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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 <[log in to unmask]>
> 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:[log in to unmask]]
>>> On Behalf Of Aryeh Weiss
>>> Sent: Friday, 18 March 2016 11:38 PM
>>> To: [log in to unmask]
>>> 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
>>
--
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
|
