An integrated single- and two-photon non-diffracting light-sheet microscope

Abstract

We describe a fluorescence optical microscope with both single-photon and two-photon non-diffracting light-sheet excitations for large volume imaging. With a special design to accommodate two different wavelength ranges (visible: 400-700 nm and near infrared: 800-1200 nm), we combine the line-Bessel sheet (LBS, for single-photon excitation) and the scanning Bessel beam (SBB, for two-photon excitation) light sheet together in a single microscope setup. For a transparent thin sample where the scattering can be ignored, the LBS single-photon excitation is the optimal imaging solution. When the light scattering becomes significant for a deep-cell or deep-tissue imaging, we use SBB light-sheet two-photon excitation with a longer wavelength. We achieved nearly identical lateral/axial resolution of about 350/270 nm for both imagings. This integrated light-sheet microscope may have a wide application for live-cell and live-tissue three-dimensional high-speed imaging.

Simultaneous multiple-level magnification selective plane illumination microscopy (sMx-SPIM) imaging system

“…Since the first technological advent of LSFM in 1993 [1], there have been significant advances [6][7][8][9][10][11][12][13] in order to meet the demands for biological studies and its applications. Several research studies have been reported for improving the obtainable resolution (both temporal [12,14,15] and spatial [10,[16][17][18][19]), simultaneous recovery of multi-physiological activities [20][21][22][23], and signal contrast [3]. Recently, studies on development of multiview based SPIM-that primarily aim to enhance the achievable imaging depth and image quality of SPIM by fusing or stitching sequential multiview imageswere reported [6,[24][25][26].…”

Light sheets with extended length

“…The phototoxicity can be further reduced by spreading the light energy uniformly into the sheet. We recently demonstrated a simple and robust method to generate ultrathin line Bessel sheets (LBS) [9,10]. Using this technique, we create smooth and long light sheets without dithering the beam, which can have much less phototoxicity than structured light sheets.…”