Two-photon light-sheet live imaging at kilohertz frame rate using birefringence-based pulse splitting

TL;DR

This work tackles the bottleneck of slow frame rates and photodamage in two-photon live imaging by introducing a passive, birefringence-based pulse-splitting scheme that splits each laser pulse into multiple collinear sub-pulses using cascaded YVO$_4$ crystals. The method decouples pulse frequency from wavelength, enabling tunable average pulse frequencies and preserving beam quality, with minimal impact on resolution and photobleaching, while enabling high-speed 2P light-sheet imaging of zebrafish heart and neural activity. By optimizing excitation at $1070$ nm and achieving effective pulse frequencies up to $16$ MHz (via pulse splitting of a fixed $4$ MHz source), the authors demonstrate kilohertz frame rates and pixel rates exceeding $150$ MHz, with subcellular resolution and manageable heating ($\Delta HBR$ reduced at 1070 nm). The approach is low-cost, compact, alignment-friendly, and adaptable to various wavelengths, representing a practical route to fast, in vivo 2P imaging with reduced photodamage and heating, especially for red fluorophores.

Abstract

Multiphoton microscopy is widely used for live imaging. However, its acquisition speed remains limited by fluorophore emission rates and photodamage. To increase the pixel rate of a two-photon microscope beyond a few megahertz (MHz), multi-point parallelized schemes have been proposed. Two-photon (2P) light-sheet microscopy emerges as an effective approach for high-speed multiphoton imaging of live specimens, as it enables parallelized excitation while minimizing the required laser power. However, optimizing the signal-to-photodamage ratio in 2P light-sheet microscopy requires to precisely control the illumination parameters, including both wavelength and pulse frequency. Since conventional femtosecond laser sources generally do not allow independent modulation of these parameters, the development of low-cost, efficient and robust strategies to modulate the temporal excitation profile is essential to fully exploit the advantages of 2P light-sheet microscopy. Here, we introduce a compact pulse splitting scheme that meets these criteria. We used cascaded birefringent crystals to convert each excitation laser pulse into an adjustable sequence of collinear sub-pulses. We demonstrate its effectiveness in optimizing 2P light-sheet imaging of live zebrafish embryos. We analyze the impact of pulse splitting on photobleaching, nonlinear photodamage, and imaging performance. Additionally, we demonstrate high-speed 2P imaging of the beating heart and brain calcium dynamics using red fluorophores in live embryos. We achieve kilohertz imaging frame rate, reaching more than 150 MHz pixel rates with fluorescent signal levels above 10 $photons.pixel^{-1}$ using a laser mean power and a peak intensity in the range of 100 mW and 0.1 $TW.cm^{-2}$ at the sample, respectively. This adjustable pulse-splitting scheme allows full advantage to be taken of light-sheet illumination for fast in vivo 2P imaging.

Figures (7)

• Figure 1: (a) Principle of birefringence-based pulse splitting with linearly polarized output: (i) When the input pulse polarization is set to $0^\circ$, and the OA of the first birefringent crystal (BC1), the second birefringent crystal (BC2), the half-wave plate (HWP), and the polarizer (P) both are aligned at $0^\circ$, the output pulse frequency remains unchanged; (ii) Adjusting the OA of BC2 to $45 ^\circ$ and the OA of the HWP to $45 ^\circ$ while maintaining others parameters unchanged doubles the output pulse frequency; (iii) Setting the OA of BC1 to $45 ^\circ$, the OA of BC2 to $0^\circ$, and the OA of HWP to $22.5 ^\circ$ results in a fourfold increase in the output pulse frequency. All OA angles of optical elements are defined relative to the vertical direction in this work. (b) Schematic of the 2P light-sheet microscopy setup. A wavelength-tunable laser beam passes through a pulse-splitting (PS) unit, then an electro-optic modulator (EOM), and is subsequently directed to a galvanometric mirror (GM). The beam is relayed through two scanning lenses, which together conjugate the GM plane to the back focal plane of the objective (OB) with appropriate magnification. The laser then illuminates the sample through OB1, while OB2 provides white-light illumination. The emitted 2PEF signals pass through OB3 and tube lens (TL) before being recorded by a camera. (c) Laser parameters for configurations with pulse frequencies of 2 $MHz$ (conventional), 2$\times$2 $MHz$, 2$\times$2$\times$2 $MHz$, 4 $MHz$ (conventional), 2$\times$4 $MHz$, 2$\times$2$\times$4 $MHz$ and 8 $MHz$ (conventional), along with the corresponding pulse train temporal profiles. The laser frequency denotes the source pulse repetition rate, laser source specifies the employed laser system, splitting indicates the optical splitter configuration, average pulse frequency represents the average pulse frequency at the output of the splitter, and the notation refers to the corresponding labeling used in this work.
• Figure 2: Analysis of 2PEF signals and photon bleaching effect. (a) Violin plots comparing the 2PEF signals with and without the pulse splitter under varying pulse frequency conditions. (b) Normalized mean 2PEF signal across multiple cells over time under different pulse frequency conditions. (c) Violin plots depicting the decay of 2PEF signals from selected cells, illustrating the bleaching effect over time at each pulse frequency condition. $\mu$ indicates the mean value, $N$ represents the number of cells analyzed, and $p$ indicates the $P$-value of t-tests, representing statistical significance between different groups of decay rates. The experiments (c-d) were conducted under laser illumination at a wavelength of $1030 \ nm$.
• Figure 3: Experimental investigation of wavelength-dependent linear heating based on water absorption and zebrafish embryonic heart beat rate (HBR) measurements. (a) Representative instantaneous HBR in zebrafish over time under femtosecond laser illumination at $1030 \ nm$ (orange) and $1070 \ nm$ (red), showing the changes in $\Delta$HBR relative to the baseline $\text{HBR}_{0}$ when the laser is on (pink time window). (b) Violin plot of $\Delta$HBR/$\text{HBR}_{0}$ ratios under $1030 \ nm$ (orange) and $1070 \ nm$ (red) femtosecond laser illumination, where each data point represents an independent measurement from a different individual zebrafish embryo. The violin plots show the median and the first and third quartiles. $\lambda_{illu.}$ stands for the excitation wavelength, $N$ indicates the number of zebrafish examined, and $\mu$ represents the mean value.
• Figure 4: Quantification of nonlinear photodamage threshold using zebrafish HBR in vivo. The mean power at the nonlinear photodamage threshold ($P_{NL}$) is measured in zebrafish under femtosecond laser exposure across different conditions: no pulse splitting, 2$\times$ pulse splitting, and 2$\times$2$\times$ pulse splitting, at pulse frequency of $4 \ MHz$, $8 \ MHz$, and $16 \ MHz$, with illumination wavelengths of $1030\ nm$ and $1070\ nm$.
• Figure 5: High-speed 2P light-sheet imaging of beating heart and neuronal activity in live zebrafish embryos. (a) 4D reconstruction of the atrio-ventricular canal in the beating heart of a 3-day post-fertilization zebrafish embryo labeled with Histone-mCherry, captured at 1 kHz frame rate (2$\times$2$\times$4 $MHz$ average pulse frequency, $110 \ mW$ mean power at sample, $147 \ MHz$ pixel rate). (b) 4D reconstruction of the entire beating heart of a 4-day post-fertilization zebrafish embryo labeled with Histone-mCherry, captured at 465 Hz frame rate (2$\times$2$\times$4 $MHz$ average pulse frequency, $110 \ mW$ mean power at sample, $165 \ MHz$ pixel rate). (c) in vivo calcium imaging in zebrafish embryo showing spontaneous activity using the jRGECO1b indicator (2$\times$2$\times$4 $MHz$ average pulse frequency, $91 \ mW$ mean power at sample), recorded at 465 Hz frame rate ($165 \ MHz$ pixel rate). (d) Neuronal activity profiles ($\Delta F/F_0$) for selected regions of interest (ROIs) from (c), demonstrating detailed activity patterns at $465 \ Hz$ frame rate. The experiments (a-d) were launched under laser illumination at a wavelength of $1070 \ nm$. Grid spacing $100 ~~\mu m$ in (a,b). See Methods section \ref{['Image visualization']} for the image visualization details.