Improving axial resolution of Bessel beam light-sheet fluorescence microscopy by photobleaching imprinting

Abstract

Light-sheet microscopy has been widely used in high-speed fluorescence imaging with low phototoxicity, while the trade-off between the field-of-view and optical sectioning capability limits its application in large-scale imaging. Although Bessel beam light-sheet microscopy greatly enhances the light-sheet length with the self-healing ability, it suffers from the strong side-lobe effect. To solve these problems, we introduce the photobleaching imprinting technique in Bessel beam light-sheet microscopy. By extracting the non-linear photobleaching-induced fluorescence decay, we get rid of the large concentric side lobe structures of the Bessel beam to achieve uniform isotropic resolution across a large field-of-view for large-scale fluorescence imaging. Both numerical simulations and experimental results on various samples are demonstrated to show our enhanced resolution and contrast over traditional Bessel-beam light-sheet microscopy.

Multicolor light‐sheet microscopy for a large field of view imaging: A comparative study between Bessel and Gaussian light‐sheets configurations

“…A detailed characterization of the Bessel beams and their multicolor LSFM performance is compared with two Gaussian beams‐based LSFM configurations (DSLM and SPIM). The implications on the effective FOV for the multiple color‐Bessel and ‐Gaussian excitation light sheets are discussed when they are implemented under the same excitation path (i.e., the same excitation objective), as commonly implemented elsewhere for single‐color light‐sheets excitations [1, 3, 6, 10, 23, 24]. Therefore, this study complements previous comparative studies between Gaussian and Bessel LSFM configurations that only discussed monochromatic (or different colors but not simultaneous) excitation [13, 24].…”

“…In addition to compromising the optical sectioning slightly (and thus the axial resolution), these side lobes introduce a small background signal at the acquired images provoking a slight image contrast reduction and blurring [10,11,[14][15][16]. Therefore, for 3D imaging of small samples, say slightly larger than the light-sheet thickness, the out-of-focus fluorescence can be reduced by using confocal line-scanning detection, spatial filtering techniques, or beams subtraction methods, among others [2,6,10,14]. Nonlinear two-photon fluorescence excitation process has also been utilized to overcome such drawbacks [1,7,15,16].…”

“…Bessel beams have already been implemented in LSFM using complex optical systems, such as spatial light modulators [2,4,11,17] and pupil filters of different phase distributions [9,10]. Other more straightforward schemes utilize commercially available axicons (conical lenses) to form the Bessel beams resulting in a less expensive and easy-to-use alternative [1,[5][6][7], or even design custom-made micro-axicons [18].…”

Photobleaching Imprinting Enhanced Background Rejection in Line-Scanning Temporal Focusing Microscopy

“…For practical implementation, the LTFM-PIM image was obtained by processing time-lapse images with the following steps (Xiong et al, 2020): 1. Take M time-lapsed LTFM images with the same exposure time: F 1 , F 2 .…”

Период Капельного Квазибесселева Пучка, Генерируемого Аксиконом Со Скругленной Вершиной

“…На практике распределение интенсивности падающего на аксикон пучка, как правило, описывается гауссовой функцией [2], в результате чего формируется квазибесселев пучок с конечной длиной распространения, которая зависит от апертуры образующего пучка. Формируемые таким образом квазибесселевы пучки могут быть эффективно использованы для захвата и манипулирования микрообъектами [3], оптической визуализации [4], обработки материалов [5], микроскопии плоскостного освещения [6] и терагерцевой фотоники [7]. Возможности их практического использования значительно расширились после демонстрации генерации квазибесселевых пучков при помощи полупроводниковых лазеров и светодиодов [8,9].…”