Fluorescence Phase Microscopy (FPM) and Nanoscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 383

spectral fringes. The severeness of this effect increases for larger oscillation periods of the
spectral fringe, i.e., for increased distances between the fluorescent layer and the z 0 .
Axial Localization Precision of Individual Fluorophores
As discussed in the previous sections, FPM can localize individual fluorophores with
nanometer-level accuracy in all 3D. The lateral position can be retrieved by adequately
analyzing the acquired diffraction-limited PSF image. In general, lateral localization can be
performed by fitting a 2D Gaussian to the image of individual fluorophores. However, to
incorporate the effect of the dielectric mirror in the total-internal-reflection-fluorescence
(TIRF)-based FPM system on the recorded PSF images, we developed a novel PSF model
for fluorescent dipoles, which expresses the emission pattern as a superposition of three
orthogonal dipoles with different radiation weights (see Ref. [39] for more details). Using
this approach, we achieved a lateral localization precision below 10 nm [42] . The axial
localization can be obtained by detecting the phase of the self-interference signal. To
measure the 3D localization precision of FPM, we dried a low concentration of fluorescent
QDs on a layered custom-made slide comprising a glass slip, dielectric mirror, and SiO 2
spacer [39,42] . The image and the self-interference spectrum of a single QD were acquired
by a homemade objective-type TIRF microscope combined with a spectrometer as shown in
Figure 18.3B . Figure 18.5 (top-left panel) presents a conventional measured image of a
single QD. The resulting diffraction-limited PSF of the single QD was next analyzed to
evaluate its centroid and consequently its lateral position with high precision. A typical
cluster of multiple lateral position determinations (referred as localizations) resulting from
repetitive localization of the QD is shown in the top-right panel of Figure 18.5 . The
standard deviation of this cluster was computed to be B 8 nm suggesting a lateral resolution
below 10 nm in FWHM. The bottom-left panel in Figure 18.5 shows the measured (solid
line) and fitted (dashed line) self-interference spectrum of a single QD. The spectral
measurements lasted for a few seconds to a minute depending on the resolution and the lens
aperture of the spectrometer. The resulting spectral interference pattern together with the a
priori knowledge about the spacer thickness were next Fourier analyzed to yield an axial
localization precision below 10 nm as shown in the bottom-right panel of Figure 18.5 .
These 3D localization accuracy levels are in accordance with other methods employing
fluorescence self-interference for the development of novel optical imaging systems with a
high resolution in all 3D [2,4] .
18.4.2 Optical Sectioning Imaging with Mesoscopic Resolution by FPM
One of the unique abilities of FPM is to acquire images deep inside the sample and across a
wide field of view with a conventional collinear excitation and detection geometry at
mesoscopic resolution. To demonstrate this capability of FPM, we first used a calibrated
sample comprising a dual-layered fluorescent sample and recorded the tomogram of the