We propose a framework to quantify photodamage in multiphoton light-sheet microscopy. Using cardiac imaging in live zebrafish embryos, we demonstrate an order of magnitude signal enhancement is safely obtained by adjusting the laser repetition rate.
Light-sheet illumination enables major increase in multiphoton imaging speed for in vivo studies. However, photoperturbation in multiphoton light-sheet microscopy remains poorly investigated. We show here that the heart beat rate of zebrafish embryos is a sensitive probe of linear and nonlinear photoperturbations. By analyzing its behavior with respect to laser power, pulse frequency and wavelength, we derive guidelines to balance signal and photoperturbation. We then demonstrate one order-of-magnitude signal enhancement over previous implementations by optimizing the laser pulse frequency. These results open new opportunities for fast in vivo imaging.
Light-sheet fluorescence microscopy is a method of choice for multiscale live imaging. Indeed, its orthogonal geometry results in high acquisition speed, large field-of-view and low photodamage. Its combination with multiphoton excited fluorescence improves its imaging depth in biological tissues. However, it appears femtosecond laser sources commonly used in multiphoton microscopy at an 80 MHz repetition rate may not be optimized to take full advantage of light-sheet illumination during live imaging. Hence, we investigated the nature of induced photodamage in multiphoton light-sheet microscopy and the influence of laser parameters on the signal-to-photodamage ratio. To this end, we used zebrafish embryonic heart beat rate and fluorophore photobleaching as sensitive reporters of photoperturbations. We characterized linear and nonlinear disruptions depending on laser parameters such as laser mean power, pulse frequency or wavelength, and determine their order and relative impact. We found an optimal pulse frequency of ~10 MHz for imaging mCherry labeled beating hearts at 1030 nm excitation wavelength. Thus, we achieved high-speed imaging without inducing additional linear heating or reaching nonlinear photodamage compared to previous implementation. We reach an order-of-magnitude enhancement in two-photon excited fluorescence signal by optimizing the laser pulse frequency while maintaining low both the laser average power and its peak irradiance. It is possible to reach even larger enhancement of 3- photon excited fluorescence using such laser parameters. More generally, using low laser pulse frequency in multiphoton light-sheet microscopy results in a drastic improvement in signal level without compromising live sample, which opens new opportunities for fast in vivo imaging.
Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We address this issue by introducing chromatic multiphoton serial (ChroMS) microscopy, a method combining multicolor multiphoton excitation through wavelength mixing and microtome-assisted serial block-face image acquisition. This approach delivers large-scale micrometric imaging of spectrally distinct fluorescent proteins with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We achieve multicolor 3D imaging over several cubic millimeters and brain-wide serial 2D multicolor imaging. We illustrate the potential of this method for several novel types of measurements interesting for region-scale or whole brain studies: (i) color-based analysis of astrocyte morphology and spatial interactions in the mouse cerebral cortex, (ii) tracing of densely labeled neurons, and (iii) brain-wide mapping of axonal projections labeled with distinct tracers.
Single-cell dry mass measurement is used in biology to follow cell cycle, to address effects of drugs, or to investigate cell metabolism. Quantitative phase imaging technique with quadriwave lateral shearing interferometry (QWLSI) allows measuring cell dry mass. The technique is very simple to set up, as it is integrated in a camera-like instrument. It simply plugs onto a standard microscope and uses a white light illumination source. Its working principle is first explained, from image acquisition to automated segmentation algorithm and dry mass quantification. Metrology of the whole process, including its sensitivity, repeatability, reliability, sources of error, over different kinds of samples and under different experimental conditions, is developed. We show that there is no influence of magnification or spatial light coherence on dry mass measurement; effect of defocus is more critical but can be calibrated. As a consequence, QWLSI is a well-suited technique for fast, simple, and reliable cell dry mass study, especially for live cells.
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