pubmed/3900106 ... Verschueren
>
>
>
> George
>
>
>
> On 3/18/2016 9:00 PM, Guy Cox wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> This is a little bit oversimplified.   The Rayleigh criterion does not
>> apply to a widefield image, but does apply in fluorescence.  The
>> condenser NA normally IS the objective NA, since they are usually one
>> and the same thing, but condenser NA only affects brightness, not resolution.
>>
>> The Abbe criterion r = 0.5 lambda / NA applies in transmitted light,
>> but ONLY if the condenser aperture equals or exceeds the objective NA.
>> Reducing the condenser NA does not have the same effect as reducing
>> the objective NA.  Reducing the condenser NA to 0 (parallel
>> illumination)  worsens  the resolution to r = lambda/NA - ie 50% of
>> what the objective should give.
>>
>>                                                Guy
>>
>> Guy Cox, Honorary Associate Professor School of Medical Sciences
>>
>> Australian Centre for Microscopy and Microanalysis, Madsen, F09,
>> University of Sydney, NSW 2006
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]]
>> On Behalf Of Aryeh Weiss
>> Sent: Friday, 18 March 2016 11:38 PM
>> To: [hidden email]
>> Subject: Re: Condenser lens choice for a given objective
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> In a transmitted-light brightfield image, the Rayleigh criterion
>> includes both the objective NA and the condenser NA
>> (1.22 lambda/(NA_obj +NA_cond)) . The makes sense because even a very
>> small NA objective can receive light scattered at a large angle if
>> the NA of the condenser is large. (This is how dark-field works).
>> So it would appear that in principle, you benefit from having a
>> condenser with as large as NA as possible  (although you may not have
>> much contrast on that brightfield image).
>>
>> BTW, you can have a "poor" man's dark field scope by using a low-NA
>> objective with a phase ring made for a higher NA objective.
>> For example, in my teaching lab, the students get very nice darkfield
>> images using our 4x/NA=0.1 objective with the ph2 phase ring.
>>
>> --aryeh
>>
>>
>>
>> On 18/03/2016 10:26 AM, Kyle Douglass wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>>> posting.
>>> *****
>>>
>>> Thanks for the feedback, Barbara. It is very helpful.
>>>
>>> I have heard the advice before about the condenser NA needing to be
>>> greater than or equal to the objective NA. Can you offer some
>>> physical explanation or intuition for why this is?
>>>
>>> One admittedly incomplete explanation I can think of for the
>>> recommendation goes like this: the light collected by the objective
>>> consists of two parts. One part is the transmitted light that is not
>>> scattered by the sample. The other part is the light scattered by
>>> the sample. If the condenser's working NA is smaller than the
>>> objective's, then the unscattered, transmitted light fills only a
>>> portion of the objective's back focal plane. However, the light
>>> scattered by the sample will probably be dispersed across the entire
>>> back focal plane because it will encode all the spatial frequencies of the sample.
>>>
>>> I wonder if it's the inhomogeneous distribution of light from the
>>> two components in the objective's back focal plane that leads to the
>>> matched NA requirements of the condenser and objective. Does this
>>> make sense?
>>>
>>> Thanks!
>>> Kyle
>>>
>>> On 03/17/2016 08:22 PM, Barbara Foster wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> Post images on http://www.imgur.com and include the link in your
>>>> posting.
>>>> *****
>>>>
>>>> Hi, Kyle
>>>>
>>>> The general rule of thumb is that the NA on the condenser should
>>>> meet or exceed that of the objective.
>>>>
>>>> If you are using oil immersion objectives, ideally, to achieve that
>>>> goal, you should use an oil immersion condenser, otherwise you are
>>>> limited to an NA of 0.9.
>>>>
>>>> Also, remember that the aperture iris in the condenser adjusts the
>>>> condenser's WORKING  numerical aperture.  Just because the
>>>> condenser is marked 1.4 NA doesn't mean that, in a practical
>>>> experiment, it will be operating at 1.4.  I follow the guidelines
>>>> set down by Frits Zernicke (inventor of Phase contrast):  gently
>>>> close the aperture iris to the "Oomph" position: that delicate
>>>> balance between sufficient edge definition and optimum resolution
>>>> (Yes, the condenser does contribute to resolution).
>>>>
>>>> As for planning for growth:
>>>> You might want to invest in a turret condenser early on.  That will
>>>> give you the option to add those other contrast techniques as you
>>>> grow into them.
>>>>
>>>> And just one more reminder, specifically regarding DIC:
>>>> If you are going to use plastic vessels (petri dishes, multi-well
>>>> plates, growth flasks), use Hoffman Modulation Contrast instead of
>>>> DIC.  DIC uses polarized light.  The plastic will affectt the shear
>>>> and cause effects that will be hard to interpret.  Some HMC set-ups
>>>> do use pol to control the width of the slit in the condenser, but
>>>> all of that is on the incoming side of the sample and will not be
>>>> affected by plastic containers.
>>>>
>>>> Good hunting!
>>>> Barbara Foster, President & Chief Consultant Microscopy/Microscopy
>>>> Education  ... "Education, not Training"
>>>> 7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P:
>>>> 972-924-5310 www.MicroscopyEducation.com
>>>>
>>>> Microscopy/Microscopy Education is a division of The Microscopy &
>>>> Imaging Place, Inc.
>>>>
>>>>
>>>> NEW!   Getting involved in Raman or FTIR?
>>>> MME is now offering courses in these areas specifically for
>>>> microscopists!
>>>> Now scheduling courses through the mid 2016.  We can customize a
>>>> course on nearly any topic, from fluorescence to confocal to image
>>>> analysis to SEM/TEM.
>>>> Call today for a free training evaluation.
>>>>
>>>>
>>>>
>>>>
>>>> At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> Post images on http://www.imgur.com and include the link in your
>>>>> posting.
>>>>> *****
>>>>>
>>>>> Hello listers,
>>>>>
>>>>>
>>>>> I have a couple of questions about condensers for you. I'd like to
>>>>> do some transmitted light imaging in an inverted microscope using
>>>>> high magnification, oil-immersion objectives. For the moment, I
>>>>> don't need to do anything other than brightfield with a high power
>>>>> LED light source. It might be nice to do phase contrast or DIC in
>>>>> the future, but I don't need it now.
>>>>>
>>>>>
>>>>> My questions are:
>>>>>
>>>>>
>>>>> 1) What are the rules of thumb for matching a brightfield
>>>>> condenser to an objective? I won't be using anything but oil
>>>>> immersion objectives with NA's greater than 1.4.
>>>>>
>>>>>
>>>>> 2) If I do want to do phase contrast or DIC in the future, should
>>>>> I put special consideration into the condenser lens selection now?
>>>>> I imagine the condenser NA will determine what phase contrast
>>>>> rings I can use, but does it impact DIC?
>>>>>
>>>>>
>>>>> Thanks!
>>>>>
>>>>> Kyle
>>>>>
>>>>>
>>>>> Dr. Kyle M. Douglass
>>>>> Post-doctoral Researcher
>>>>> EPFL - The Laboratory of Experimental Biophysics
>>>>> http://leb.epfl.ch/ http://kmdouglass.github.io
>>>>>
>>>>
>> --
>> Aryeh Weiss
>> Faculty of Engineering
>> Bar Ilan University
>> Ramat Gan 52900 Israel
>>
>> Ph:  972-3-5317638
>> FAX: [phone]
>>
>>
>
> --
>
>
>
> George McNamara, Ph.D.
> Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab) University of
> Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales
> http://works.bepress.com/gmcnamara/42
> http://works.bepress.com/gmcnamara/75
> https://www.linkedin.com/in/georgemcnamara
>

[George McNamara]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Michael,

Contrast limited? ... only if using eyes.

Video (1970s+, Reynolds, Shinya Inoue, Robert & Nina Allen, later
"Nanovid" folks, Dodt NIR-VEC-DIC),
computer frame grabber background subtraction + offset (late 1980s, ex.
Matrox MVP-AT board and Image-1/AT) and more recently (very late 1980s,
early 1990s)
scientific digital CCD cameras (ex. Photometrics Star-1, Hamamatsu
digital CCDs [C4742?]) and background subtraction in computer memory.

Reference (which can correct any of my dates): Shinya Inoue, 1986 Video
Microscopy (or more recently Inoue & Spring).

//

An early review on video (brightfield is not mentioned in the abstract)
- Reynolds 1972 PubMed 4404351

George T. Reynolds


      Image intersification applied to biological problems


Quarterly Reviews of Biophysics
<http://journals.cambridge.org/action/displayJournal?jid=QRB> /Volume  5
<http://journals.cambridge.org/action/displayBackIssues?jid=QRB&volumeId=5> /Issue 03
<http://journals.cambridge.org/action/displayIssue?jid=QRB&volumeId=5&seriesId=0&issueId=03> /
August 1972, pp 295 - 347

DOI:http://dx.doi.org/10.1017/S0033583500000974(About DOI
<http://journals.cambridge.org/action/stream?pageId=3624&level=2&sessionId=F32BC6B4AAE6DB50F8D0214BB5DF4743.journals#30>),


In many important types of observations in biological research, the
information provided by the specimens is in the form of photons-quanta
of visible light, u.v., or X-rays. The process of observation becomes
one of recording this information in useful form, with as high an
efficiency as possible. The problem becomes particularly important when
for some reason or other the total number or rate of quanta provided by
the specimen is small. Examples of such limitations are included in the
following: (i) Processes permitting only low-intensity illumination in
order not to interfere with the biological processes under observation.
(ii) Processes changing very rapidly and requiring rapid sequence
recording. (iii) Processes providing only a limited number of photons
per event, such as bioluminescence. (iv) Processes in which radioactive
tracers are utilized, and observation of radioactivity is desirable at
low specific activity or within short time intervals. (v) X-ray
diffraction processes where the specimen is weakly diffracting or where
the X-ray intensity must be kept low in order not to damage the
specimen. (vi) Processes involving the observation of fluorescence,
where the intensity is low because of limitations on the amount of
tagging material.

A
George

On 3/20/2016 7:33 AM, Michael Model wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> In my opinion, visibility in bright field is more limited by contrast than
> by theoretical resolution...
>
> As for brightness, "in the absence of strong scattering, brightness in
> transmitted illumination depends mostly on direct, and not on diffracted,
> light (as the popular formula (NAob/M)2 assumes) and thus on the smallest
> NA between the objective and condenser; this can be easily verified by
> using an objective with a variable numerical aperture. "
>
> Mike Model
>
> On Sat, Mar 19, 2016 at 1:07 PM, George McNamara <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Hi Kyle,
>>
>> Your post indicates that for now you just want to get a brightfield image
>> through your 100x objective lens. The purpose of an objective lens is two
>> fold:
>> 1. resolution ... emphasized in the replies below ... high NA condenser
>> results in higher spatial resolution.
>> 2. brightness ... get enough photon flux so you can do your experiment.
>>
>> NA: at Guy's limit of zero NA on the illumination side, you will few
>> photons reaching the objective lens and detector(s). You will also have
>> (practically) infinite depth of focus with respect to imaging dust and
>> other things on every optical surface. Many of these could be cleaned up by
>> background subtraction (plus constant).
>>
>> Brightness is proportional to:
>>
>>          NA^4
>> B ~ ----------
>>           M^2
>>
>> The numerator is assuming equal NA (ex. epi-illumination with a single
>> objective lens). I assume this could be re-written as
>>
>> (NAcond^2)*(NAobj^2)
>>
>> which is ok until Guy's limit of NAcond = 0. I'll also mention that you do
>> not even need a transmitted light LED, condenser, or condenser arm if you
>> have a sensitive enough detector(s): room lights, desk lamps, computer
>> monitors can provide enough light (I first encountered this problem/feature
>> working with James Sabry in Jim Spudich's lab using a back illuminated CCD
>> on an inverted microscope, no recalling what objective lens, but was 19
>> years ago and available in their published papers).
>>
>> More importantly, M^2 means that your 100x lens is putting 1% as much
>> photon flux onto a pixel as a 10x lens would.
>>
>> My advice: go find a long working distance objective lens that gets enough
>> light onto your specimen to get you a useful brightfield image. You could
>> later figure out if you need a phase contrast/DIC turret, what NA and
>> working distance you need etc.
>> I also suggest instead of hardware contrast (DIC requires polarizers and
>> prisms ... can avoid illumination side polarizer if using a laser as in
>> confocal microscope stand DIC using the transmitted light pathway in
>> reverse; phase contrast requires a phase ring in the objective lens -
>> usually with a lower NA for a given price point), you start looking into
>> synthetic contrast options. The simplest is to just go with digital
>> contrast by background subtraction.
>> Software to get quantitative phase microscopy data of wet mass (leading to
>> dry mass) - a couple of links and comments:
>>
>> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3555125/  ... McCarty ...
>> uses 0.1 NA on condenser side.
>>
>> http://www.jove.com/video/50988/quantitative-optical-microscopy-measurement-cellular-biophysical
>> ... McCarty, see downloads (.M files).
>>
>> Nugent / IATIA (now Ultima Capital) developed the first software only
>> solution
>> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624349/.
>> http://www.ncbi.nlm.nih.gov/pubmed/15800856   ... Nugent / IATIA on
>> confocal
>>
>> http://onlinelibrary.wiley.com/doi/10.1111/j.1440-1681.2004.04100.x/abstract
>> http://aups.org.au/Proceedings/34/121-127/121-127.pdf   ... fig 3
>> illustrates synthetic phase and DIC; fig 4 shows improves segmentation.
>>
>> http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging/applicationNotes/comparisonWithOpticalPhaseContrastModalities.pdf
>>
>> http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging/applicationNotes/measurementOfAreaChanges.pdf
>> (I think you can ignore the "Confidence-Publication Pending" at top - this
>> appnote was posted by the manufacturer and has been online for years).
>>
>>
>> Hardware assisted (not a complete list - some use holography, others
>> interferometry):
>> Ovizio
>> Gabriel Popescu
>> Graham Dunn
>>
>> PubMed has more - a simple search is:   "quantitative phase microscopy"
>>
>> McCarty's JoVE article now has downloadable .M (MatLab) files. If Anne
>> Carpenter or anyone on the Cellprofiler team is reading this (or someone
>> send is it their way), I encourage Anne to work with McCarty and their
>> University to get "MaCarty QPm" into Cellprofiler.
>>
>> not QPm, this may still be of interest to listservites:
>> Direct imaging of phase objects enables conventional deconvolution in
>> bright field light microscopy
>> http://www.ncbi.nlm.nih.gov/pubmed/24558478
>>
>> //
>>
>> Getting more out of high NA objective lens ... confocal or widefield
>> interference reflection microscopy (IRM) provides data on cell-substratum
>> adhesion ... including contact area. In reflection confocal (ok, for Jim
>> Pawley and Guy Cox: scattered confocal), you can get optical sections of
>> the cell, "label free". Some IRM data I posted online:
>>
>> http://works.bepress.com/gmcnamara/10/
>> http://works.bepress.com/gmcnamara/7/
>>
>> Widefield IRM is very simple if your filter cube does not have an exciter
>> filter (best to do this with a wavelength and intensity selectable LED
>> illuminator than a broad spectrum arc lamp): just turn on a wavelength(s)
>> that enable some light to bounce from the cells/coverglass through the
>> dichroic and emission filter (could do even better with a dichroic only,
>> and even better with a 50/50 beamsplitter only). My thanks to Tom DiMatteo,
>> Epi Technology, for telling me about single LED control on my early gen
>> SOLA. I had a long chat with Tom at his ABRF booth a couple of years ago.
>> IRM can be quantitative
>> http://www.ncbi.nlm.nih.gov/pubmed/23024911
>> http://www.ncbi.nlm.nih.gov/pubmed/20013754
>> http://www.ncbi.nlm.nih.gov/pubmed/3900106 ... Verschueren
>>
>>
>>
>> George
>>
>>
>>
>> On 3/18/2016 9:00 PM, Guy Cox wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your posting.
>>> *****
>>>
>>> This is a little bit oversimplified.   The Rayleigh criterion does not
>>> apply to a widefield image, but does apply in fluorescence.  The condenser
>>> NA normally IS the objective NA, since they are usually one and the same
>>> thing, but condenser NA only affects brightness, not resolution.
>>>
>>> The Abbe criterion r = 0.5 lambda / NA applies in transmitted light, but
>>> ONLY if the condenser aperture equals or exceeds the objective NA.
>>> Reducing the condenser NA does not have the same effect as reducing the
>>> objective NA.  Reducing the condenser NA to 0 (parallel illumination)
>>>   worsens  the resolution to r = lambda/NA - ie 50% of what the objective
>>> should give.
>>>
>>>                                                 Guy
>>>
>>> Guy Cox, Honorary Associate Professor
>>> School of Medical Sciences
>>>
>>> Australian Centre for Microscopy and Microanalysis,
>>> Madsen, F09, University of Sydney, NSW 2006
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]]
>>> On Behalf Of Aryeh Weiss
>>> Sent: Friday, 18 March 2016 11:38 PM
>>> To: [hidden email]
>>> Subject: Re: Condenser lens choice for a given objective
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your posting.
>>> *****
>>>
>>> In a transmitted-light brightfield image, the Rayleigh criterion includes
>>> both the objective NA and the condenser NA
>>> (1.22 lambda/(NA_obj +NA_cond)) . The makes sense because even a very
>>> small NA objective can receive light scattered at a large angle if the NA
>>> of the condenser is large. (This is how dark-field works).
>>> So it would appear that in principle, you benefit from having a condenser
>>> with as large as NA as possible  (although you may not have much contrast
>>> on that brightfield image).
>>>
>>> BTW, you can have a "poor" man's dark field scope by using a low-NA
>>> objective with a phase ring made for a higher NA objective.
>>> For example, in my teaching lab, the students get very nice darkfield
>>> images using our 4x/NA=0.1 objective with the ph2 phase ring.
>>>
>>> --aryeh
>>>
>>>
>>>
>>> On 18/03/2016 10:26 AM, Kyle Douglass wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> Post images on http://www.imgur.com and include the link in your
>>>> posting.
>>>> *****
>>>>
>>>> Thanks for the feedback, Barbara. It is very helpful.
>>>>
>>>> I have heard the advice before about the condenser NA needing to be
>>>> greater than or equal to the objective NA. Can you offer some physical
>>>> explanation or intuition for why this is?
>>>>
>>>> One admittedly incomplete explanation I can think of for the
>>>> recommendation goes like this: the light collected by the objective
>>>> consists of two parts. One part is the transmitted light that is not
>>>> scattered by the sample. The other part is the light scattered by the
>>>> sample. If the condenser's working NA is smaller than the objective's,
>>>> then the unscattered, transmitted light fills only a portion of the
>>>> objective's back focal plane. However, the light scattered by the
>>>> sample will probably be dispersed across the entire back focal plane
>>>> because it will encode all the spatial frequencies of the sample.
>>>>
>>>> I wonder if it's the inhomogeneous distribution of light from the two
>>>> components in the objective's back focal plane that leads to the
>>>> matched NA requirements of the condenser and objective. Does this make
>>>> sense?
>>>>
>>>> Thanks!
>>>> Kyle
>>>>
>>>> On 03/17/2016 08:22 PM, Barbara Foster wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> Post images on http://www.imgur.com and include the link in your
>>>>> posting.
>>>>> *****
>>>>>
>>>>> Hi, Kyle
>>>>>
>>>>> The general rule of thumb is that the NA on the condenser should meet
>>>>> or exceed that of the objective.
>>>>>
>>>>> If you are using oil immersion objectives, ideally, to achieve that
>>>>> goal, you should use an oil immersion condenser, otherwise you are
>>>>> limited to an NA of 0.9.
>>>>>
>>>>> Also, remember that the aperture iris in the condenser adjusts the
>>>>> condenser's WORKING  numerical aperture.  Just because the condenser
>>>>> is marked 1.4 NA doesn't mean that, in a practical experiment, it
>>>>> will be operating at 1.4.  I follow the guidelines set down by Frits
>>>>> Zernicke (inventor of Phase contrast):  gently close the aperture
>>>>> iris to the "Oomph" position: that delicate balance between
>>>>> sufficient edge definition and optimum resolution (Yes, the condenser
>>>>> does contribute to resolution).
>>>>>
>>>>> As for planning for growth:
>>>>> You might want to invest in a turret condenser early on.  That will
>>>>> give you the option to add those other contrast techniques as you
>>>>> grow into them.
>>>>>
>>>>> And just one more reminder, specifically regarding DIC:
>>>>> If you are going to use plastic vessels (petri dishes, multi-well
>>>>> plates, growth flasks), use Hoffman Modulation Contrast instead of
>>>>> DIC.  DIC uses polarized light.  The plastic will affectt the shear
>>>>> and cause effects that will be hard to interpret.  Some HMC set-ups
>>>>> do use pol to control the width of the slit in the condenser, but all
>>>>> of that is on the incoming side of the sample and will not be
>>>>> affected by plastic containers.
>>>>>
>>>>> Good hunting!
>>>>> Barbara Foster, President & Chief Consultant Microscopy/Microscopy
>>>>> Education  ... "Education, not Training"
>>>>> 7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P:
>>>>> 972-924-5310 www.MicroscopyEducation.com
>>>>>
>>>>> Microscopy/Microscopy Education is a division of The Microscopy &
>>>>> Imaging Place, Inc.
>>>>>
>>>>>
>>>>> NEW!   Getting involved in Raman or FTIR?
>>>>> MME is now offering courses in these areas specifically for
>>>>> microscopists!
>>>>> Now scheduling courses through the mid 2016.  We can customize a
>>>>> course on nearly any topic, from fluorescence to confocal to image
>>>>> analysis to SEM/TEM.
>>>>> Call today for a free training evaluation.
>>>>>
>>>>>
>>>>>
>>>>>
>>>>> At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:
>>>>>
>>>>>> *****
>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>> Post images on http://www.imgur.com and include the link in your
>>>>>> posting.
>>>>>> *****
>>>>>>
>>>>>> Hello listers,
>>>>>>
>>>>>>
>>>>>> I have a couple of questions about condensers for you. I'd like to
>>>>>> do some transmitted light imaging in an inverted microscope using
>>>>>> high magnification, oil-immersion objectives. For the moment, I
>>>>>> don't need to do anything other than brightfield with a high power
>>>>>> LED light source. It might be nice to do phase contrast or DIC in
>>>>>> the future, but I don't need it now.
>>>>>>
>>>>>>
>>>>>> My questions are:
>>>>>>
>>>>>>
>>>>>> 1) What are the rules of thumb for matching a brightfield condenser
>>>>>> to an objective? I won't be using anything but oil immersion
>>>>>> objectives with NA's greater than 1.4.
>>>>>>
>>>>>>
>>>>>> 2) If I do want to do phase contrast or DIC in the future, should I
>>>>>> put special consideration into the condenser lens selection now? I
>>>>>> imagine the condenser NA will determine what phase contrast rings I
>>>>>> can use, but does it impact DIC?
>>>>>>
>>>>>>
>>>>>> Thanks!
>>>>>>
>>>>>> Kyle
>>>>>>
>>>>>>
>>>>>> Dr. Kyle M. Douglass
>>>>>> Post-doctoral Researcher
>>>>>> EPFL - The Laboratory of Experimental Biophysics http://leb.epfl.ch/
>>>>>> http://kmdouglass.github.io
>>>>>>
>>> --
>>> Aryeh Weiss
>>> Faculty of Engineering
>>> Bar Ilan University
>>> Ramat Gan 52900 Israel
>>>
>>> Ph:  972-3-5317638
>>> FAX: [phone]
>>>
>>>
>> --
>>
>>
>>
>> George McNamara, Ph.D.
>> Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab)
>> University of Texas M.D. Anderson Cancer Center
>> Houston, TX 77054
>> Tattletales http://works.bepress.com/gmcnamara/42
>> http://works.bepress.com/gmcnamara/75
>> https://www.linkedin.com/in/georgemcnamara
>>

--



George McNamara, Ph.D.
Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab)
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42
http://works.bepress.com/gmcnamara/75
https://www.linkedin.com/in/georgemcnamara

[Kyle Michael Douglass]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Thanks everyone for your input. Here's the summary of what I learned
(please correct me if I am wrong):

Resolution in transmitted light bright field microscopy depends just as
much on the condenser NA as on the objective's NA. The light that is
scattered from the sample into the objective by light waves traveling
off-axis encode the fine details in the specimen. Illuminating the
sample at large angles results in scattered waves entering the objective
that otherwise would not have been within the objective's NA had the
sample been illuminated with on-axis, parallel light.

On the other hand, to improve contrast, there are numerous digital means
offering background subtraction, etc. Optically, contrast can be
improved by illuminating the sample with on-axis, collimated light.

I would conclude based on the discussion that, if one performs no
post-processing, there is a trade-off between resolution and contrast in
transmitted light bright field microscopy. One must decide what is best
for his or her experiment and set the illumination accordingly.

Thanks again!
Kyle

On 03/20/2016 03:51 PM, George McNamara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Michael,
>
> Contrast limited? ... only if using eyes.
>
> Video (1970s+, Reynolds, Shinya Inoue, Robert & Nina Allen, later
> "Nanovid" folks, Dodt NIR-VEC-DIC),
> computer frame grabber background subtraction + offset (late 1980s,
> ex. Matrox MVP-AT board and Image-1/AT) and more recently (very late
> 1980s, early 1990s)
> scientific digital CCD cameras (ex. Photometrics Star-1, Hamamatsu
> digital CCDs [C4742?]) and background subtraction in computer memory.
>
> Reference (which can correct any of my dates): Shinya Inoue, 1986
> Video Microscopy (or more recently Inoue & Spring).
>
> //
>
> An early review on video (brightfield is not mentioned in the
> abstract) - Reynolds 1972 PubMed 4404351
>
> George T. Reynolds
>
>
>      Image intersification applied to biological problems
>
>
> Quarterly Reviews of Biophysics
> <http://journals.cambridge.org/action/displayJournal?jid=QRB> /Volume  
> 5
> <http://journals.cambridge.org/action/displayBackIssues?jid=QRB&volumeId=5>
> /Issue 03
> <http://journals.cambridge.org/action/displayIssue?jid=QRB&volumeId=5&seriesId=0&issueId=03>
> / August 1972, pp 295 - 347
>
> DOI:http://dx.doi.org/10.1017/S0033583500000974(About DOI
> <http://journals.cambridge.org/action/stream?pageId=3624&level=2&sessionId=F32BC6B4AAE6DB50F8D0214BB5DF4743.journals#30>),
>
>
> In many important types of observations in biological research, the
> information provided by the specimens is in the form of photons-quanta
> of visible light, u.v., or X-rays. The process of observation becomes
> one of recording this information in useful form, with as high an
> efficiency as possible. The problem becomes particularly important
> when for some reason or other the total number or rate of quanta
> provided by the specimen is small. Examples of such limitations are
> included in the following: (i) Processes permitting only low-intensity
> illumination in order not to interfere with the biological processes
> under observation. (ii) Processes changing very rapidly and requiring
> rapid sequence recording. (iii) Processes providing only a limited
> number of photons per event, such as bioluminescence. (iv) Processes
> in which radioactive tracers are utilized, and observation of
> radioactivity is desirable at low specific activity or within short
> time intervals. (v) X-ray diffraction processes where the specimen is
> weakly diffracting or where the X-ray intensity must be kept low in
> order not to damage the specimen. (vi) Processes involving the
> observation of fluorescence, where the intensity is low because of
> limitations on the amount of tagging material.
>
> A
> George
>
> On 3/20/2016 7:33 AM, Michael Model wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your
>> posting.
>> *****
>>
>> In my opinion, visibility in bright field is more limited by contrast
>> than
>> by theoretical resolution...
>>
>> As for brightness, "in the absence of strong scattering, brightness in
>> transmitted illumination depends mostly on direct, and not on
>> diffracted,
>> light (as the popular formula (NAob/M)2 assumes) and thus on the
>> smallest
>> NA between the objective and condenser; this can be easily verified by
>> using an objective with a variable numerical aperture. "
>>
>> Mike Model
>>
>> On Sat, Mar 19, 2016 at 1:07 PM, George McNamara
>> <[hidden email]>
>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>>> posting.
>>> *****
>>>
>>> Hi Kyle,
>>>
>>> Your post indicates that for now you just want to get a brightfield
>>> image
>>> through your 100x objective lens. The purpose of an objective lens
>>> is two
>>> fold:
>>> 1. resolution ... emphasized in the replies below ... high NA condenser
>>> results in higher spatial resolution.
>>> 2. brightness ... get enough photon flux so you can do your experiment.
>>>
>>> NA: at Guy's limit of zero NA on the illumination side, you will few
>>> photons reaching the objective lens and detector(s). You will also have
>>> (practically) infinite depth of focus with respect to imaging dust and
>>> other things on every optical surface. Many of these could be
>>> cleaned up by
>>> background subtraction (plus constant).
>>>
>>> Brightness is proportional to:
>>>
>>>          NA^4
>>> B ~ ----------
>>>           M^2
>>>
>>> The numerator is assuming equal NA (ex. epi-illumination with a single
>>> objective lens). I assume this could be re-written as
>>>
>>> (NAcond^2)*(NAobj^2)
>>>
>>> which is ok until Guy's limit of NAcond = 0. I'll also mention that
>>> you do
>>> not even need a transmitted light LED, condenser, or condenser arm
>>> if you
>>> have a sensitive enough detector(s): room lights, desk lamps, computer
>>> monitors can provide enough light (I first encountered this
>>> problem/feature
>>> working with James Sabry in Jim Spudich's lab using a back
>>> illuminated CCD
>>> on an inverted microscope, no recalling what objective lens, but was 19
>>> years ago and available in their published papers).
>>>
>>> More importantly, M^2 means that your 100x lens is putting 1% as much
>>> photon flux onto a pixel as a 10x lens would.
>>>
>>> My advice: go find a long working distance objective lens that gets
>>> enough
>>> light onto your specimen to get you a useful brightfield image. You
>>> could
>>> later figure out if you need a phase contrast/DIC turret, what NA and
>>> working distance you need etc.
>>> I also suggest instead of hardware contrast (DIC requires polarizers
>>> and
>>> prisms ... can avoid illumination side polarizer if using a laser as in
>>> confocal microscope stand DIC using the transmitted light pathway in
>>> reverse; phase contrast requires a phase ring in the objective lens -
>>> usually with a lower NA for a given price point), you start looking
>>> into
>>> synthetic contrast options. The simplest is to just go with digital
>>> contrast by background subtraction.
>>> Software to get quantitative phase microscopy data of wet mass
>>> (leading to
>>> dry mass) - a couple of links and comments:
>>>
>>> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3555125/  ... McCarty ...
>>> uses 0.1 NA on condenser side.
>>>
>>> http://www.jove.com/video/50988/quantitative-optical-microscopy-measurement-cellular-biophysical 
>>>
>>> ... McCarty, see downloads (.M files).
>>>
>>> Nugent / IATIA (now Ultima Capital) developed the first software only
>>> solution
>>> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624349/.
>>> http://www.ncbi.nlm.nih.gov/pubmed/15800856   ... Nugent / IATIA on
>>> confocal
>>>
>>> http://onlinelibrary.wiley.com/doi/10.1111/j.1440-1681.2004.04100.x/abstract 
>>>
>>> http://aups.org.au/Proceedings/34/121-127/121-127.pdf   ... fig 3
>>> illustrates synthetic phase and DIC; fig 4 shows improves segmentation.
>>>
>>> http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging/applicationNotes/comparisonWithOpticalPhaseContrastModalities.pdf 
>>>
>>>
>>> http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imaging/applicationNotes/measurementOfAreaChanges.pdf 
>>>
>>> (I think you can ignore the "Confidence-Publication Pending" at top
>>> - this
>>> appnote was posted by the manufacturer and has been online for years).
>>>
>>>
>>> Hardware assisted (not a complete list - some use holography, others
>>> interferometry):
>>> Ovizio
>>> Gabriel Popescu
>>> Graham Dunn
>>>
>>> PubMed has more - a simple search is:   "quantitative phase microscopy"
>>>
>>> McCarty's JoVE article now has downloadable .M (MatLab) files. If Anne
>>> Carpenter or anyone on the Cellprofiler team is reading this (or
>>> someone
>>> send is it their way), I encourage Anne to work with McCarty and their
>>> University to get "MaCarty QPm" into Cellprofiler.
>>>
>>> not QPm, this may still be of interest to listservites:
>>> Direct imaging of phase objects enables conventional deconvolution in
>>> bright field light microscopy
>>> http://www.ncbi.nlm.nih.gov/pubmed/24558478
>>>
>>> //
>>>
>>> Getting more out of high NA objective lens ... confocal or widefield
>>> interference reflection microscopy (IRM) provides data on
>>> cell-substratum
>>> adhesion ... including contact area. In reflection confocal (ok, for
>>> Jim
>>> Pawley and Guy Cox: scattered confocal), you can get optical
>>> sections of
>>> the cell, "label free". Some IRM data I posted online:
>>>
>>> http://works.bepress.com/gmcnamara/10/
>>> http://works.bepress.com/gmcnamara/7/
>>>
>>> Widefield IRM is very simple if your filter cube does not have an
>>> exciter
>>> filter (best to do this with a wavelength and intensity selectable LED
>>> illuminator than a broad spectrum arc lamp): just turn on a
>>> wavelength(s)
>>> that enable some light to bounce from the cells/coverglass through the
>>> dichroic and emission filter (could do even better with a dichroic
>>> only,
>>> and even better with a 50/50 beamsplitter only). My thanks to Tom
>>> DiMatteo,
>>> Epi Technology, for telling me about single LED control on my early gen
>>> SOLA. I had a long chat with Tom at his ABRF booth a couple of years
>>> ago.
>>> IRM can be quantitative
>>> http://www.ncbi.nlm.nih.gov/pubmed/23024911
>>> http://www.ncbi.nlm.nih.gov/pubmed/20013754
>>> http://www.ncbi.nlm.nih.gov/pubmed/3900106 ... Verschueren
>>>
>>>
>>>
>>> George
>>>
>>>
>>>
>>> On 3/18/2016 9:00 PM, Guy Cox wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> Post images on http://www.imgur.com and include the link in your
>>>> posting.
>>>> *****
>>>>
>>>> This is a little bit oversimplified.   The Rayleigh criterion does not
>>>> apply to a widefield image, but does apply in fluorescence. The
>>>> condenser
>>>> NA normally IS the objective NA, since they are usually one and the
>>>> same
>>>> thing, but condenser NA only affects brightness, not resolution.
>>>>
>>>> The Abbe criterion r = 0.5 lambda / NA applies in transmitted
>>>> light, but
>>>> ONLY if the condenser aperture equals or exceeds the objective NA.
>>>> Reducing the condenser NA does not have the same effect as reducing
>>>> the
>>>> objective NA.  Reducing the condenser NA to 0 (parallel illumination)
>>>>   worsens  the resolution to r = lambda/NA - ie 50% of what the
>>>> objective
>>>> should give.
>>>>
>>>>                                                 Guy
>>>>
>>>> Guy Cox, Honorary Associate Professor
>>>> School of Medical Sciences
>>>>
>>>> Australian Centre for Microscopy and Microanalysis,
>>>> Madsen, F09, University of Sydney, NSW 2006
>>>>
>>>> -----Original Message-----
>>>> From: Confocal Microscopy List
>>>> [mailto:[hidden email]]
>>>> On Behalf Of Aryeh Weiss
>>>> Sent: Friday, 18 March 2016 11:38 PM
>>>> To: [hidden email]
>>>> Subject: Re: Condenser lens choice for a given objective
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> Post images on http://www.imgur.com and include the link in your
>>>> posting.
>>>> *****
>>>>
>>>> In a transmitted-light brightfield image, the Rayleigh criterion
>>>> includes
>>>> both the objective NA and the condenser NA
>>>> (1.22 lambda/(NA_obj +NA_cond)) . The makes sense because even a very
>>>> small NA objective can receive light scattered at a large angle if
>>>> the NA
>>>> of the condenser is large. (This is how dark-field works).
>>>> So it would appear that in principle, you benefit from having a
>>>> condenser
>>>> with as large as NA as possible  (although you may not have much
>>>> contrast
>>>> on that brightfield image).
>>>>
>>>> BTW, you can have a "poor" man's dark field scope by using a low-NA
>>>> objective with a phase ring made for a higher NA objective.
>>>> For example, in my teaching lab, the students get very nice darkfield
>>>> images using our 4x/NA=0.1 objective with the ph2 phase ring.
>>>>
>>>> --aryeh
>>>>
>>>>
>>>>
>>>> On 18/03/2016 10:26 AM, Kyle Douglass wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> Post images on http://www.imgur.com and include the link in your
>>>>> posting.
>>>>> *****
>>>>>
>>>>> Thanks for the feedback, Barbara. It is very helpful.
>>>>>
>>>>> I have heard the advice before about the condenser NA needing to be
>>>>> greater than or equal to the objective NA. Can you offer some
>>>>> physical
>>>>> explanation or intuition for why this is?
>>>>>
>>>>> One admittedly incomplete explanation I can think of for the
>>>>> recommendation goes like this: the light collected by the objective
>>>>> consists of two parts. One part is the transmitted light that is not
>>>>> scattered by the sample. The other part is the light scattered by the
>>>>> sample. If the condenser's working NA is smaller than the
>>>>> objective's,
>>>>> then the unscattered, transmitted light fills only a portion of the
>>>>> objective's back focal plane. However, the light scattered by the
>>>>> sample will probably be dispersed across the entire back focal plane
>>>>> because it will encode all the spatial frequencies of the sample.
>>>>>
>>>>> I wonder if it's the inhomogeneous distribution of light from the two
>>>>> components in the objective's back focal plane that leads to the
>>>>> matched NA requirements of the condenser and objective. Does this
>>>>> make
>>>>> sense?
>>>>>
>>>>> Thanks!
>>>>> Kyle
>>>>>
>>>>> On 03/17/2016 08:22 PM, Barbara Foster wrote:
>>>>>
>>>>>> *****
>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>> Post images on http://www.imgur.com and include the link in your
>>>>>> posting.
>>>>>> *****
>>>>>>
>>>>>> Hi, Kyle
>>>>>>
>>>>>> The general rule of thumb is that the NA on the condenser should
>>>>>> meet
>>>>>> or exceed that of the objective.
>>>>>>
>>>>>> If you are using oil immersion objectives, ideally, to achieve that
>>>>>> goal, you should use an oil immersion condenser, otherwise you are
>>>>>> limited to an NA of 0.9.
>>>>>>
>>>>>> Also, remember that the aperture iris in the condenser adjusts the
>>>>>> condenser's WORKING  numerical aperture.  Just because the condenser
>>>>>> is marked 1.4 NA doesn't mean that, in a practical experiment, it
>>>>>> will be operating at 1.4.  I follow the guidelines set down by Frits
>>>>>> Zernicke (inventor of Phase contrast):  gently close the aperture
>>>>>> iris to the "Oomph" position: that delicate balance between
>>>>>> sufficient edge definition and optimum resolution (Yes, the
>>>>>> condenser
>>>>>> does contribute to resolution).
>>>>>>
>>>>>> As for planning for growth:
>>>>>> You might want to invest in a turret condenser early on.  That will
>>>>>> give you the option to add those other contrast techniques as you
>>>>>> grow into them.
>>>>>>
>>>>>> And just one more reminder, specifically regarding DIC:
>>>>>> If you are going to use plastic vessels (petri dishes, multi-well
>>>>>> plates, growth flasks), use Hoffman Modulation Contrast instead of
>>>>>> DIC.  DIC uses polarized light.  The plastic will affectt the shear
>>>>>> and cause effects that will be hard to interpret.  Some HMC set-ups
>>>>>> do use pol to control the width of the slit in the condenser, but
>>>>>> all
>>>>>> of that is on the incoming side of the sample and will not be
>>>>>> affected by plastic containers.
>>>>>>
>>>>>> Good hunting!
>>>>>> Barbara Foster, President & Chief Consultant Microscopy/Microscopy
>>>>>> Education  ... "Education, not Training"
>>>>>> 7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P:
>>>>>> 972-924-5310 www.MicroscopyEducation.com
>>>>>>
>>>>>> Microscopy/Microscopy Education is a division of The Microscopy &
>>>>>> Imaging Place, Inc.
>>>>>>
>>>>>>
>>>>>> NEW!   Getting involved in Raman or FTIR?
>>>>>> MME is now offering courses in these areas specifically for
>>>>>> microscopists!
>>>>>> Now scheduling courses through the mid 2016.  We can customize a
>>>>>> course on nearly any topic, from fluorescence to confocal to image
>>>>>> analysis to SEM/TEM.
>>>>>> Call today for a free training evaluation.
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:
>>>>>>
>>>>>>> *****
>>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>> Post images on http://www.imgur.com and include the link in your
>>>>>>> posting.
>>>>>>> *****
>>>>>>>
>>>>>>> Hello listers,
>>>>>>>
>>>>>>>
>>>>>>> I have a couple of questions about condensers for you. I'd like to
>>>>>>> do some transmitted light imaging in an inverted microscope using
>>>>>>> high magnification, oil-immersion objectives. For the moment, I
>>>>>>> don't need to do anything other than brightfield with a high power
>>>>>>> LED light source. It might be nice to do phase contrast or DIC in
>>>>>>> the future, but I don't need it now.
>>>>>>>
>>>>>>>
>>>>>>> My questions are:
>>>>>>>
>>>>>>>
>>>>>>> 1) What are the rules of thumb for matching a brightfield condenser
>>>>>>> to an objective? I won't be using anything but oil immersion
>>>>>>> objectives with NA's greater than 1.4.
>>>>>>>
>>>>>>>
>>>>>>> 2) If I do want to do phase contrast or DIC in the future, should I
>>>>>>> put special consideration into the condenser lens selection now? I
>>>>>>> imagine the condenser NA will determine what phase contrast rings I
>>>>>>> can use, but does it impact DIC?
>>>>>>>
>>>>>>>
>>>>>>> Thanks!
>>>>>>>
>>>>>>> Kyle
>>>>>>>
>>>>>>>
>>>>>>> Dr. Kyle M. Douglass
>>>>>>> Post-doctoral Researcher
>>>>>>> EPFL - The Laboratory of Experimental Biophysics
>>>>>>> http://leb.epfl.ch/
>>>>>>> http://kmdouglass.github.io
>>>>>>>
>>>> --
>>>> Aryeh Weiss
>>>> Faculty of Engineering
>>>> Bar Ilan University
>>>> Ramat Gan 52900 Israel
>>>>
>>>> Ph:  972-3-5317638
>>>> FAX: [phone]
>>>>
>>>>
>>> --
>>>
>>>
>>>
>>> George McNamara, Ph.D.
>>> Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab)
>>> University of Texas M.D. Anderson Cancer Center
>>> Houston, TX 77054
>>> Tattletales http://works.bepress.com/gmcnamara/42
>>> http://works.bepress.com/gmcnamara/75
>>> https://www.linkedin.com/in/georgemcnamara
>>>
>
>

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch

[Guy Cox-2]

OMG, can nobody here read?

Resolution in transmitted light bright field microscopy depends just as much on the condenser NA as on the objective's NA.

NO, NO, NO!!!!!!!!   I refuse to repeat myself, just go back and reread this thread.

                                             Guy


Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kyle Douglass
Sent: Monday, 21 March 2016 6:17 PM
To: [hidden email]
Subject: Re: Condenser lens choice for a given objective

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Thanks everyone for your input. Here's the summary of what I learned (please correct me if I am wrong):

Resolution in transmitted light bright field microscopy depends just as much on the condenser NA as on the objective's NA. The light that is scattered from the sample into the objective by light waves traveling off-axis encode the fine details in the specimen. Illuminating the sample at large angles results in scattered waves entering the objective that otherwise would not have been within the objective's NA had the sample been illuminated with on-axis, parallel light.

On the other hand, to improve contrast, there are numerous digital means offering background subtraction, etc. Optically, contrast can be improved by illuminating the sample with on-axis, collimated light.

I would conclude based on the discussion that, if one performs no post-processing, there is a trade-off between resolution and contrast in transmitted light bright field microscopy. One must decide what is best for his or her experiment and set the illumination accordingly.

Thanks again!
Kyle

On 03/20/2016 03:51 PM, George McNamara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Michael,
>
> Contrast limited? ... only if using eyes.
>
> Video (1970s+, Reynolds, Shinya Inoue, Robert & Nina Allen, later
> "Nanovid" folks, Dodt NIR-VEC-DIC), computer frame grabber background
> subtraction + offset (late 1980s, ex. Matrox MVP-AT board and
> Image-1/AT) and more recently (very late 1980s, early 1990s)
> scientific digital CCD cameras (ex. Photometrics Star-1, Hamamatsu
> digital CCDs [C4742?]) and background subtraction in computer memory.
>
> Reference (which can correct any of my dates): Shinya Inoue, 1986
> Video Microscopy (or more recently Inoue & Spring).
>
> //
>
> An early review on video (brightfield is not mentioned in the
> abstract) - Reynolds 1972 PubMed 4404351
>
> George T. Reynolds
>
>
>      Image intersification applied to biological problems
>
>
> Quarterly Reviews of Biophysics
> <http://journals.cambridge.org/action/displayJournal?jid=QRB> /Volume
> 5
> <http://journals.cambridge.org/action/displayBackIssues?jid=QRB&volume
> Id=5>
> /Issue 03
> <http://journals.cambridge.org/action/displayIssue?jid=QRB&volumeId=5&
> seriesId=0&issueId=03>
> / August 1972, pp 295 - 347
>
> DOI:http://dx.doi.org/10.1017/S0033583500000974(About DOI
> <http://journals.cambridge.org/action/stream?pageId=3624&level=2&sessi
> onId=F32BC6B4AAE6DB50F8D0214BB5DF4743.journals#30>),
>
>
> In many important types of observations in biological research, the
> information provided by the specimens is in the form of photons-quanta
> of visible light, u.v., or X-rays. The process of observation becomes
> one of recording this information in useful form, with as high an
> efficiency as possible. The problem becomes particularly important
> when for some reason or other the total number or rate of quanta
> provided by the specimen is small. Examples of such limitations are
> included in the following: (i) Processes permitting only low-intensity
> illumination in order not to interfere with the biological processes
> under observation. (ii) Processes changing very rapidly and requiring
> rapid sequence recording. (iii) Processes providing only a limited
> number of photons per event, such as bioluminescence. (iv) Processes
> in which radioactive tracers are utilized, and observation of
> radioactivity is desirable at low specific activity or within short
> time intervals. (v) X-ray diffraction processes where the specimen is
> weakly diffracting or where the X-ray intensity must be kept low in
> order not to damage the specimen. (vi) Processes involving the
> observation of fluorescence, where the intensity is low because of
> limitations on the amount of tagging material.
>
> A
> George
>
> On 3/20/2016 7:33 AM, Michael Model wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your
>> posting.
>> *****
>>
>> In my opinion, visibility in bright field is more limited by contrast
>> than by theoretical resolution...
>>
>> As for brightness, "in the absence of strong scattering, brightness
>> in transmitted illumination depends mostly on direct, and not on
>> diffracted, light (as the popular formula (NAob/M)2 assumes) and thus
>> on the smallest NA between the objective and condenser; this can be
>> easily verified by using an objective with a variable numerical
>> aperture. "
>>
>> Mike Model
>>
>> On Sat, Mar 19, 2016 at 1:07 PM, George McNamara
>> <[hidden email]>
>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>>> posting.
>>> *****
>>>
>>> Hi Kyle,
>>>
>>> Your post indicates that for now you just want to get a brightfield
>>> image through your 100x objective lens. The purpose of an objective
>>> lens is two
>>> fold:
>>> 1. resolution ... emphasized in the replies below ... high NA
>>> condenser results in higher spatial resolution.
>>> 2. brightness ... get enough photon flux so you can do your experiment.
>>>
>>> NA: at Guy's limit of zero NA on the illumination side, you will few
>>> photons reaching the objective lens and detector(s). You will also
>>> have
>>> (practically) infinite depth of focus with respect to imaging dust
>>> and other things on every optical surface. Many of these could be
>>> cleaned up by background subtraction (plus constant).
>>>
>>> Brightness is proportional to:
>>>
>>>          NA^4
>>> B ~ ----------
>>>           M^2
>>>
>>> The numerator is assuming equal NA (ex. epi-illumination with a
>>> single objective lens). I assume this could be re-written as
>>>
>>> (NAcond^2)*(NAobj^2)
>>>
>>> which is ok until Guy's limit of NAcond = 0. I'll also mention that
>>> you do not even need a transmitted light LED, condenser, or
>>> condenser arm if you have a sensitive enough detector(s): room
>>> lights, desk lamps, computer monitors can provide enough light (I
>>> first encountered this problem/feature working with James Sabry in
>>> Jim Spudich's lab using a back illuminated CCD on an inverted
>>> microscope, no recalling what objective lens, but was 19 years ago
>>> and available in their published papers).
>>>
>>> More importantly, M^2 means that your 100x lens is putting 1% as
>>> much photon flux onto a pixel as a 10x lens would.
>>>
>>> My advice: go find a long working distance objective lens that gets
>>> enough light onto your specimen to get you a useful brightfield
>>> image. You could later figure out if you need a phase contrast/DIC
>>> turret, what NA and working distance you need etc.
>>> I also suggest instead of hardware contrast (DIC requires polarizers
>>> and prisms ... can avoid illumination side polarizer if using a
>>> laser as in confocal microscope stand DIC using the transmitted
>>> light pathway in reverse; phase contrast requires a phase ring in
>>> the objective lens - usually with a lower NA for a given price
>>> point), you start looking into synthetic contrast options. The
>>> simplest is to just go with digital contrast by background
>>> subtraction.
>>> Software to get quantitative phase microscopy data of wet mass
>>> (leading to dry mass) - a couple of links and comments:
>>>
>>> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3555125/  ... McCarty ...
>>> uses 0.1 NA on condenser side.
>>>
>>> http://www.jove.com/video/50988/quantitative-optical-microscopy-meas
>>> urement-cellular-biophysical
>>>
>>> ... McCarty, see downloads (.M files).
>>>
>>> Nugent / IATIA (now Ultima Capital) developed the first software
>>> only solution http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624349/.
>>> http://www.ncbi.nlm.nih.gov/pubmed/15800856   ... Nugent / IATIA on
>>> confocal
>>>
>>> http://onlinelibrary.wiley.com/doi/10.1111/j.1440-1681.2004.04100.x/
>>> abstract
>>>
>>> http://aups.org.au/Proceedings/34/121-127/121-127.pdf   ... fig 3
>>> illustrates synthetic phase and DIC; fig 4 shows improves segmentation.
>>>
>>> http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imagi
>>> ng/applicationNotes/comparisonWithOpticalPhaseContrastModalities.pdf
>>>
>>>
>>> http://www.ultimacapital.net/iatiaimaging/Publications/Iatia%20Imagi
>>> ng/applicationNotes/measurementOfAreaChanges.pdf
>>>
>>> (I think you can ignore the "Confidence-Publication Pending" at top
>>> - this
>>> appnote was posted by the manufacturer and has been online for years).
>>>
>>>
>>> Hardware assisted (not a complete list - some use holography, others
>>> interferometry):
>>> Ovizio
>>> Gabriel Popescu
>>> Graham Dunn
>>>
>>> PubMed has more - a simple search is:   "quantitative phase microscopy"
>>>
>>> McCarty's JoVE article now has downloadable .M (MatLab) files. If
>>> Anne Carpenter or anyone on the Cellprofiler team is reading this
>>> (or someone send is it their way), I encourage Anne to work with
>>> McCarty and their University to get "MaCarty QPm" into Cellprofiler.
>>>
>>> not QPm, this may still be of interest to listservites:
>>> Direct imaging of phase objects enables conventional deconvolution
>>> in bright field light microscopy
>>> http://www.ncbi.nlm.nih.gov/pubmed/24558478
>>>
>>> //
>>>
>>> Getting more out of high NA objective lens ... confocal or widefield
>>> interference reflection microscopy (IRM) provides data on
>>> cell-substratum adhesion ... including contact area. In reflection
>>> confocal (ok, for Jim Pawley and Guy Cox: scattered confocal), you
>>> can get optical sections of the cell, "label free". Some IRM data I
>>> posted online:
>>>
>>> http://works.bepress.com/gmcnamara/10/
>>> http://works.bepress.com/gmcnamara/7/
>>>
>>> Widefield IRM is very simple if your filter cube does not have an
>>> exciter filter (best to do this with a wavelength and intensity
>>> selectable LED illuminator than a broad spectrum arc lamp): just
>>> turn on a
>>> wavelength(s)
>>> that enable some light to bounce from the cells/coverglass through
>>> the dichroic and emission filter (could do even better with a
>>> dichroic only, and even better with a 50/50 beamsplitter only). My
>>> thanks to Tom DiMatteo, Epi Technology, for telling me about single
>>> LED control on my early gen SOLA. I had a long chat with Tom at his
>>> ABRF booth a couple of years ago.
>>> IRM can be quantitative
>>> http://www.ncbi.nlm.nih.gov/pubmed/23024911
>>> http://www.ncbi.nlm.nih.gov/pubmed/20013754
>>> http://www.ncbi.nlm.nih.gov/pubmed/3900106 ... Verschueren
>>>
>>>
>>>
>>> George
>>>
>>>
>>>
>>> On 3/18/2016 9:00 PM, Guy Cox wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> Post images on http://www.imgur.com and include the link in your
>>>> posting.
>>>> *****
>>>>
>>>> This is a little bit oversimplified.   The Rayleigh criterion does not
>>>> apply to a widefield image, but does apply in fluorescence. The
>>>> condenser NA normally IS the objective NA, since they are usually
>>>> one and the same thing, but condenser NA only affects brightness,
>>>> not resolution.
>>>>
>>>> The Abbe criterion r = 0.5 lambda / NA applies in transmitted
>>>> light, but ONLY if the condenser aperture equals or exceeds the
>>>> objective NA.
>>>> Reducing the condenser NA does not have the same effect as reducing
>>>> the objective NA.  Reducing the condenser NA to 0 (parallel
>>>> illumination)
>>>>   worsens  the resolution to r = lambda/NA - ie 50% of what the
>>>> objective should give.
>>>>
>>>>                                                 Guy
>>>>
>>>> Guy Cox, Honorary Associate Professor School of Medical Sciences
>>>>
>>>> Australian Centre for Microscopy and Microanalysis, Madsen, F09,
>>>> University of Sydney, NSW 2006
>>>>
>>>> -----Original Message-----
>>>> From: Confocal Microscopy List
>>>> [mailto:[hidden email]]
>>>> On Behalf Of Aryeh Weiss
>>>> Sent: Friday, 18 March 2016 11:38 PM
>>>> To: [hidden email]
>>>> Subject: Re: Condenser lens choice for a given objective
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> Post images on http://www.imgur.com and include the link in your
>>>> posting.
>>>> *****
>>>>
>>>> In a transmitted-light brightfield image, the Rayleigh criterion
>>>> includes both the objective NA and the condenser NA
>>>> (1.22 lambda/(NA_obj +NA_cond)) . The makes sense because even a
>>>> very small NA objective can receive light scattered at a large
>>>> angle if the NA of the condenser is large. (This is how dark-field
>>>> works).
>>>> So it would appear that in principle, you benefit from having a
>>>> condenser with as large as NA as possible  (although you may not
>>>> have much contrast on that brightfield image).
>>>>
>>>> BTW, you can have a "poor" man's dark field scope by using a low-NA
>>>> objective with a phase ring made for a higher NA objective.
>>>> For example, in my teaching lab, the students get very nice
>>>> darkfield images using our 4x/NA=0.1 objective with the ph2 phase ring.
>>>>
>>>> --aryeh
>>>>
>>>>
>>>>
>>>> On 18/03/2016 10:26 AM, Kyle Douglass wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> Post images on http://www.imgur.com and include the link in your
>>>>> posting.
>>>>> *****
>>>>>
>>>>> Thanks for the feedback, Barbara. It is very helpful.
>>>>>
>>>>> I have heard the advice before about the condenser NA needing to
>>>>> be greater than or equal to the objective NA. Can you offer some
>>>>> physical explanation or intuition for why this is?
>>>>>
>>>>> One admittedly incomplete explanation I can think of for the
>>>>> recommendation goes like this: the light collected by the
>>>>> objective consists of two parts. One part is the transmitted light
>>>>> that is not scattered by the sample. The other part is the light
>>>>> scattered by the sample. If the condenser's working NA is smaller
>>>>> than the objective's, then the unscattered, transmitted light
>>>>> fills only a portion of the objective's back focal plane. However,
>>>>> the light scattered by the sample will probably be dispersed
>>>>> across the entire back focal plane because it will encode all the
>>>>> spatial frequencies of the sample.
>>>>>
>>>>> I wonder if it's the inhomogeneous distribution of light from the
>>>>> two components in the objective's back focal plane that leads to
>>>>> the matched NA requirements of the condenser and objective. Does
>>>>> this make sense?
>>>>>
>>>>> Thanks!
>>>>> Kyle
>>>>>
>>>>> On 03/17/2016 08:22 PM, Barbara Foster wrote:
>>>>>
>>>>>> *****
>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>> Post images on http://www.imgur.com and include the link in your
>>>>>> posting.
>>>>>> *****
>>>>>>
>>>>>> Hi, Kyle
>>>>>>
>>>>>> The general rule of thumb is that the NA on the condenser should
>>>>>> meet or exceed that of the objective.
>>>>>>
>>>>>> If you are using oil immersion objectives, ideally, to achieve
>>>>>> that goal, you should use an oil immersion condenser, otherwise
>>>>>> you are limited to an NA of 0.9.
>>>>>>
>>>>>> Also, remember that the aperture iris in the condenser adjusts
>>>>>> the condenser's WORKING  numerical aperture.  Just because the
>>>>>> condenser is marked 1.4 NA doesn't mean that, in a practical
>>>>>> experiment, it will be operating at 1.4.  I follow the guidelines
>>>>>> set down by Frits Zernicke (inventor of Phase contrast):  gently
>>>>>> close the aperture iris to the "Oomph" position: that delicate
>>>>>> balance between sufficient edge definition and optimum resolution
>>>>>> (Yes, the condenser does contribute to resolution).
>>>>>>
>>>>>> As for planning for growth:
>>>>>> You might want to invest in a turret condenser early on.  That
>>>>>> will give you the option to add those other contrast techniques
>>>>>> as you grow into them.
>>>>>>
>>>>>> And just one more reminder, specifically regarding DIC:
>>>>>> If you are going to use plastic vessels (petri dishes, multi-well
>>>>>> plates, growth flasks), use Hoffman Modulation Contrast instead
>>>>>> of DIC.  DIC uses polarized light.  The plastic will affectt the
>>>>>> shear and cause effects that will be hard to interpret.  Some HMC
>>>>>> set-ups do use pol to control the width of the slit in the
>>>>>> condenser, but all of that is on the incoming side of the sample
>>>>>> and will not be affected by plastic containers.
>>>>>>
>>>>>> Good hunting!
>>>>>> Barbara Foster, President & Chief Consultant
>>>>>> Microscopy/Microscopy Education  ... "Education, not Training"
>>>>>> 7101 Royal Glen Trail, Suite A  - McKinney, TX 75070 - P:
>>>>>> 972-924-5310 www.MicroscopyEducation.com
>>>>>>
>>>>>> Microscopy/Microscopy Education is a division of The Microscopy &
>>>>>> Imaging Place, Inc.
>>>>>>
>>>>>>
>>>>>> NEW!   Getting involved in Raman or FTIR?
>>>>>> MME is now offering courses in these areas specifically for
>>>>>> microscopists!
>>>>>> Now scheduling courses through the mid 2016.  We can customize a
>>>>>> course on nearly any topic, from fluorescence to confocal to
>>>>>> image analysis to SEM/TEM.
>>>>>> Call today for a free training evaluation.
>>>>>>
>>>>>>
>>>>>>
>>>>>>
>>>>>> At 08:36 AM 3/17/2016, Kyle Michael Douglass wrote:
>>>>>>
>>>>>>> *****
>>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>> Post images on http://www.imgur.com and include the link in your
>>>>>>> posting.
>>>>>>> *****
>>>>>>>
>>>>>>> Hello listers,
>>>>>>>
>>>>>>>
>>>>>>> I have a couple of questions about condensers for you. I'd like
>>>>>>> to do some transmitted light imaging in an inverted microscope
>>>>>>> using high magnification, oil-immersion objectives. For the
>>>>>>> moment, I don't need to do anything other than brightfield with
>>>>>>> a high power LED light source. It might be nice to do phase
>>>>>>> contrast or DIC in the future, but I don't need it now.
>>>>>>>
>>>>>>>
>>>>>>> My questions are:
>>>>>>>
>>>>>>>
>>>>>>> 1) What are the rules of thumb for matching a brightfield
>>>>>>> condenser to an objective? I won't be using anything but oil
>>>>>>> immersion objectives with NA's greater than 1.4.
>>>>>>>
>>>>>>>
>>>>>>> 2) If I do want to do phase contrast or DIC in the future,
>>>>>>> should I put special consideration into the condenser lens
>>>>>>> selection now? I imagine the condenser NA will determine what
>>>>>>> phase contrast rings I can use, but does it impact DIC?
>>>>>>>
>>>>>>>
>>>>>>> Thanks!
>>>>>>>
>>>>>>> Kyle
>>>>>>>
>>>>>>>
>>>>>>> Dr. Kyle M. Douglass
>>>>>>> Post-doctoral Researcher
>>>>>>> EPFL - The Laboratory of Experimental Biophysics
>>>>>>> http://leb.epfl.ch/ http://kmdouglass.github.io
>>>>>>>
>>>> --
>>>> Aryeh Weiss
>>>> Faculty of Engineering
>>>> Bar Ilan University
>>>> Ramat Gan 52900 Israel
>>>>
>>>> Ph:  972-3-5317638
>>>> FAX: [phone]
>>>>
>>>>
>>> --
>>>
>>>
>>>
>>> George McNamara, Ph.D.
>>> Single Cells Analyst, T-Cell Therapy Lab (Cooper Lab) University of
>>> Texas M.D. Anderson Cancer Center Houston, TX 77054 Tattletales
>>> http://works.bepress.com/gmcnamara/42
>>> http://works.bepress.com/gmcnamara/75
>>> https://www.linkedin.com/in/georgemcnamara
>>>
>
>

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch

[Kyle Michael Douglass]

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Post images on http://www.imgur.com and include the link in your posting.
*****

Hi all,

On 03/21/2016 11:35 AM, Guy Cox wrote:
> OMG, can nobody here read?
>
> Resolution in transmitted light bright field microscopy depends just as much on the condenser NA as on the objective's NA.
>
> NO, NO, NO!!!!!!!!   I refuse to repeat myself, just go back and reread this thread.
>
>                                               Guy
>

Thanks for your correction, Guy. I do in fact see your e-mail that
states that the condenser NA only affects brightness and not resolution.
Would you happen to have a reference, either an article or a text book,
that discusses this so I can try to understand your argument better? You
can e-mail it to me off list if you'd like.

Thanks again everyone for your help,
Kyle

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch

[Zdenek Svindrych-2]

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*****

Hi,



See e.g. Handbook of biological confocal microscopy (2nd ed), ed. J.B.
Pawley, chapter 1 page 1 equation 1:

d_min = lambda / (NA_obj + NA_cond)

 - this is the spacing of a grating that is just resolvable in trasmitted-
light microscopy, so condenser and objective NA have equal impact on the
resolution (this has been used to get gigapixel images with low mag low NA
large FOV objective and high NA illumination + some tricks).




equation 2 at the same page:

r_Airy = 0.61*Lambda/NA_obj

 - this is the airy disk radius of an image of an infinitely small luminous
point, does not matter whether trans-illuminated or fluorescent. This
changes as there are more 'points', their 'airy patterns' interfere in trans
-illuminated microscopy and equation 2 cannot be used. It's more useful in
fluorescence microscopy.




Disclaimer: I only understand fluorescence, to some extend. Not the rest...




zdenek




---------- Původní zpráva ----------
Od: Kyle Douglass <[hidden email]>
Komu: [hidden email]
Datum: 21. 3. 2016 7:35:42
Předmět: Re: Condenser lens choice for a given objective

"*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi all,

On 03/21/2016 11:35 AM, Guy Cox wrote:
> OMG, can nobody here read?
>
> Resolution in transmitted light bright field microscopy depends just as
much on the condenser NA as on the objective's NA.
>
> NO, NO, NO!!!!!!!! I refuse to repeat myself, just go back and reread this
thread.
>
> Guy
>

Thanks for your correction, Guy. I do in fact see your e-mail that
states that the condenser NA only affects brightness and not resolution.
Would you happen to have a reference, either an article or a text book,
that discusses this so I can try to understand your argument better? You
can e-mail it to me off list if you'd like.

Thanks again everyone for your help,
Kyle

--
Kyle M. Douglass, PhD
Post-doctoral researcher
The Laboratory of Experimental Biophysics
EPFL, Lausanne, Switzerland
http://kmdouglass.github.io
http://leb.epfl.ch"

[Stanislav Vitha-2]

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*****

My understanding is that in brightfield mode with axial light (condenser NA
= 0), resolution d=lambda/NA

With strictly oblique illumination where the angle of illumination equals the
acceptance angle of the objective (0th order light travels through one edge
of the lens, while the 1st order light travels through the opposite edge), the
resolution is doubled: d=0.5*lambda/NA.
 
With Kohler illumination, i.e. illuminating with a solid cone of light at a
variety of angles, where the condenser NA = objective NA, the resolution is
somewhere in between:
0.5*lambda /NA < d < lambda/NA
So for this setup, the Rayleigh formula (0.61*lambda/NA) is actually closer
to reality than the Abbe formula (0.5*lambda/NA), in my opinion.

For a standard brightfield setup, lateral resolution depends on the total NA
of the system, i.e. the average of the objective NA and the condenser NA,
where the effective condenser NA is equal or less than the objective NA.  

This is what Guy was indicating in his earlier post, I think.

Since the effective NA of illumination is only as large as the NA of the
objective, increasing the condenser NA beyond the NA of the objective
(e.g.,  using a 20x/0.5 objective, condenser aperture opened to NA=0.9) is
not going to increase resolution.

Source:
R. Wayne: Light and Video Microscopy. Academic Press, New York, 2009.
ISBN 978-0-12-374234-6



Stan Vitha
Texas A&M University
Microscopy and Imaging Center

[Julio Vazquez-2]

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Not much has been said about the practical aspects. If you will be imaging slides, then oil immersion condenser is best, although a high NA dry condenser (0.95) may work just fine for your needs and may be more practical. If you will be imaging dishes, chambers, plates, and such, then you can't use an oil immersion condenser, and you may not even be able to properly set Koehler illumination with a dry high NA condenser because their working distance is quite small. We use long working distance (and therefore low NA) condensers on all our inverted scopes, simply because they work with a broader range of samples. Illumination intensity is generally not limiting in bright field, and you often don't need to achieve maximum theoretical resolution either. The only times a low NA long working distance condenser has been a problem for us is when using a 100x/1.4 Phase contrast objective (but we were imaging bacteria in a flow chamber, and a high NA condenser wouldn't work too well either), and also when trying to collect single harmonic signal on a transmitted light detector on a multi photon system, where the collection efficiency of a high NA oil immersion condenser would be much greater. On our upright microscopes that we use primarily for slides, we use high NA (0.95) dry condensers. If you are doing dark field, then NA of the condenser also needs to be greater than NA of objective.

Julio Vazquez, PhD
Fred Hutchinson Cancer Research Center
Seattle, WA 98109
fredhutch.org

On Mar 17, 2016, at 10:50 AM, Kyle Michael Douglass wrote:

> Hello listers,
>
>
> I have a couple of questions about condensers for you. I'd like to do some transmitted light imaging in an inverted microscope using high magnification, oil-immersion objectives. For the moment, I don't need to do anything other than brightfield with a high power LED light source. It might be nice to do phase contrast or DIC in the future, but I don't need it now.
>
>
> My questions are:
>
>
> 1) What are the rules of thumb for matching a brightfield condenser to an objective? I won't be using anything but oil immersion objectives with NA's greater than 1.4.
>
>
> 2) If I do want to do phase contrast or DIC in the future, should I put special consideration into the condenser lens selection now? I imagine the condenser NA will determine what phase contrast rings I can use, but does it impact DIC?
>
>
> Thanks!
>
> Kyle

[Michael Model]

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*****

As I understand, the effect of condenser on theoretical resolution comes
from the fact that its opening makes illumination less coherent and closing
makes it more coherent. There is no simple formula for that. There was a
paper that from the 1950s where this was explained and calculated,
 Unfortunately I don't have the reference with me

Mike

On Mon, Mar 21, 2016 at 11:47 AM, Stanislav Vitha <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> My understanding is that in brightfield mode with axial light (condenser NA
> = 0), resolution d=lambda/NA
>
> With strictly oblique illumination where the angle of illumination equals
> the
> acceptance angle of the objective (0th order light travels through one edge
> of the lens, while the 1st order light travels through the opposite edge),
> the
> resolution is doubled: d=0.5*lambda/NA.
>
> With Kohler illumination, i.e. illuminating with a solid cone of light at a
> variety of angles, where the condenser NA = objective NA, the resolution is
> somewhere in between:
> 0.5*lambda /NA < d < lambda/NA
> So for this setup, the Rayleigh formula (0.61*lambda/NA) is actually closer
> to reality than the Abbe formula (0.5*lambda/NA), in my opinion.
>
> For a standard brightfield setup, lateral resolution depends on the total
> NA
> of the system, i.e. the average of the objective NA and the condenser NA,
> where the effective condenser NA is equal or less than the objective NA.
>
> This is what Guy was indicating in his earlier post, I think.
>
> Since the effective NA of illumination is only as large as the NA of the
> objective, increasing the condenser NA beyond the NA of the objective
> (e.g.,  using a 20x/0.5 objective, condenser aperture opened to NA=0.9) is
> not going to increase resolution.
>
> Source:
> R. Wayne: Light and Video Microscopy. Academic Press, New York, 2009.
> ISBN 978-0-12-374234-6
>
>
>
> Stan Vitha
> Texas A&M University
> Microscopy and Imaging Center
>
>