Analytic method to optimize aperture design in focal modulation microscopy

Abstract

Focal modulation microscopy (FMM) has been demonstrated more effective than confocal microscopy for imaging of thick biological tissues. To improve its penetration depth further, we propose a simple analytical method to enlarge the modulation depth, the unique property of FMM directly linked to its signal-to-noise ratio. The modulation depth increases as the excitation intensity of the binary phase aperture status is pushed further away from the focal region of the detection optics, thereby creating a dark region in the focal volume, which we call maximally flat crater (MFC). By direct algebraic manipulation, MFCs are achieved for both scalar and vector diffraction optics. Numerical results show that the modulation depth from MFC is very close to the maximum values, with a small difference less than 3% for the same number of subapertures. Applications of bifocus produced by MFC apertures are also discussed.

Augmented line-scan focal modulation microscopy for multi-dimensional imaging of zebrafish heart in vivo

“…The noise level in an imaging system depends on many factors such its work principle, configuration, and operation mode. While LSFMM images enjoy a much better signal to background ratio than that of LSCM images, their noise level is generally higher due to limited modulation depth (typically 30-60%) and coherent speckles generated by the aberrated ballistic excitation photons [23,24]. In addition, the LSCM images are usually much brighter as scattered illumination photons also contribute to the excitation of focal plane fluorophores.…”

Effects of pupil filter patterns in line-scan focal modulation microscopy

“…The first is the maximally flat cater (MFC) method, which was previously developed for point-scanning FMM. 20 The second is simply the equal-area (EA) patterns, which guarantee that the total areas of modulated and nonmodulated zones are identical and all off-center zones are equal in size. According to MFC, for a binary phase pupil filter with nðn ≥ 2Þ zones and n − 1 boundaries at m 1 ; m 2 ; : : : ; m n−1 , the amplitude in the focal region can be expanded into a Taylor series with coefficients given by the moment of the aperture, where the p'th moment is defined as 21 E Q -T A R G E T ; t e m p : i n t r a l i n k -; e 0 0 9 ; 6 3 ; 6 3 1…”

“…x vanish due to the rotational symmetry of annular MFC filter. 20 Therefore, MFC filters are expected to be less effective in pushing the intensity distribution away from the center in an LSFMM system. Given that EA outperforms MFC in LSFMM in terms of modulation depth, our further investigation is confined to EA pupil filters.…”

Line-scan focal modulation microscopy

“…[14][15][16] It employs a spatiotemporal phase modulator in the excitation light path to modulate the phase of the excitation beam spatially and temporally (at a fixed frequency). The spatially and temporally phase modulated beam, after being condensed by an objective, is subjected to intensity modulation that is essentially confined to the focal plane.…”