http://confocal-microscopy-list.275.s1.nabble.com/Condenser-lens-choice-for-a-given-objective-tp7584901p7584920.html
Thanks everyone for your input. Here's the summary of what I learned
much on the condenser NA as on the objective's NA. The light that is
off-axis encode the fine details in the specimen. Illuminating the
offering background subtraction, etc. Optically, contrast can be
transmitted light bright field microscopy. One must decide what is best
> *****
> 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: 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>>>
>
>
Kyle M. Douglass, PhD