Depth-extended, high-resolution fluorescence microscopy: whole-cell imaging with double-ring phase (DRiP) modulation

Abstract

We report a depth-extended, high-resolution fluorescence microscopy system based on interfering Bessel beams generated with double-ring phase (DRiP) modulation. The DRiP method effectively suppresses the Bessel side lobes, exhibiting a high resolution of the main lobe throughout a four-to five-fold improved depth of focus (DOF), compared to conventional wide-field microscopy. We showed both theoretically and experimentally the generation and propagation of a DRiP point-spread function (DRiP-PSF) of the imaging system. We further developed an approach for creating an axially-uniform DRiP-PSF and successfully demonstrated diffraction-limited, depth-extended imaging of cellular structures. We expect the DRiP method to contribute to the fast-developing field of non-diffractingbeam-enabled optical microscopy and be useful for various types of imaging modalities.

Non-Diffracting Light Wave: Fundamentals and Biomedical Applications

“…The combination of the Bessel beam and the three-photon excitation was confirmed to increase the SNR in the 3PM, owing to the higher-order nonlinearity in the 3PM than that in the 2PM [101,130]. The axial superposition of two Bessel beams for a 0 th -order Bessel beam was also demonstrated in fluorescence microscopes using beams interfered with specific conical wave vectors [23,161]. The interfered Bessel beam is produced by creating two concentric annular apertures (Figure 15E) [23].…”

“…In terms of the application aspect, we mainly Side-view profiles of positively translated (z 0 7 mm), original (z 0 0 mm), negatively translated (z 0 −7 mm) double-ring PSFs, and the average of all the three PSFs. Reproduced from OSA [23,133].…”

“…The axial superposition of two Bessel beams for a 0 th -order Bessel beam was also demonstrated in fluorescence microscopes using beams interfered with specific conical wave vectors [23,161]. The interfered Bessel beam is produced by creating two concentric annular apertures (Figure 15E) [23]. The radii and thicknesses of the two annuli are determined by varying SLM phase patterns for optimized performance.…”

“…One possible means to overcome scattering is using the nondiffracting beam, which preserves the shape over a distance longer than the Gaussian Rayleigh range. Here, the theory, generation, and significant features of many "non-diffracting" beams are reviewed, with an emphasis on the biomedical applications of those fascinating beams, including the multimodal biomedical imaging [11,[23][24][25][26], optical micromanipulation [10,27,28], and optical transfection [29][30][31]. Specifically, we introduce the use of non-diffracting beams to overcome the challenges arising from various applications, including two-photon microscopy, Raman spectroscopy, and optical manipulation.…”

Bessel-droplet foci enable high-resolution and high-contrast volumetric imaging of synapses and circulation in the brainin vivo

“…Our approach thus allowed us to utilize the excitation by multiple droplets to probe a much larger volume. Together with its high throughput, our method is also ideally suited for single-photon fluorescence microscopy modalities including in the fluorescence emission path for high-contrast extended depth-of-field widefield imaging (28).…”