Fluorescence Phase Microscopy (FPM) and Nanoscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 374

function (PSF) of single fluorophores [1,3,5,9,10] , (ii) stimulated emission depletion of
fluorescence for engineering of 3D PSFs that are narrowed down to subdiffraction
dimensions [6] , and (iii) structured illumination to obtain a 3D optical resolution of
B 100 nm [11] . In 3D microscopy with mesoscopic resolution, fluorescence-discrimination
mechanisms involve selective axial (depth) illumination and off-axis collection of light to
improve the background suppression of fluorescence in 3D extended specimens
[12,13,15,16] and multiple-view imaging that allows for 3D image reconstruction of the
sample by using backprojection processing techniques [12,17] or mathematical models of
photon propagation in turbid media together with application of inversion theory [12,14] .
Common to most existing and proposed fluorescence imaging methods is their reliance on
the detection of the fluorescence intensity (rather than the fluorescent field) to provide axial
(depth) information about the specimen. The reason for these intensity-based approaches is
simple: photodetectors can extract only the magnitude of the incident optical field. As we
discuss later, however, the phase of the fluorescent wave can also encode information,
which would be useful for a broad spectrum of application in the natural and life sciences.
In this chapter, we introduce the concept of fluorescence interferometry and present its
usefulness in converting phase variations of fluorescent light into amplitude variations. As a
result, this amplitude-to-phase conversion offers a new form of fluorescence imaging that
encodes axial (depth) information about the sample in the phase of the fluorescent waves
and yields a new paradigm of 3D imaging at the nanoscale and mesoscale levels. Here, we
demonstrate that the treatment of fluorescent waves as low-temporal coherence optical
fields and their manipulation by self-referencing interferometry is useful for metrology and
imaging at the mesoscopic to nanoscopic resolution scales. Interestingly, manipulation of
low-temporal coherence optical fields is largely employed in imaging through tissue and
cells and has had a profound impact on the field of biomedicine. Examples include optical
coherence tomography (OCT; [18 23] ) that uses coherence gating to simultaneously
provide micron-scale optical sectioning along penetration depths of a few millimeters and
quantitative phase microscopy [24 28] that enables measurements with nanometer-level
accuracy. We point out that the phenomenon of fluorescence interference has been
investigated in fundamental studies of molecules in front of reflecting surfaces [29,30] ,
applied to the assessment of nanometer-level displacements of a fluorescent molecule
above a reflector [31 33] , employed in 4Pi and I5M microscopy for achieving
subdiffraction-limited resolution along the axial (depth) dimension [34 37] , and also used
in spectroscopy [38] .
This chapter is organized as follows. We first describe the process of fluorescence self-
interference and its potential use for providing new measurement capabilities, such as
fluorescence tomography with mesoscopic resolution and nanometer-level localization of
individual fluorescent markers in all 3D. Next, we present time-domain (TD) and spectral-
domain (SD) experimental realizations of fluorescence phase microscopy (FPM) and