Differential Interference Contrast Microscopy (DIC) - Biomedical Optical Phase Microscopy and Nanoscopy - page 37

computing phase (dry mass) and retardance images (middle top and bottom, accordingly).
On the far right is a color combination of the phase (dry mass) and retardance modes, in
which red and green colors correspond to dry mass distribution and retardance, respectively.
Morphological structures, such as chromosomes, are especially prominent in the phase
mode image. The birefringent spindle fibers (actually bundles of microtubules) exhibit
much better contrast in the retardance mode. The combined picture provides clear evidence
of our notion that the proposed technique can reveal architecture (morphology) of live cells
without fluorescent labeling using 1.4NA optics.
2.6 Combination of OI-DIC and Fluorescence Imaging
In recent years, advances in fluorescent biosensors have made fluorescence imaging of
living cells a key tool for cell biologists. However, quantitative data such as refractive
indices and birefringences of whole specimens provide important information about protein
concentration, density, and structural organization inside cells, but cannot be measured
using fluorescence imaging. Although OI-DIC microscopy provides an extraordinary level
of detail, the technique is more informative of cellular structures than of specific molecules.
Fluorescence microscopy, on the other hand, provides detailed information on the
distribution patterns of specific molecules and ions, but this information is often hard to
interpret in the absence of additional structural information.
Combining fluorescence microscopy with OI-DIC imaging is an ideal technology that
provides specificity of fluorescence and quantification of OI-DIC. For example, the type of
organelles in a live specimen could be scored using fluorescence markers, and subsequent
organelle development followed during a long time series using the OI-DIC with minimal
phototoxicity.
Two schematics of the OI-DIC in combination with fluorescence microscopy were built:
(1) simultaneous OI-DIC and fluorescence with two CCD cameras and (2) sequential
OI-DIC and fluorescence with a switchable beamsplitter cube. Both approaches have been
used successfully.
Figure 2.10 shows simultaneous images of chromosomes in a live Spisula oocyte treated
with Hoechst 33342 (DNA-specific fluorescent dye) obtained by OI-DIC microscopy and
wide field fluorescence microscopy. The micrographs were taken with a new Olympus
30 3 /1.05NA silicon oil objective lens UPLSAPO 30XS and photo eyepiece PE2.5 3 . The
fluorescence image contains four selected square areas with chromosomes. These areas are
enhanced in the computed quantitative gradient and phase images. The white level is
0.1 nm/nm in the enhanced areas and 0.3 nm/nm in other areas of the gradient picture.
Brighter OI-DIC signal in the phase image indicates higher optical density, which
corresponds to higher dry mass or concentration of the objects. The dry mass image of