Implementation of spatial overlap modulation nonlinear optical microscopy using an electro-optic deflector

Abstract

A spatial overlap modulation (SPOM) technique is a nonlinear optical microscopy technique which enhances the three-dimensional spatial resolution and rejects the out-of-focus background limiting the imaging depth inside a highly scattering sample. Here, we report on the implementation of SPOM in which beam pointing modulation is achieved by an electro-optic deflector. The modulation and demodulation frequencies are enhanced to 200 kHz and 400 kHz, respectively, resulting in a 200-fold enhancement compared with the previously reported system. The resolution enhancement and suppression of the out-of-focus background are demonstrated by sum-frequency-generation imaging of pounded granulated sugar and deep imaging of fluorescent beads in a tissue-like phantom, respectively.

Modulated alignment dual-axis (MAD) confocal microscopy for deep optical sectioning in tissues

“…In the context of DAC microscopy, focal modulation microscopy is not ideal in its original form [21] since the Rayleigh range of the low-NA beams utilized in DAC microscopy is large and would therefore result in a modulated point spread function that extends over a large area, rather than being confined to the focal volume defined by the intersection of the DAC illumination and collection beams. Alternatively, the spatial overlap modulation technique reported by Isobe et al [22,23] relies on a slight spatial modulation in the lateral direction between two coaxial beams, which generates a strong modulation in the signal generated at the focus of a nonlinear microscope but negligible modulation of the out-of-focus background signals.…”

Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination

“…This background fluorescence limits the maximum imaging depth [5]. Indeed, extensive efforts have been made to overcome this limitation [6][7][8][9][10][11][12][13][14][15][16]. The decreased scattering of excitation light in the sample, which has been achieved by using TPEF at 1280 nm [6,7] and three-photon excited fluorescence at 1700 nm [8], has extended the maximum imaging depth.…”

“…Adaptive optics can be used to reject the background by differential aberration imaging [12] and to recover diffraction-limited performance by compensating for wavefront distortion of the excitation pulse [13] in TPEF microscopy. The out-of-focus background in various point-scanning nonlinear microscopies has been rejected by spatial overlap modulation of two-color pulses, which creates spatial nonlinear intensity modulation near the focal point [14,15]. The suppression of the background fluorescence in TPEF microscopy has been also achieved by utilizing photoactivatable fluorophores, which remain in a non-fluorescent state until optically triggered [16].…”

Spatial Overlap Modulation Nonlinear Optical Microscopy for Background-Free Deep Imaging

“…(b, c) Sum frequency generation (SFG) images of granulated sugar pounded in a mortar obtained by (b) conventional SFG microscopy and (c) SPOMNOM 20) . (d, e) Maximumintensity x projections of the image stacks of fixed mouse brain tissues, which express a YFP in a subset of neurons, obtained by (d) conventional TPEF microscopy and (e) SPOMNOM 19) .…”