http://confocal-microscopy-list.275.s1.nabble.com/Condenser-lens-choice-for-a-given-objective-tp7584901p7584916.html
using an objective with a variable numerical aperture. "
> *****
> 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: 972-3-7384051
>>
>>
>
> --
>
>
>
> 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>