Biomedical Engineering Reference
In-Depth Information
where n is the refractive index of the specimen, M is the combined lateral magnification of
the objective and zoom lenses, and e is the smallest distance that can be resolved by the
image detector (measured on the detector's face plate). The wave optical depth of field is
determined by one quarter of the distance between the first two diffraction minima, above
and below focus. The geometrical optical depth is a result of the “circle of confusion.” For
example, a bright-field microscope using a 40 3 /0.95NA objective lens and a CCD camera
with 6.45
m at wavelength
546 nm. As it was shown by Inou ´ [10] , the thickness of optical section (depth of field) of a
conventional DIC microscope equipped with the same objective lens could be as little as
0.25
μ
m square pixels would have the field depth in water 1.0
μ
μ
m.
The contrast in DIC is produced by the optical path difference in a small in-focus volume
where two interfering beams are spatially separated. Here, the beams travel through the
different areas of the specimen under investigation. The out-of-focus object introduces
practically the same phase disturbance in both the beams because the beams go through
almost the same area of the specimen. Therefore, the out-of-focus disturbance is suppressed
by optical subtraction. The optical section depth becomes thinner if the shear amount is
smaller, and the objective and condenser NAs are larger. The narrow optical sectioning DIC
phenomenon is similar to removing an out-of-focus haze in the structured illumination
microscopy (SIM) [9] . The SIM employs a single-spatial-frequency grid pattern, which is
projected onto the object under investigation. Raw images are taken at three spatial
positions of the grid. The out-of-focus picture of the object does not depend on the pattern
position. As a result, the out-of-focus haze in SIM is subtracted computationally.
Application of computation subtraction in the DIC would be expected to improve its
sectioning capability even further. The computation subtraction of images with different
biases is employed in various techniques, such as polarization modulation DIC (PM-DIC)
[11,12] , differential detection DIC (D-DIC) with polarizing beamsplitter [13] , phase-shifting
DIC (PS-DIC) [14
19] , and
orientation-independent DIC (OI-DIC) [6,20,21] . In particular, the PM-DIC removes a
background contribution that is insensitive to defocus [12] . It is shown theoretically and
confirmed experimentally that an RM-DIC microscope has stronger optical sectioning than
a conventional DIC microscope, and the optical section depth is thinner if a Nomarski
prism with smaller shear amount is used [19] . Our experiments with the OI-DIC microscope
using 100 3 /1.3NA oil immersion objective lens demonstrated the optical section depth
about 0.1 μ m. The corresponding field depth of a bright-field microscope would be 0.5 μ m.
16] , retardation modulation DIC (RM-DIC) [17
2.2 Measuring Shear Angle of DIC Prism
Shear amount (distance) is the critical parameter of a DIC microscope that determines its
contrast, sensitivity, resolution, and optical section depth. Another issue with DIC
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