Differential Interference Contrast Microscopy (DIC) - Biomedical Optical Phase Microscopy and Nanoscopy - page 35

Prof. James LaFountain (State University of New York at Buffalo, Buffalo, NY). The phase
image was computed by Dr. David Biggs (KB Imaging Solutions, Waterford, NY) by
employing the iterative deconvolution approach mentioned earlier.

The following are the comments made on this picture by MBL Distinguished Scientist
Shinya Inou´:

The image is a real WOW! That is so striking; I have never seen such a view of a divid-
ing cell, ever! Absolutely; have a huge blow up of this image for your poster at ASCB.
The crane fly spermatocyte meiosis image is just mind blowing. You see the many thin-
thread-shaped mitochondria surrounding the spindle, the chromosomes themselves and
even the spindle fibers, all in striking 3-D. Also the scattered dyctiosomes show as promi-
nent bright spots. But even more, the image shows some cytoplasmic structures that I
had never seen in my life. Those must relate to Keith Porter's endoplasmic reticulum
(membrane-related structures?) but in a different form. Their contrast is low but they def-
initely appear to be indented where astral rays would be expected. I don't think anyone
has shown such structural differentiation of the cytoplasm until now, using any mode of
microscopy whether in live or fixed and stained cells! In a nutshell this image shows the
unusual capability of your orientation independent DIC system exceptionally well.

2.5 Combination of OI-DIC and Orientation-Independent
Polarization Imaging

DIC microscopy produces images of optical phase gradients in a transparent specimen,
which is caused by variation of refractive index within thin optical section. Polarized light
microscopy reveals structural or internal anisotropy due to form birefringence, intrinsic
birefringence, stress birefringence, and other factors. An image in DIC microscope is
determined by the optical phase distribution in the specimen, while an image in a polarized
light microscope is produced by the polarization splitting of the optical phase caused
by the specimen anisotropy. Thus, polarization microscopy data and DIC results are
complementary. Both methods, however, have the same shortcomings: they require the
proper orientation of a specimen in relation to the optical system in order to achieve high
quality results, and the images are not quantitative. Hence, it would be beneficial to
combine the OI-DIC microscope with an orientation-independent differential polarization
(OI-Pol) system [45] .

The differential OI-Pol microscope captures several images of the specimen with slightly
different polarization settings of illumination or imaging beam [29,45 47] . Then, the

images are subtracted one from another and further processed in order to obtain the
quantitative picture of retardance and fast axis azimuth distribution.

The combined system is unique, providing complementary phase images of thin optical
sections of the specimen that display a distribution of refractive index gradient and