DIC or Phase contrast for image morphometry mesasurements?

[Monique Vasseur]

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Dear All,

I wonder what is better for yeast morphometry shape and size image analysis:
should we better use phase contrast images or DIC images? Is one more
accurate  for measures? )Which threshold will be more precise: the one of DIC
shear or the one of phase contrast halo?)  Do some of you have experience on
this question? Any input is welcome  Thanks a lot!
Monique

[Theresa Swayne]

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Hi Monique,

If fluorescence is a possibility, the work of the Ohya and Morishita labs (using fluorescent ConA to mark the cell wall) might be helpful. They also wrote a program called CalMorph to analyze many cell size and shape parameters.

Good luck,
Theresa

On Dec 6, 2011, at 3:23 PM, Vasseur Monique wrote:

>
> I wonder what is better for yeast morphometry shape and size image analysis:
> should we better use phase contrast images or DIC images?

------------------------------------
Theresa C. Swayne, Ph.D.
Manager, Confocal and Specialized Microscopy Shared Resource
Herbert Irving Comprehensive Cancer Center, Columbia University
1130 Saint Nicholas Ave, 222A
New York, NY 10032

212-851-4613

[hidden email]
http://hiccc.columbia.edu/research/sharedresources/confocal

[mmodel]

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Hi Monique, what exactly would you like to measure? We have a method for volume measurements, if this would be of interest. Some of it has been published, but I can send you the latest information. - Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Vasseur Monique
Sent: Tuesday, December 06, 2011 3:23 PM
To: [hidden email]
Subject: DIC or Phase contrast for image morphometry mesasurements?

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Dear All,

I wonder what is better for yeast morphometry shape and size image analysis:
should we better use phase contrast images or DIC images? Is one more
accurate  for measures? )Which threshold will be more precise: the one of DIC
shear or the one of phase contrast halo?)  Do some of you have experience on
this question? Any input is welcome  Thanks a lot!
Monique

[G. Esteban Fernandez]

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I did a lot of yeast imaging with DIC in grad. school and it was a
pain for morphometry because of the "shadow" effect of DIC; one side
of the cells was bright while the other was dark and that was
problematic for thresholding.  Now I'd use phase, or even brightfield
or darkfield.  From your question, though, it sounds like getting the
cell outline is no problem for you with DIC and you're just wondering
about accuracy, which I don't know.

-Esteban


On Tue, Dec 6, 2011 at 12:23 PM, Vasseur Monique
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> I wonder what is better for yeast morphometry shape and size image analysis:
> should we better use phase contrast images or DIC images? Is one more
> accurate  for measures? )Which threshold will be more precise: the one of DIC
> shear or the one of phase contrast halo?)  Do some of you have experience on
> this question? Any input is welcome  Thanks a lot!
> Monique

[George McNamara]

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Hi Monique,

Brightfield with optimized Kohler illumination (condenser field aperture
diaphragm focused and centered, numerical aperture diaphragm optimized
for the NA of your objective lens). You did not specify what detector -
I will assume a good scientific grade monochrome digital camera (ex.
Hamamatsu ORCA-ER). Use monochromatic light (ex. 546 nm narrow bandpass
filter), or the emission filter of a green or blue fluorescence bandpass
filter cube (ex. GFP or DAPI). Shorter wavelength is better. Higher NA
objective AND condenser lenses better. Ideally use a 1.4 NA oil
immersion objective lens and oil immersion objective lens. I recommend X
and Y pixel size 3.5x smaller than the resolution equation of d =
1.22*lambda/(NAobj+NAcond), that is pixel size ~ d/3.5 (note; if you
cannot get to 3.5x, try to get close).

Refractive index of the mounting medium (culture medium if live cells)
is tricky. Higher NA, closer to that of the lens immersion medium is
better, but:
1. may be higher osmolarity, resulting in cell shrinkage, which may
defeat the goal of making the measurement.
2. the cells will become closer to invisible, the closer to R.I. matched
and best focus you reach (reference is F. Zernike, Science 121 (Issue
314, Mar. 11, 1955: 345-349, http ://www.jstor.org).

With respect to #2, a good digital camera and understanding of
acquisition settings, contrast adjustments, and shading correction, will
get you excellent and reproducible images, even if contrast by eye is
minimal. Some DAPI or Hoechst (or GFP positive cells) may help find the
cells!

If you have access to a Diatom 'test plate' - Carolina Biological Supply
(good luck with CBS's search engine) - see


  Fundamentals of light microscopy and electronic imaging

By Douglas B. Murphy
pages 93-94

http://books.google.com/books?id=UFgdjxTULJMC&pg=PA93&lpg=PA93&dq=diatom+test+plate&source=bl&ots=vkHltkLVvi&sig=L399EbcUIx3ebJac54Hfvce5vxM&hl=en&ei=887eTsjnNpTAtgeT0cmFBA&sa=X&oi=book_result&ct=result&resnum=2&ved=0CCQQ6AEwAQ#v=onepage&q=diatom%20test%20plate&f=false 


g and h specimens(h is Amphipleura pellucida, 0.27 um/stria) are hard to
resolve with white light, better with green (GFP emission filter),
better with blue light (DAPI bandpass emission filter ... typically
halogen lamp should be at max voltage and will likely need to optimize
the exposure time).

This slide as well as a thin unstained muscle tissue section slide were
great slides to work with at UIC (video contrast adjustment days). For a
while Richardson (Corp?) in Canada had a nice resolution target. Other
companies still offer them. Measuring the performance on the diatom
slide or other test target and stating that measurement in your methods
section should help the review process when you go to publish.


Plan R: reflection mode confocal microscopy with a short wavelength
laser line (405 nm or more practically on most Argon ion laser systems,
458 nm). Be careful to record the PMT gain and offset settings. Note
that if a cell is very close to the coverglass, (or slide) you can get
interference reflection contrast (helpful or not depending on goals).
IRM is useful to find the coverglass, but may cause confusion for cells
on or near the coverglass.

Plan S: plasma membrane (or cell wall) fluorescent label using a
fluorophore or fluorescent protein that you can use on your nearest STED
nanoscope. Alternatively, PALm, STORM, FPALM, 3D-SIM, etc.

Best wishes,

George


On 12/6/2011 3:23 PM, Vasseur Monique wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All,
>
> I wonder what is better for yeast morphometry shape and size image analysis:
> should we better use phase contrast images or DIC images? Is one more
> accurate  for measures? )Which threshold will be more precise: the one of DIC
> shear or the one of phase contrast halo?)  Do some of you have experience on
> this question? Any input is welcome  Thanks a lot!
> Monique
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[James Pawley]

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>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>I did a lot of yeast imaging with DIC in grad. school and it was a
>pain for morphometry because of the "shadow" effect of DIC; one side
>of the cells was bright while the other was dark and that was
>problematic for thresholding.  Now I'd use phase, or even brightfield
>or darkfield.  From your question, though, it sounds like getting the
>cell outline is no problem for you with DIC and you're just wondering
>about accuracy, which I don't know.
>
>-Esteban
>
>
>On Tue, Dec 6, 2011 at 12:23 PM, Vasseur Monique
><[hidden email]> wrote:
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>>
>>  Dear All,
>>
>>  I wonder what is better for yeast morphometry shape and size image analysis:
>>  should we better use phase contrast images or DIC images? Is one more
>>  accurate  for measures? )Which threshold will be more precise: the
>>one of DIC
>>  shear or the one of phase contrast halo?)  Do some of you have experience on
>>  this question? Any input is welcome  Thanks a lot!
>>  Monique

Dear Monique,

As everyone is chipping in, have you thought about using darkfield?

You would need a good darkfield condenser and they can be a bother to
set up (though a lot cheaper than DIC and not too bad setting up on
such a thin specimen.) and an objective with an NA about 1.15 (or
larger but with an iris diaphragm. Of course, you would lose a little
on the resolution because of the lower NA (but not that much if your
specimen is in water as you don't get many rays propagating past NA
1.25 anyway because of reflection losses.

What you do gain is a very simple image (objects having a different
RI from their surroundings look white on a black background). And, on
such an image, it might be much easier to come up with an accurate
and  reproducible measurement scheme.

It would probably not be hard to get your microscope salesman to come
around and set up a system for you to see how well it works.

Jim Pawley
--
***************************************************************************
Prof. James B. Pawley,                          Ph.
608-238-3953                        
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 9-21, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/        Applications accepted after 11/16/12
               "If it ain't diffraction, it must be statistics." Anon.

[Peter Gabriel Pitrone]

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Hello Monique,

I'd recommend you listen to Michael Model on this one... I know that what
he has to offer may be very useful for you!!

Another possibility would be to use Dark Field, but you would need a
special setup: an Iris oil objective of very high N.A. and a oil Dark
Field condensor with the highest N.A. you can get your hands on. One
problem would be any thing in the sample that will scatter light WILL show
up (dirt & grime on your coverslips for example).

Good luck!!

Pete

On Tue, December 6, 2011 9:52 pm, MODEL, MICHAEL wrote:
| *****
| To join, leave or search the confocal microscopy listserv, go to:
| http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
| *****
|
| Hi Monique, what exactly would you like to measure? We have a method for
| volume measurements, if this would be of interest. Some of it has been
| published, but I can send you the latest information. - Mike
|
| -----Original Message-----
| From: Confocal Microscopy List [mailto:[hidden email]]
| On Behalf Of Vasseur Monique
| Sent: Tuesday, December 06, 2011 3:23 PM
| To: [hidden email]
| Subject: DIC or Phase contrast for image morphometry mesasurements?
|
| *****
| To join, leave or search the confocal microscopy listserv, go to:
| http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
| *****
|
| Dear All,
|
| I wonder what is better for yeast morphometry shape and size image
| analysis:
| should we better use phase contrast images or DIC images? Is one more
| accurate  for measures? )Which threshold will be more precise: the one of
| DIC
| shear or the one of phase contrast halo?)  Do some of you have experience
| on
| this question? Any input is welcome  Thanks a lot!
| Monique
|


--
Peter Gabriel Pitrone - TechRMS
Microscopy/Imaging Specialist
Prof. Dr. Pavel Tomancak group
Max Planck Institute for
Molecular Biology and Genetics
Pfotenhauerstr. 108
01307 Dresden

"If a straight line fit is required, obtain only two data points." - Anon.

[Monique Vasseur]

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Hi Mike,
We are trying to caracterize and classify different yeast mutants by their morphology appearance (morphotype) and size.  Actually, we are trying to caracterize in 2D with the different shape factors like circularity, elongation, aspect ratio, sphericity... and so on, and by the area. For your volume measurements do you need 3D acquisition? I guess volume should be more precise than area. Yes, your publication interests me.  Thanks  a lot
All the best,

Monique Vasseur
-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de MODEL, MICHAEL
Envoyé : 6 décembre 2011 15:53
À : [hidden email]
Objet : Re: DIC or Phase contrast for image morphometry mesasurements?

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Hi Monique, what exactly would you like to measure? We have a method for volume measurements, if this would be of interest. Some of it has been published, but I can send you the latest information. - Mike

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Vasseur Monique
Sent: Tuesday, December 06, 2011 3:23 PM
To: [hidden email]
Subject: DIC or Phase contrast for image morphometry mesasurements?

*****
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Dear All,

I wonder what is better for yeast morphometry shape and size image analysis:
should we better use phase contrast images or DIC images? Is one more
accurate  for measures? )Which threshold will be more precise: the one of DIC
shear or the one of phase contrast halo?)  Do some of you have experience on
this question? Any input is welcome  Thanks a lot!
Monique

[Stanislav Vitha]

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Use blue or green filter for imaging to minimize chromatic aberration.
Brightfield with properly set up Köhler illumination (open the condenser
diaphragm as much as you can while still getting acceptable contrast) should
provide for best resolution without the edge artifacts of DIC or phase
contrast.  
With a 100x/1.3 or 1.4  oil immersion objective and a typical interline
transfer CCD camera (6.45 um pixel size), you should be OK as far as Nyquist
sampling.

I find DIC quite tricky for measurements of cell outlines, given the
shadowing effects.

If you are OK with somewhat lower resolution of the phase contrast
technique, you can minimize the phase halo around the cells by increasing
the refractive index of the medium - you could add some BSA  (if I remember
correctly, something like 10 % is used by some).

Stan Vitha

On Tue, 6 Dec 2011 14:23:26 -0600, Vasseur Monique
<[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear All,
>
>I wonder what is better for yeast morphometry shape and size image analysis:
>should we better use phase contrast images or DIC images? Is one more
>accurate  for measures? )Which threshold will be more precise: the one of DIC
>shear or the one of phase contrast halo?)  Do some of you have experience on
>this question? Any input is welcome  Thanks a lot!
>Monique

[Pascal Weber]

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Have you test any color camera to do that ? May be it is possible to do that
whit a bw camera with a green filter. Most of the time the filter increase
contrast !
At yet i measure colonies on agarose substract and it's realy easy to do that.
I'm using MetaMorph software and make threshold using size, circularity,
shape and any filter you want...

[Barbara Foster]

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Hi, Monique

Several basic fundamentals to consider:
a. RE: Phase
Phase suffers from haloing, an artifact which can obscure edges.
Having said that, the technique can be optimized in two ways.  Both
depend on the fact that Phase is engineered to work best when there
is a lambda/4 shift in light passing through the mountant
("background) versus the light passing through the sample.  The
further you deviate from this engineering requirement, the worse the
halo will become.  Without going into all the physics, as to the
source of this artifact, here are the practicalities: you can
optimize the RI match between the sample and the mountant AND you can
optimize the wavelength (lambda). Since you are working with a living
organism, you will be restricted on what mountant will work.  Water
(RI 1.33) is probably preferable, but you might want to also try
something like a glucose solution (Caution: yeast love sugar and will
produce CO2 which, in small quantities, should just dissolve, but in
larger quantities will cause bubbles in your sample). On the
wavelength side, your phase kit should have come with a green
filter.  Check with your microscope vendor as to which wavelength
(typically 546nm or 589nm) your phase kit was engineered to use.  If
the filter is just a glass filter, you can improve the situation by
using a narrow band pass interference filter.  Here again, there will
be a trade-off... you may need more light going in and that might not
be good for your yeast.

b. RE: Darkfield
Two drawbacks here:
Darkfield has infinitely deep depth of field, so if your preps are
not really clean, you may get interference from objects above and
below the real plane of focus.
Depending on the diffraction at the edge, you may also get thicker
boundaries, making exact location of the edge difficult to
determine.  Again, you might be able to optimize by playing with the
mounting medium.

c. RE: DIC
I agree with the earlier posting that the bright edge/dark edge can
be a problem, but as I understand it, some of the newer software
programs have algorithms to address this issue.  It's been a while
since I worked in this area, but I seem to remember edge filters
(Sobel? Roberts?) which might also help.  Alternatively, if your DIC
is tunable to the point that you can change the background from
"first order gray" to "first order red", you can get away from the
bright/dark problem and just get "color contrast" (one edge blue, one
edge yellow).  Because DIC is typically used to detect gradients and
the most sensitive setting is when the background is dove gray, we
typically don't advise folks to work in this range, but it is
certainly doable.  Of all the techniques, this one will provide the
best, crispest edge definition and the most shallow depth of field
(tunable using your aperture iris).  For all the details as to why
this works, see my article in American Lab from April 1988.  (I can
email you a PDF, although it is not a very good copy).

Good hunting... and let us know how you make out!

Best regards,
Barbara Foster, President and Sr. Consultant

Microscopy/Microscopy Education
P: [phone]
W: www.MicroscopyEducation.com

We are now scheduling courses through June 2012




At 09:33 AM 12/7/2011, you wrote:

>*****
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>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Have you test any color camera to do that ? May be it is possible to do that
>whit a bw camera with a green filter. Most of the time the filter increase
>contrast !
>At yet i measure colonies on agarose substract and it's realy easy to do that.
>I'm using MetaMorph software and make threshold using size, circularity,
>shape and any filter you want...

[George McNamara]

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Hi Monique,

You're welcome. I see that you are interested in shape factors. If you
have access to MetaMorph software, the ones you mentioned are in there
(plus fiber length and breadth, though yeast are probably not snake like
enough to make these useful) and 40+ of my favorites: Elliptical Fourier
Analysis (EFA). Page 7 of
http://sydney.edu.au/medicine/bosch/facilities/microscopy/ImagingSoftware/IMA_Measurement_Parameters.pdf 

is not a complete list of MetaMorph's EFA parameters (I take no credit
or blame for any of the drawings).
There is an option to turn them on in Preferences. You can also combine
interesting coefficients, as Tom Coates did in a 1993 paper (before I
went to work for him) - PubMed 8243213. See also the classic paper by
Ferson, Rohlf  et al 1985 - http://www.jstor.org/pss/2413345 (I turned
Rohlf's Fortran code into Turbo Pascal, which the MetaMorph programmers
turned into long forgotten MPL code, which morphed into something that
still works in MM7.7). Rohlf's web site is still up, and even has 3D EFA
- http://life.bio.sunysb.edu/morph/soft-outlines.html  (I only
implemented 2D - also Rohlf's code and mine were able to average
harmonics over all inputted perimeters and then do the inverse FFT to
make an averaged shape - never implemented in MetaMorph ... when I
learned of TypIC - see below - I liked the approach of picking the
median, since that would be a real shape (if odd number of cells
inputted)).

Robert Murphy's PSLID and related software/database at  
http://murphylab.web.cmu.edu/ may or may not have EFA built in to it. I
don't see whether PSLID or SLIC still allows uploading ~100 images and
calculate results for you. The ancestor of PSLID was TypIC:

Toward objective selection of representative microscope images.
</pubmed/10096918> Markey MK, *Boland* MV, *Murphy* RF. Biophys J. 1999
Apr;76(4):2230-7. PMID: 10096918.

Scientists wishing to communicate the essential characteristics of a
pattern (such as an immunofluorescence distribution) currently must make
a subjective choice of one or two images to publish. We therefore
developed methods for objectively choosing a typical image from a set,
with emphasis on images from cell biology. The methods involve
calculation of numerical features to describe each image, calculation of
similarity between images as a distance in feature space, and ranking of
images by distance from the center of the feature distribution. Two
types of features were explored, image texture measures and Zernike
polynomial moments, and various distance measures were utilized.
Criteria for evaluating methods for assigning typicality were proposed
and applied to sets of images containing more than one pattern. The
results indicate the importance of using distance measures that are
insensitive to the presence of outliers. For collections of images of
the distributions of a lysosomal protein, a Golgi protein, and nuclear
DNA, the images chosen as most typical were in good agreement with the
conventional understanding of organelle morphologies. The methods
described here have been implemented in a web server
(http://murphylab.web.cmu.edu/services/TyplC).
{web link long dead}.


Best wishes,

George
p.s. if you can find a MetaMorph CD, there should be an EFA bibliography
on it - probably inside a zip file. I can send it to anyone interested
(may be a few days due to travel).

*****

Hi Mike,
We are trying to caracterize and classify different yeast mutants by their morphology appearance (morphotype) and size.  Actually, we are trying to caracterize in 2D with the different shape factors like circularity, elongation, aspect ratio, sphericity... and so on, and by the area. For your volume measurements do you need 3D acquisition? I guess volume should be more precise than area. Yes, your publication interests me.  Thanks  a lot
All the best,

Monique Vasseur

On 12/7/2011 10:13 AM, Vasseur Monique wrote:

> Hi George,
>
> Thanks so much for the advices. I didn't think about blue monochromatic illumination to improve resolution neither to use a test plate.  You comments are always much appreciated and so useful.  Thank you!
> Best wishes,
>
> Monique
> -----Message d'origine-----
> De : Confocal Microscopy List [mailto:[hidden email]] De la part de George McNamara
> Envoyé : 6 décembre 2011 21:45
> À : [hidden email]
> Objet : Re: DIC or Phase contrast for image morphometry mesasurements?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Monique,
>
> Brightfield with optimized Kohler illumination (condenser field aperture
> diaphragm focused and centered, numerical aperture diaphragm optimized
> for the NA of your objective lens). You did not specify what detector -
> I will assume a good scientific grade monochrome digital camera (ex.
> Hamamatsu ORCA-ER). Use monochromatic light (ex. 546 nm narrow bandpass
> filter), or the emission filter of a green or blue fluorescence bandpass
> filter cube (ex. GFP or DAPI). Shorter wavelength is better. Higher NA
> objective AND condenser lenses better. Ideally use a 1.4 NA oil
> immersion objective lens and oil immersion objective lens. I recommend X
> and Y pixel size 3.5x smaller than the resolution equation of d =
> 1.22*lambda/(NAobj+NAcond), that is pixel size ~ d/3.5 (note; if you
> cannot get to 3.5x, try to get close).
>
> Refractive index of the mounting medium (culture medium if live cells)
> is tricky. Higher NA, closer to that of the lens immersion medium is
> better, but:
> 1. may be higher osmolarity, resulting in cell shrinkage, which may
> defeat the goal of making the measurement.
> 2. the cells will become closer to invisible, the closer to R.I. matched
> and best focus you reach (reference is F. Zernike, Science 121 (Issue
> 314, Mar. 11, 1955: 345-349, http ://www.jstor.org).
>
> With respect to #2, a good digital camera and understanding of
> acquisition settings, contrast adjustments, and shading correction, will
> get you excellent and reproducible images, even if contrast by eye is
> minimal. Some DAPI or Hoechst (or GFP positive cells) may help find the
> cells!
>
> If you have access to a Diatom 'test plate' - Carolina Biological Supply
> (good luck with CBS's search engine) - see
>
>
>    Fundamentals of light microscopy and electronic imaging
>
> By Douglas B. Murphy
> pages 93-94
>
> http://books.google.com/books?id=UFgdjxTULJMC&pg=PA93&lpg=PA93&dq=diatom+test+plate&source=bl&ots=vkHltkLVvi&sig=L399EbcUIx3ebJac54Hfvce5vxM&hl=en&ei=887eTsjnNpTAtgeT0cmFBA&sa=X&oi=book_result&ct=result&resnum=2&ved=0CCQQ6AEwAQ#v=onepage&q=diatom%20test%20plate&f=false
>
>
> g and h specimens(h is Amphipleura pellucida, 0.27 um/stria) are hard to
> resolve with white light, better with green (GFP emission filter),
> better with blue light (DAPI bandpass emission filter ... typically
> halogen lamp should be at max voltage and will likely need to optimize
> the exposure time).
>
> This slide as well as a thin unstained muscle tissue section slide were
> great slides to work with at UIC (video contrast adjustment days). For a
> while Richardson (Corp?) in Canada had a nice resolution target. Other
> companies still offer them. Measuring the performance on the diatom
> slide or other test target and stating that measurement in your methods
> section should help the review process when you go to publish.
>
>
> Plan R: reflection mode confocal microscopy with a short wavelength
> laser line (405 nm or more practically on most Argon ion laser systems,
> 458 nm). Be careful to record the PMT gain and offset settings. Note
> that if a cell is very close to the coverglass, (or slide) you can get
> interference reflection contrast (helpful or not depending on goals).
> IRM is useful to find the coverglass, but may cause confusion for cells
> on or near the coverglass.
>
> Plan S: plasma membrane (or cell wall) fluorescent label using a
> fluorophore or fluorescent protein that you can use on your nearest STED
> nanoscope. Alternatively, PALm, STORM, FPALM, 3D-SIM, etc.
>
> Best wishes,
>
> George
>
>
> On 12/6/2011 3:23 PM, Vasseur Monique wrote:
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear All,
>>
>> I wonder what is better for yeast morphometry shape and size image analysis:
>> should we better use phase contrast images or DIC images? Is one more
>> accurate  for measures? )Which threshold will be more precise: the one of DIC
>> shear or the one of phase contrast halo?)  Do some of you have experience on
>> this question? Any input is welcome  Thanks a lot!
>> Monique
>>
>>
>>      
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[Peter Werner]

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Vasseur Monique wrote:

> I wonder what is better for yeast morphometry shape and size image  
> analysis:
> should we better use phase contrast images or DIC images? Is one more
> accurate  for measures? )Which threshold will be more precise: the  
> one of DIC
> shear or the one of phase contrast halo?)  Do some of you have  
> experience on
> this question? Any input is welcome  Thanks a lot!
> Monique

I'd say whatever technique you use, avoid phase contrast for  
morphometry due to haloing. In my experience, it's often practically  
impossible to define an "edge" in a phase contrast image. As stated  
elsewhere, simple bright-field can provide very accurate morphometry  
if the cells have sufficient innate contrast, or if they can be  
stained, though I'm not sure if your protocol allows for that.

Peter G. Werner
Merritt College Microscopy Program