DIC condensers

[Jeff Spector]

*****
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.
*****

Greetings,
   This isn't exactly a "confocal" questions but I know a lot of "micoscopy
gurus" live on this list so I thought it a good place to ask this. I have a
colleague who is trying to image individual (i.e. small and diffraction
limited) microtubules in a flow chamber by using DIC. They are currently
using a 100x 1.45 oil Objective, but only a .52 LWD condenser. They were
using a solid state light source but couldn't get good image so we switched
to a lamp for illumination and the images are much better, and we can now
see the microtbules but there still isn't a lot of contrast.  My question
is, is it worth it to go to a high NA (perhaps oil immersion) condenser,
and can anyone think of why the lamp would give a better DIC image than a
solid state light source?
thanks in advance for the help...
 -Jeff

[David Claypool]

*****
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.
*****

Non-commercial response:

Jeff,

In my experience, use a Planapochromat 60/1.4 or 100/1.45 oil objective, 1.4NA oil condenser, precision interference line green filter(546/10), and "standard" or "high" contrast DIC objectives & DIC condenser prisms. You will need to carefully test which combination of DIC prisms work best for you. Be sure the back aperture of the objective is completely filled. You will probably need to introduce a 1.5x-2.5x magnification factor in the camera light path, and, in this case, a solid state illuminator should give you more light than a 100W halogen lamp. Some microscope systems offer a high NA illuminator adapter that will accept a solid state illuminator liquid light guide, and this works well for this application. You will probably need to adjust your image histogram for the best image.

I recently used the above combination on an inverted microscope  and achieved excellent results with a 5MP sCMOS camera.

David

David J. Claypool
Digital Imaging Product Manager
Micro Video Instruments
Office: 800-875-2041 x5221
Cell: 603-809-5342
[hidden email]

> On Nov 28, 2014, at 9:30 PM, "Jeff Spector" <[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.
> *****
>
> Greetings,
>   This isn't exactly a "confocal" questions but I know a lot of "micoscopy
> gurus" live on this list so I thought it a good place to ask this. I have a
> colleague who is trying to image individual (i.e. small and diffraction
> limited) microtubules in a flow chamber by using DIC. They are currently
> using a 100x 1.45 oil Objective, but only a .52 LWD condenser. They were
> using a solid state light source but couldn't get good image so we switched
> to a lamp for illumination and the images are much better, and we can now
> see the microtbules but there still isn't a lot of contrast.  My question
> is, is it worth it to go to a high NA (perhaps oil immersion) condenser,
> and can anyone think of why the lamp would give a better DIC image than a
> solid state light source?
> thanks in advance for the help...
> -Jeff

[Guy Cox-2]

Jeff,

        If you want to get decent contrast in DIC you need a high NA condenser with a high NA objective.  Of course the plate must be correct for the objective, and Köhler illumination must be correctly set up.  My guess is that that wasn't true for your LED source, and you weren't filling the BFP.  Don't take this personally but the widespread use of fluorescence has made many microscopists become slack about setting up Köhler illumination!  

                                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 Jeff Spector
Sent: Saturday, 29 November 2014 1:22 PM
To: [hidden email]
Subject: DIC condensers

*****
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.
*****

Greetings,
   This isn't exactly a "confocal" questions but I know a lot of "micoscopy gurus" live on this list so I thought it a good place to ask this. I have a colleague who is trying to image individual (i.e. small and diffraction
limited) microtubules in a flow chamber by using DIC. They are currently using a 100x 1.45 oil Objective, but only a .52 LWD condenser. They were using a solid state light source but couldn't get good image so we switched to a lamp for illumination and the images are much better, and we can now see the microtbules but there still isn't a lot of contrast.  My question is, is it worth it to go to a high NA (perhaps oil immersion) condenser, and can anyone think of why the lamp would give a better DIC image than a solid state light source?
thanks in advance for the help...
 -Jeff

[Johannes Helm]

*****
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.
*****

Dear Jeff,

Guy has already sent an answer and provided good information for you.

I would like to send some additional information:

In case of transmitted light wide field microscopy, as a rule of thumb
the (lateral!) resolution is

d = (1.28 * lambda) / (NAobj + NAcond)

This formula is quite useful, and shows pretty well, to which degree,
indeed, resolution is dependent on the numerical aperture of the
condenser.  "Of course", it is never a 100% correct description of
reality, since you will never be able to attain illumination by "just
one" wavelength; in the very best case you might get a Lorentzian
profile of a good single mode laserbeam.

Also, in DIC microscopy, when, as Guy already has written, close to
perfect Koehler illumnation is a sine qua non for a "good image" -
whatever THIS would be, :-), another issue is that you can trade
contrast for resolution. I.e.: By closing the condenser aperture
diaphragm - often referred to as the "A diaphragm" as opposed to the "F
diaphragm", which is the Field Diaphragm - you will increase contrast
while at the same time reduce resolution. In "manuals for microscopists"
of the sixties and seventies as published in Germany, one will sometimes
be instructed to do the following: "First, attain perfect Koehler
illumation, then lower the condenser somewhat to attain better
contrast." Yes, it works, and it is a kind of rather un-controlled
oblique illumination, which one fakes in that way. But: As a real
physicist, you would, for ideological reasons, never do this. If you are
a pragmatic life scientist: Well, you have a better and easier life,
anyway.

Also note: Make sure that your light source and your polarizer and
analyzer are useful in the same wavelength band! At least on older DIC
systems, polarizers and analyzers will do a good job in the visible
wavelength range, while they will act as a mere 80% Transmission ND
filter in IR and do hardly polarize! For IR, you might need separate and
special polarizers (and analyzers, of course, which are nothing else
than polarizers in another functional position).

Best wishes and have a good 1st of advent,

Johannes

On 2014-11-28 21:21, Jeff Spector 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.
> *****
>
> Greetings,
>    This isn't exactly a "confocal" questions but I know a lot of
> "micoscopy
> gurus" live on this list so I thought it a good place to ask this. I
> have a
> colleague who is trying to image individual (i.e. small and
> diffraction
> limited) microtubules in a flow chamber by using DIC. They are
> currently
> using a 100x 1.45 oil Objective, but only a .52 LWD condenser. They
> were
> using a solid state light source but couldn't get good image so we
> switched
> to a lamp for illumination and the images are much better, and we can
> now
> see the microtbules but there still isn't a lot of contrast.  My
> question
> is, is it worth it to go to a high NA (perhaps oil immersion)
> condenser,
> and can anyone think of why the lamp would give a better DIC image
> than a
> solid state light source?
> thanks in advance for the help...
>  -Jeff

--
P. Johannes Helm

Voice: ([phone] (office)
Fax: [phone] (office)

[James Pawley]

*****
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.
*****

Dear Jeff,

I agree with Guy about the Kohler but there is
also the matter of wavelength. DIC prisms do work
with "white" light but they are usually designed
to work best at a particular wavelength (often
the green line of the Hg source, 546nm). I don't
know the emission spectrum of your LED (or how
hot you have your tungsten-halogen, as this
affects it spectrum, IR filter? UV filter?) and I
don't know the response of your detector, but if
seeing the greatest possible contrast is
important, you might optimize the wavelength of
the operation as well as being sure that Kohler
is working properly

Another thing to keep in mind is that DIC depends
on keeping two light paths separate by having
their polarization be at 90 degrees to each
other. Problems can stem from the fact that the
degree to which light is reflected when passing
through a glass-air surface varies with the pol
axis. This is not a major problem on low-NA
lenses because the incidence angle is near normal
and reflection is low (especially on modern
coated optics). However, on high-NA lenses, rays
near the edge of the aperture impinge at angles
where the differential reflection of the two ray
bundles becomes significant. This can be seen by
looking at the BFP using the phase telescope
(Bertrand lens?) with only the polarizer and
analyzer in place and adjusted for maximum
extinction (no DIC prisms and a clear glass
specimen, but an oiled condenser open to the same
NA as the objective).

If the problem is significant, you will see what
is called The Maltese Cross, which comes about
because 4 symmetrically located, blurry,
more-or-less circular lighter blobs occur in the
NA regions where particular ray-bundles are
depleted. Of course, this also happens when you
are using the same optics with the DIC prisms in
place.  The result is that the two bundles are no
longer quite independent and this reduces the
contrast. Shinya Inoue developed a corrector lens
that Nikon used to sell to reduce this problem,
but I don't know if it was ever upgraded to the
newer scopes (maybe someone from Nikon can tell
us?). And while we are on the topic of pol, DIC
is best performed using Pol (strain-free)
objectives because strain-birefringence can also
mess up pol performance. I am not clear as to
whether your 1.45 lens meets this requirement.

Finally, if you interested in seeing individual
microtubules, you will need more than eyeballs
and good optics.  MTs are very small and don't
produce much contrast to start off with. They
were initially only made visible by virtue of
video-rate electronic contrast enhancement that
involved not only using a very bright light
source (to increase signal levels and reduce the
relative effect of Poisson Noise in the detector,
and to reduce this further, one could
exponentially average the image electronically
over many video frames. Usually one used a
mercury source, but if you do this, be sure to
include a UV filter (and perhaps an
"interference-green" to isolate the 546nm line)
so you don't damage your polarizer.). The
resulting high "average brightness" was then
electronically subtracted off so that one could
expand the contrast of the signal that remained.

This procedure invariably produced a very blotchy
image. Small imperfections in the optics (dust,
bubbles, scratches) create low contrast and
generally out-of-focus features referred to as
mottle. The contrast of mottle is so low that it
is only visible after the sort of contrast
enhancement just described. What made DIC so
suitable for viewing small features by
video-enhancment was its very narrow depth of
field. By slightly changing the focus, one could
form an image in which the ONLY features recorded
where those due to mottle. This image could then
be averaged, stored and subtracted from the live
image (often this difference image was also
averaged to further reduce noise). The result was
essentially the image contrast caused only by the
"now-in-focus" MT, a feature that might have had
only 1% contrast before enhancement.

Good luck,

Jim Pawley

>Jeff,
>
> If you want to get decent contrast in DIC
>you need a high NA condenser with a high NA
>objective.  Of course the plate must be correct
>for the objective, and Köhler illumination must
>be correctly set up.  My guess is that that
>wasn't true for your LED source, and you weren't
>filling the BFP.  Don't take this personally but
>the widespread use of fluorescence has made many
>microscopists become slack about setting up
>Köhler illumination!
>
> 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 Jeff Spector
>Sent: Saturday, 29 November 2014 1:22 PM
>To: [hidden email]
>Subject: DIC condensers
>
>*****
>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.
>*****
>
>Greetings,
>    This isn't exactly a "confocal" questions but
>I know a lot of "micoscopy gurus" live on this
>list so I thought it a good place to ask this. I
>have a colleague who is trying to image
>individual (i.e. small and diffraction
>limited) microtubules in a flow chamber by using
>DIC. They are currently using a 100x 1.45 oil
>Objective, but only a .52 LWD condenser. They
>were using a solid state light source but
>couldn't get good image so we switched to a lamp
>for illumination and the images are much better,
>and we can now see the microtbules but there
>still isn't a lot of contrast.  My question is,
>is it worth it to go to a high NA (perhaps oil
>immersion) condenser, and can anyone think of
>why the lamp would give a better DIC image than
>a solid state light source?
>thanks in advance for the help...
>  -Jeff

--
               ****************************************
James and Christine Pawley, 5446 Burley Place (PO
Box 2348), Sechelt, BC, Canada, V0N3A0,
Phone [phone], email <[hidden email]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[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 Johannes,

You do not need to add Nomarski (or Smith or anyone else's) DIC
components. You do need a high NA objective lens and digital image
processing. See

http://web.stanford.edu/group/blocklab/GutierrezAJP2010.pdf

Also helps to be in one of the top ten labs in the world for this kind
of work.

Enjoy,

George
p.s. see http://www.ncbi.nlm.nih.gov/pubmed/17381703  for an example of
LED-DIC.
Recent IR-DIC "Dodt contrast", http://www.ncbi.nlm.nih.gov/pubmed/24298032
Earlier IR-DIC http://www.ncbi.nlm.nih.gov/pubmed/2085783
1984 paper using AVEC-DIC to image single microtubules in live cells
http://www.ncbi.nlm.nih.gov/pubmed/6333427

On 11/29/2014 10:33 AM, Johannes Helm 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.
> *****
>
> Dear Jeff,
>
> Guy has already sent an answer and provided good information for you.
>
> I would like to send some additional information:
>
> In case of transmitted light wide field microscopy, as a rule of thumb
> the (lateral!) resolution is
>
> d = (1.28 * lambda) / (NAobj + NAcond)
>
> This formula is quite useful, and shows pretty well, to which degree,
> indeed, resolution is dependent on the numerical aperture of the
> condenser.  "Of course", it is never a 100% correct description of
> reality, since you will never be able to attain illumination by "just
> one" wavelength; in the very best case you might get a Lorentzian
> profile of a good single mode laserbeam.
>
> Also, in DIC microscopy, when, as Guy already has written, close to
> perfect Koehler illumnation is a sine qua non for a "good image" -
> whatever THIS would be, :-), another issue is that you can trade
> contrast for resolution. I.e.: By closing the condenser aperture
> diaphragm - often referred to as the "A diaphragm" as opposed to the
> "F diaphragm", which is the Field Diaphragm - you will increase
> contrast while at the same time reduce resolution. In "manuals for
> microscopists" of the sixties and seventies as published in Germany,
> one will sometimes be instructed to do the following: "First, attain
> perfect Koehler illumation, then lower the condenser somewhat to
> attain better contrast." Yes, it works, and it is a kind of rather
> un-controlled oblique illumination, which one fakes in that way. But:
> As a real physicist, you would, for ideological reasons, never do
> this. If you are a pragmatic life scientist: Well, you have a better
> and easier life, anyway.
>
> Also note: Make sure that your light source and your polarizer and
> analyzer are useful in the same wavelength band! At least on older DIC
> systems, polarizers and analyzers will do a good job in the visible
> wavelength range, while they will act as a mere 80% Transmission ND
> filter in IR and do hardly polarize! For IR, you might need separate
> and special polarizers (and analyzers, of course, which are nothing
> else than polarizers in another functional position).
>
> Best wishes and have a good 1st of advent,
>
> Johannes
>
> On 2014-11-28 21:21, Jeff Spector 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.
>> *****
>>
>> Greetings,
>>    This isn't exactly a "confocal" questions but I know a lot of
>> "micoscopy
>> gurus" live on this list so I thought it a good place to ask this. I
>> have a
>> colleague who is trying to image individual (i.e. small and diffraction
>> limited) microtubules in a flow chamber by using DIC. They are currently
>> using a 100x 1.45 oil Objective, but only a .52 LWD condenser. They were
>> using a solid state light source but couldn't get good image so we
>> switched
>> to a lamp for illumination and the images are much better, and we can
>> now
>> see the microtbules but there still isn't a lot of contrast.  My
>> question
>> is, is it worth it to go to a high NA (perhaps oil immersion) condenser,
>> and can anyone think of why the lamp would give a better DIC image
>> than a
>> solid state light source?
>> thanks in advance for the help...
>>  -Jeff
>

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

[Jeff Spector]

*****
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,
 Thanks for the input. We've got a high NA oil condenser on the way so
hopefully this solves our problem. When using both the LED and the Lamp we
did make sure that we were in Kohler illumination in both cases.  We are in
fact able to get images using the low NA condenser but we have to stop down
the F-stop all the way and open the A-stop all the way. We can get images
but the contrast just isn't good enough to perform our measurements so
hopefully going to a higher NA  condenser will solve this problem..
thanks for the input..
 -Jeff

On Sat, Nov 29, 2014 at 4:26 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 Johannes,
>
> You do not need to add Nomarski (or Smith or anyone else's) DIC
> components. You do need a high NA objective lens and digital image
> processing. See
>
> http://web.stanford.edu/group/blocklab/GutierrezAJP2010.pdf
>
> Also helps to be in one of the top ten labs in the world for this kind of
> work.
>
> Enjoy,
>
> George
> p.s. see http://www.ncbi.nlm.nih.gov/pubmed/17381703  for an example of
> LED-DIC.
> Recent IR-DIC "Dodt contrast", http://www.ncbi.nlm.nih.gov/pubmed/24298032
> Earlier IR-DIC http://www.ncbi.nlm.nih.gov/pubmed/2085783
> 1984 paper using AVEC-DIC to image single microtubules in live cells
> http://www.ncbi.nlm.nih.gov/pubmed/6333427
>
>
> On 11/29/2014 10:33 AM, Johannes Helm 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.
>> *****
>>
>> Dear Jeff,
>>
>> Guy has already sent an answer and provided good information for you.
>>
>> I would like to send some additional information:
>>
>> In case of transmitted light wide field microscopy, as a rule of thumb
>> the (lateral!) resolution is
>>
>> d = (1.28 * lambda) / (NAobj + NAcond)
>>
>> This formula is quite useful, and shows pretty well, to which degree,
>> indeed, resolution is dependent on the numerical aperture of the
>> condenser.  "Of course", it is never a 100% correct description of reality,
>> since you will never be able to attain illumination by "just one"
>> wavelength; in the very best case you might get a Lorentzian profile of a
>> good single mode laserbeam.
>>
>> Also, in DIC microscopy, when, as Guy already has written, close to
>> perfect Koehler illumnation is a sine qua non for a "good image" - whatever
>> THIS would be, :-), another issue is that you can trade contrast for
>> resolution. I.e.: By closing the condenser aperture diaphragm - often
>> referred to as the "A diaphragm" as opposed to the "F diaphragm", which is
>> the Field Diaphragm - you will increase contrast while at the same time
>> reduce resolution. In "manuals for microscopists" of the sixties and
>> seventies as published in Germany, one will sometimes be instructed to do
>> the following: "First, attain perfect Koehler illumation, then lower the
>> condenser somewhat to attain better contrast." Yes, it works, and it is a
>> kind of rather un-controlled oblique illumination, which one fakes in that
>> way. But: As a real physicist, you would, for ideological reasons, never do
>> this. If you are a pragmatic life scientist: Well, you have a better and
>> easier life, anyway.
>>
>> Also note: Make sure that your light source and your polarizer and
>> analyzer are useful in the same wavelength band! At least on older DIC
>> systems, polarizers and analyzers will do a good job in the visible
>> wavelength range, while they will act as a mere 80% Transmission ND filter
>> in IR and do hardly polarize! For IR, you might need separate and special
>> polarizers (and analyzers, of course, which are nothing else than
>> polarizers in another functional position).
>>
>> Best wishes and have a good 1st of advent,
>>
>> Johannes
>>
>> On 2014-11-28 21:21, Jeff Spector 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.
>>> *****
>>>
>>> Greetings,
>>>    This isn't exactly a "confocal" questions but I know a lot of
>>> "micoscopy
>>> gurus" live on this list so I thought it a good place to ask this. I
>>> have a
>>> colleague who is trying to image individual (i.e. small and diffraction
>>> limited) microtubules in a flow chamber by using DIC. They are currently
>>> using a 100x 1.45 oil Objective, but only a .52 LWD condenser. They were
>>> using a solid state light source but couldn't get good image so we
>>> switched
>>> to a lamp for illumination and the images are much better, and we can now
>>> see the microtbules but there still isn't a lot of contrast.  My question
>>> is, is it worth it to go to a high NA (perhaps oil immersion) condenser,
>>> and can anyone think of why the lamp would give a better DIC image than a
>>> solid state light source?
>>> thanks in advance for the help...
>>>  -Jeff
>>>
>>
>>
>
> --
>
>
>
> George McNamara, Ph.D.
> Single Cells Analyst
> L.J.N. Cooper Lab
> University of Texas M.D. Anderson Cancer Center
> Houston, TX 77054
> Tattletales http://works.bepress.com/gmcnamara/42
>

[Craig Brideau]

*****
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.
*****

Make sure you are filling the aperture of your high-NA condenser when you
get it! The LED may have been underfilling or overfilling the lens which
could be the reason for your discrepancy between lamp and LED.

Craig Brideau

On Sun, Nov 30, 2014 at 9:07 AM, Jeff Spector <[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 all,
>  Thanks for the input. We've got a high NA oil condenser on the way so
> hopefully this solves our problem. When using both the LED and the Lamp we
> did make sure that we were in Kohler illumination in both cases.  We are in
> fact able to get images using the low NA condenser but we have to stop down
> the F-stop all the way and open the A-stop all the way. We can get images
> but the contrast just isn't good enough to perform our measurements so
> hopefully going to a higher NA  condenser will solve this problem..
> thanks for the input..
>  -Jeff
>
> On Sat, Nov 29, 2014 at 4:26 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 Johannes,
> >
> > You do not need to add Nomarski (or Smith or anyone else's) DIC
> > components. You do need a high NA objective lens and digital image
> > processing. See
> >
> > http://web.stanford.edu/group/blocklab/GutierrezAJP2010.pdf
> >
> > Also helps to be in one of the top ten labs in the world for this kind of
> > work.
> >
> > Enjoy,
> >
> > George
> > p.s. see http://www.ncbi.nlm.nih.gov/pubmed/17381703  for an example of
> > LED-DIC.
> > Recent IR-DIC "Dodt contrast",
> http://www.ncbi.nlm.nih.gov/pubmed/24298032
> > Earlier IR-DIC http://www.ncbi.nlm.nih.gov/pubmed/2085783
> > 1984 paper using AVEC-DIC to image single microtubules in live cells
> > http://www.ncbi.nlm.nih.gov/pubmed/6333427
> >
> >
> > On 11/29/2014 10:33 AM, Johannes Helm 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.
> >> *****
> >>
> >> Dear Jeff,
> >>
> >> Guy has already sent an answer and provided good information for you.
> >>
> >> I would like to send some additional information:
> >>
> >> In case of transmitted light wide field microscopy, as a rule of thumb
> >> the (lateral!) resolution is
> >>
> >> d = (1.28 * lambda) / (NAobj + NAcond)
> >>
> >> This formula is quite useful, and shows pretty well, to which degree,
> >> indeed, resolution is dependent on the numerical aperture of the
> >> condenser.  "Of course", it is never a 100% correct description of
> reality,
> >> since you will never be able to attain illumination by "just one"
> >> wavelength; in the very best case you might get a Lorentzian profile of
> a
> >> good single mode laserbeam.
> >>
> >> Also, in DIC microscopy, when, as Guy already has written, close to
> >> perfect Koehler illumnation is a sine qua non for a "good image" -
> whatever
> >> THIS would be, :-), another issue is that you can trade contrast for
> >> resolution. I.e.: By closing the condenser aperture diaphragm - often
> >> referred to as the "A diaphragm" as opposed to the "F diaphragm", which
> is
> >> the Field Diaphragm - you will increase contrast while at the same time
> >> reduce resolution. In "manuals for microscopists" of the sixties and
> >> seventies as published in Germany, one will sometimes be instructed to
> do
> >> the following: "First, attain perfect Koehler illumation, then lower the
> >> condenser somewhat to attain better contrast." Yes, it works, and it is
> a
> >> kind of rather un-controlled oblique illumination, which one fakes in
> that
> >> way. But: As a real physicist, you would, for ideological reasons,
> never do
> >> this. If you are a pragmatic life scientist: Well, you have a better and
> >> easier life, anyway.
> >>
> >> Also note: Make sure that your light source and your polarizer and
> >> analyzer are useful in the same wavelength band! At least on older DIC
> >> systems, polarizers and analyzers will do a good job in the visible
> >> wavelength range, while they will act as a mere 80% Transmission ND
> filter
> >> in IR and do hardly polarize! For IR, you might need separate and
> special
> >> polarizers (and analyzers, of course, which are nothing else than
> >> polarizers in another functional position).
> >>
> >> Best wishes and have a good 1st of advent,
> >>
> >> Johannes
> >>
> >> On 2014-11-28 21:21, Jeff Spector 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.
> >>> *****
> >>>
> >>> Greetings,
> >>>    This isn't exactly a "confocal" questions but I know a lot of
> >>> "micoscopy
> >>> gurus" live on this list so I thought it a good place to ask this. I
> >>> have a
> >>> colleague who is trying to image individual (i.e. small and diffraction
> >>> limited) microtubules in a flow chamber by using DIC. They are
> currently
> >>> using a 100x 1.45 oil Objective, but only a .52 LWD condenser. They
> were
> >>> using a solid state light source but couldn't get good image so we
> >>> switched
> >>> to a lamp for illumination and the images are much better, and we can
> now
> >>> see the microtbules but there still isn't a lot of contrast.  My
> question
> >>> is, is it worth it to go to a high NA (perhaps oil immersion)
> condenser,
> >>> and can anyone think of why the lamp would give a better DIC image
> than a
> >>> solid state light source?
> >>> thanks in advance for the help...
> >>>  -Jeff
> >>>
> >>
> >>
> >
> > --
> >
> >
> >
> > George McNamara, Ph.D.
> > Single Cells Analyst
> > L.J.N. Cooper Lab
> > University of Texas M.D. Anderson Cancer Center
> > Houston, TX 77054
> > Tattletales http://works.bepress.com/gmcnamara/42
> >
>

[Jeff Spector]

*****
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 all the input. Our high NA  oil condenser came today so I will
get to testing it out soon. A quick question I have is what pixel size
should I aim for?  If the condenser is 1.4 and the objective is 1.45 should
I still aim for ~ 90 nm pixels (as I would in fluorescence). I only ask
because we have both a 60x and a 100x that are 1.45 NA...
I understand that larger pixels will be "undersampling" my images, but will
they lead to perhaps slightly(better contrast ? Are there any references
 discussing optimization of pixel size in DIC imaging, or do we always want
to be 2-3 pixels covering the nyquist limit?
thanks...
-jeff

On Sun, Nov 30, 2014 at 8:08 PM, Craig Brideau <[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.
> *****
>
> Make sure you are filling the aperture of your high-NA condenser when you
> get it! The LED may have been underfilling or overfilling the lens which
> could be the reason for your discrepancy between lamp and LED.
>
> Craig Brideau
>
> On Sun, Nov 30, 2014 at 9:07 AM, Jeff Spector <[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 all,
> >  Thanks for the input. We've got a high NA oil condenser on the way so
> > hopefully this solves our problem. When using both the LED and the Lamp
> we
> > did make sure that we were in Kohler illumination in both cases.  We are
> in
> > fact able to get images using the low NA condenser but we have to stop
> down
> > the F-stop all the way and open the A-stop all the way. We can get images
> > but the contrast just isn't good enough to perform our measurements so
> > hopefully going to a higher NA  condenser will solve this problem..
> > thanks for the input..
> >  -Jeff
> >
> > On Sat, Nov 29, 2014 at 4:26 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 Johannes,
> > >
> > > You do not need to add Nomarski (or Smith or anyone else's) DIC
> > > components. You do need a high NA objective lens and digital image
> > > processing. See
> > >
> > > http://web.stanford.edu/group/blocklab/GutierrezAJP2010.pdf
> > >
> > > Also helps to be in one of the top ten labs in the world for this kind
> of
> > > work.
> > >
> > > Enjoy,
> > >
> > > George
> > > p.s. see http://www.ncbi.nlm.nih.gov/pubmed/17381703  for an example
> of
> > > LED-DIC.
> > > Recent IR-DIC "Dodt contrast",
> > http://www.ncbi.nlm.nih.gov/pubmed/24298032
> > > Earlier IR-DIC http://www.ncbi.nlm.nih.gov/pubmed/2085783
> > > 1984 paper using AVEC-DIC to image single microtubules in live cells
> > > http://www.ncbi.nlm.nih.gov/pubmed/6333427
> > >
> > >
> > > On 11/29/2014 10:33 AM, Johannes Helm 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.
> > >> *****
> > >>
> > >> Dear Jeff,
> > >>
> > >> Guy has already sent an answer and provided good information for you.
> > >>
> > >> I would like to send some additional information:
> > >>
> > >> In case of transmitted light wide field microscopy, as a rule of thumb
> > >> the (lateral!) resolution is
> > >>
> > >> d = (1.28 * lambda) / (NAobj + NAcond)
> > >>
> > >> This formula is quite useful, and shows pretty well, to which degree,
> > >> indeed, resolution is dependent on the numerical aperture of the
> > >> condenser.  "Of course", it is never a 100% correct description of
> > reality,
> > >> since you will never be able to attain illumination by "just one"
> > >> wavelength; in the very best case you might get a Lorentzian profile
> of
> > a
> > >> good single mode laserbeam.
> > >>
> > >> Also, in DIC microscopy, when, as Guy already has written, close to
> > >> perfect Koehler illumnation is a sine qua non for a "good image" -
> > whatever
> > >> THIS would be, :-), another issue is that you can trade contrast for
> > >> resolution. I.e.: By closing the condenser aperture diaphragm - often
> > >> referred to as the "A diaphragm" as opposed to the "F diaphragm",
> which
> > is
> > >> the Field Diaphragm - you will increase contrast while at the same
> time
> > >> reduce resolution. In "manuals for microscopists" of the sixties and
> > >> seventies as published in Germany, one will sometimes be instructed to
> > do
> > >> the following: "First, attain perfect Koehler illumation, then lower
> the
> > >> condenser somewhat to attain better contrast." Yes, it works, and it
> is
> > a
> > >> kind of rather un-controlled oblique illumination, which one fakes in
> > that
> > >> way. But: As a real physicist, you would, for ideological reasons,
> > never do
> > >> this. If you are a pragmatic life scientist: Well, you have a better
> and
> > >> easier life, anyway.
> > >>
> > >> Also note: Make sure that your light source and your polarizer and
> > >> analyzer are useful in the same wavelength band! At least on older DIC
> > >> systems, polarizers and analyzers will do a good job in the visible
> > >> wavelength range, while they will act as a mere 80% Transmission ND
> > filter
> > >> in IR and do hardly polarize! For IR, you might need separate and
> > special
> > >> polarizers (and analyzers, of course, which are nothing else than
> > >> polarizers in another functional position).
> > >>
> > >> Best wishes and have a good 1st of advent,
> > >>
> > >> Johannes
> > >>
> > >> On 2014-11-28 21:21, Jeff Spector 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.
> > >>> *****
> > >>>
> > >>> Greetings,
> > >>>    This isn't exactly a "confocal" questions but I know a lot of
> > >>> "micoscopy
> > >>> gurus" live on this list so I thought it a good place to ask this. I
> > >>> have a
> > >>> colleague who is trying to image individual (i.e. small and
> diffraction
> > >>> limited) microtubules in a flow chamber by using DIC. They are
> > currently
> > >>> using a 100x 1.45 oil Objective, but only a .52 LWD condenser. They
> > were
> > >>> using a solid state light source but couldn't get good image so we
> > >>> switched
> > >>> to a lamp for illumination and the images are much better, and we can
> > now
> > >>> see the microtbules but there still isn't a lot of contrast.  My
> > question
> > >>> is, is it worth it to go to a high NA (perhaps oil immersion)
> > condenser,
> > >>> and can anyone think of why the lamp would give a better DIC image
> > than a
> > >>> solid state light source?
> > >>> thanks in advance for the help...
> > >>>  -Jeff
> > >>>
> > >>
> > >>
> > >
> > > --
> > >
> > >
> > >
> > > George McNamara, Ph.D.
> > > Single Cells Analyst
> > > L.J.N. Cooper Lab
> > > University of Texas M.D. Anderson Cancer Center
> > > Houston, TX 77054
> > > Tattletales http://works.bepress.com/gmcnamara/42
> > >
> >
>

[Barbara Foster]

*****
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

For a quick overview of how DIC works and how to optimize it, you
might want to check out this article.  A PDF is not very good quality
(the original article was ancient!) but the information is solid and
will help answer a lot of these questions:
http://microscopyeducation.com/images/AR_AL_Apr_1988_DIC.pdf

Good hunting!
Barbara Foster, President & Chief Consultant
Microscopy/Microscopy Education*
www.MicroscopyEducation.com


Have you taken part yet in our survey, "20 Really Quick
Questions"?  If not, please give us your input.  Every data point is
important! Deadline: Dec 6.  "Thank you gift": A chance to win one of
TEN $100 Amazon gift cards
Click here or paste into your browser:
<http://www.surveygizmo.com/s3/1885625/c7c50d3c93fb>http://www.surveygizmo.com/s3/1885625/c7c50d3c93fb 




At 03:35 AM 12/2/2014, you 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.
>*****
>
>Non-commercial response:
>
>Jeff,
>
>In my experience, use a Planapochromat 60/1.4 or 100/1.45 oil
>objective, 1.4NA oil condenser, precision interference line green
>filter(546/10), and "standard" or "high" contrast DIC objectives &
>DIC condenser prisms. You will need to carefully test which
>combination of DIC prisms work best for you. Be sure the back
>aperture of the objective is completely filled. You will probably
>need to introduce a 1.5x-2.5x magnification factor in the camera
>light path, and, in this case, a solid state illuminator should give
>you more light than a 100W halogen lamp. Some microscope systems
>offer a high NA illuminator adapter that will accept a solid state
>illuminator liquid light guide, and this works well for this
>application. You will probably need to adjust your image histogram
>for the best image.
>
>I recently used the above combination on an inverted microscope  and
>achieved excellent results with a 5MP sCMOS camera.
>
>David
>
>David J. Claypool
>Digital Imaging Product Manager
>Micro Video Instruments
>Office: 800-875-2041 x5221
>Cell: 603-809-5342
>[hidden email]
>
> > On Nov 28, 2014, at 9:30 PM, "Jeff Spector" <[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.
> > *****
> >
> > Greetings,
> >   This isn't exactly a "confocal" questions but I know a lot of "micoscopy
> > gurus" live on this list so I thought it a good place to ask this. I have a
> > colleague who is trying to image individual (i.e. small and diffraction
> > limited) microtubules in a flow chamber by using DIC. They are currently
> > using a 100x 1.45 oil Objective, but only a .52 LWD condenser. They were
> > using a solid state light source but couldn't get good image so we switched
> > to a lamp for illumination and the images are much better, and we can now
> > see the microtbules but there still isn't a lot of contrast.  My question
> > is, is it worth it to go to a high NA (perhaps oil immersion) condenser,
> > and can anyone think of why the lamp would give a better DIC image than a
> > solid state light source?
> > thanks in advance for the help...
> > -Jeff