Three-photon light-sheet fluorescence microscopy

Abstract

We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single-and two-photon modes has emerged as a powerful wide-field, low photo-damage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime enhancing its penetration depth. Our present study uses a standard conventional femtosecond pulsed laser at 1000 nm wavelength for the imaging of 450 µm diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams.

Balancing signal and photoperturbation in multiphoton light-sheet microscopy by optimizing laser pulse frequency

“…A quadratic signal enhancement is obtained in the case of 3PEF, corresponding to 64-fold more signal at f = 10 MHz (Fig. S3) compared to previously reported 3P-SPIM at f = 80MHz [29].…”

“…As a consequence, nonlinear signals used in multiphoton light-sheet microscopy, such as 2-photon excited fluorescence (2PEF [4,24,27,28]), second harmonic generation (SHG [30]) or 3-photon excited fluorescence (3PEF [29]) can be enhanced by increasing T while keeping P mean constant. Using 2P-SPIM imaging zebrafish embryos expressing mCherry fluorescent proteins, KTP nanocrystals [30], and fluorospheres embedded in a gel (Supplementary methods and Table S1), we confirmed that both 2PEF and SHG signals depend linearly on T (n = 2, Fig.…”

“…In multiphoton light-sheet microscopy, adjusting the laser pulse frequency remains an attractive yet unexplored strategy to balance signal and photoperturbation. Indeed, most multiphoton light-sheet microscopes described so far [4,[10][11][12][13][24][25][26][27][28][29] were implemented using mode-locked Ti:sapphire laser at ~80 Mhz, which is the most commonly used laser source in multitphoton microscopy. However, this choice is questionable and may not take full advantage of light-sheet illumination and its orthogonal geometry.…”

Two‐ and three‐photon absorption cross‐section characterization for high‐brightness, cell‐specific multiphoton fluorescence brain imaging

“…As a general tendency, seen from the 2PEF and 3PEF images in 3AD, 3PEF images are much sharper and show a much higher contrast compared to their 2PEF counterparts. This finding agrees well with the earlier threephoton imaging studies [3][4][5][6][7].…”

“…In their seminal work, Horton et al have shown that microscopy based on three‐photon‐excited fluorescence (3PEF) enables a noninvasive, high‐resolution in vivo imaging of subcortical structures within an intact mouse brain. Significant recent milestones in the development of 3PEF imaging technology include demonstration of optical imaging of neuronal activity deep in intact mouse brain , wide‐field three‐photon optogenetic stimulation , brain structure and function detection through an intact skull , and 3PEF light‐sheet microscopy .…”