Biomedical Engineering Reference
In-Depth Information
Phase stepper
λ
/2 - Plate
Analyzer
PBS
CCD
camera
Laser
Specimen
CCD
camera
Bright field
illumination
Figure 4.5
Coniguration of the setup in bright ield mode for locating and positioning of the sample prior to
the measurement.
Confocal LSM:
Epifluorescence
Interferometer setup:
Transmission
Objective
Cover glass
Sample
Slide
Excitation
Fluorescence emission
Condenser
Figure 4.6
Comparison between the confocal laser scanning microscopy and the interferometer setup. he two
beam paths are equivalent when focused to the bottom of the specimen.
Because we are concerned with aberrations in the CFMs, we have to verify whether this experiment
actually models a CFM setup. he phase-stepping interferometer works in transmission mode* while a
typical CFM uses epi-detection where the emitted luorescence light is collected by the same lens. Both
setups are compared in Figure 4.6. If the focus is set to the bottom of the specimen, the aberrations
introduced within the two beam paths are equivalent because the wavefront leaves the sample below
the focus experiencing no further aberration. Similarly, a focal position at the top of the specimen
below the coverslip is equivalent to an epi-coniguration focused to the top of the specimen from below.
Only these two conigurations deliver results identical to the epi-setup of a confocal or multiphoton
*
It does not seem to be practical to build an interferometer that operates in epi-mode. For the interferometry, one would
need a relecting probe at the focal spot to maintain the coherence properties of the wavefront. he relection of the wave-
front would make it travel back to the lens, but it would also cause an inversion of the wavefront symmetry. herefore, the
overall propagation path of the wavefront would not be identical to the one corresponding to the confocal epi-geometry.
Solutions to this problem might include a curved mirror or a phase conjugate mirror below the sample but that is likely
to lead to serious alignment problems or technical diiculties.
