Fluorescence Phase Microscopy (FPM) and Nanoscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 376

combined. A fluorescence interference pattern is detected only when the optical path-length
difference of the clockwise and counterclockwise fluorescent beams is equal to or less than
the coherence length of the fluorophore, which is typically on the order of a few
micrometers. For a fluorescent emitter located far from the zero differential optical path-
length point (z 0 ) of the self-referencing interferometer (positions I and III), no interference
pattern is detected and the received signal is proportional to the emission intensity of the
fluorophore. As the fluorescent source comes close to z 0 (position II) to within its coherence
length, a clear interference pattern appears. The z position of a point source z (relative to z 0 )
can be extracted from the self-interference pattern with two distinct precision scales by
using white-light interferometry. For example, a mesoscopic precision scale (1 10 m) can

be achieved by spectrally resolved interferometry [39 41] or by axially scanning the
fluorescent point source through the depth of field (which is kept greater than the coherence
length of the source or the z dimension of the object under observation) followed by the
identification of the central fringe position [39 41] , whereas a nanoscopic precision scale
( , 100 nm) can be obtained by phase-shifting [2,4] or spectral interferometric techniques
[32,33,40,42] .

μ

The localization of fluorophores with mesoscopic or nanoscopic precision scales has
important implications for fluorescence phase imaging. First, small fluorescent markers
(i.e., those having width smaller than one half of the fluorophore's center emission
wavelength) which are located at different axial positions and separated by greater than one
half of the fluorophore's coherence length along the same axial line (depth) can be
distinguished unmistakably because an interference pattern is detected only for markers
residing at j z 2 z 0 j , l c /2, where z is the axial (depth) position of the fluorophore and l c is
its coherence length. As a result, this capability opens up the new possibility for depth-
resolved optical imaging of thick specimens with a large confocal length at the mesoscopic
scale. Secondly, phase-shifting analysis or spectral interferometry can yield the capability
for localizing individual fluorescent probes along the axial (depth) dimension with
nanometer-level accuracy—an important task for accomplishing fluorescence imaging with
nanoscopic resolution in all 3D.

Section 18.3 deals with various experimental designs of FPM, such as setups employing
time-resolved interferometry and spectrally resolved interferometry, and describes in detail
their capabilities and limitations.

18.3 Experimental Setups of FPM

The design and experimental realization of a fluorescence phase microscope require the
combination of a self-referencing interferometer (such as the two-opposing-lenses
interferometer employed in 4Pi and I5M microscopy systems [34 37] ) with appropriate