Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DIC-or-Phase-contrast-for-image-morphometry-mesasurements-tp7068286p7069256.html
*****
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