Imaging a large number of samples is necessary to improve statistical robustness in biological assays. Using standard multiwell plates is not possible in usual light-sheet microscopes. One solution is to use an Oblique Plane Microscope (OPM), which is a special fluorescence light sheet microscope, with a single objective near to the sample. To avoid aberrations, OPM generally uses water or silicon immersion to match the refractive index of the sample. In this work we present an oil-immersed OPM and experimentally demonstrate the possibility of using a primary objective with a higher numerical aperture.
Over the past few decades, a multitude of optical imaging techniques have emerged. Among them, full-field optical coherence tomography (FF-OCT) has gained significant importance in various biomedical applications. Indeed, FF-OCT stands out as a noninvasive and label-free imaging method capable of generating high-resolution 3D microscopic images of light-scattering biological specimens. However, FF-OCT approach is limited for in-vivo imaging and images from FF-OCT lack the specificity required for accurate diagnosis. Hence, there is a need to have access to in-vivo imaging and to incorporate additional contrast modalities, such as elastography, into the FF-OCT technique. Indeed, the combination of FF-OCT with shear wave elastography enables the quantitative assessment of tissue stiffness at a resolution of a few micrometers. In this context, we present a novel FF-OCT approach that enables single-shot acquisitions, making it well-suited for both in-vivo imaging and transient shear wave elastography.
The last few decades have seen the emergence of a huge number of optical imaging techniques. Among them, full-field optical coherence tomography (FF-OCT) has become valuable for many biomedical applications. Indeed, FF-OCT is a noninvasive and label-free imaging technique that produces high-resolution 3D microscopic images of scattering biological samples. Using FF-OCT approach for in-vivo imaging would enable the observation of cell-scale structures in living samples. Moreover, living samples have an active vascularization that can therefore be observed using Doppler imaging. We propose in this study a new FF-OCT approach that enables single-shot acquisitions which is suitable for in-vivo and Doppler imaging.
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.
Light sheet fluorescence microscopy (LSFM) enables fast 3D imaging of live cells, however the traditional LSFM geometry is not compatible with conventional multiwell plates used in high content microscopy.
We have developed an automated LSFM platereader based on an oblique plane microscope (OPM) that is compatible with multiwell plates. The system enables automated studies of cells cultured in a collagen matrix. Cells across 30 different conditions are imaged every 5 minutes for 12 hours. A custom 3D segmentation and tracking pipeline analyses cell morphological dynamics, allowing the study of a range of treatment conditions on the ability of cancer cells to change shape and invade.
Oblique plane microscopy (OPM) is a light-sheet fluorescence microscopy technique that is implemented on a standard inverted microscope frame. OPM uses a single high numerical aperture microscope objective to both produce a tilted excitation light-sheet and to image the fluorescence emitted from the tilted plane back to the cameras. It is therefore compatible with conventional sample-mounting techniques such as microscope slides and multiwell plates. Four excitation laser lines and two high-speed sCMOS cameras with separate emission filters enable the simultaneous imaging of several fluorophores and spectral ratiometric FRET acquisitions.
Previously, 3-D OPM imaging has been implemented by remote refocusing. Here, a stage-scanning approach to 3-D OPM imaging is demonstrated - enabling three-dimensional multi-channel acquisition including of multiwell plates - and the synchronization of the stage movement and camera acquisition will be described.
The ability of the stage-scanning system to image fields of view larger than the field of view of the primary microscope objective is demonstrated using fluorescently labelled limbs of crustaceans and its ability to perform time-lapse 3-D imaging over 12 hours is demonstrated using a sample of tumor spheroids with an acquisition time of 3 s for a typical spheroid providing 400x1280x1024 voxels per spheroid.
We also apply the system to spectral ratiometric Förster resonant energy transfer (FRET) measurements in tumor spheroids expressing a FRET biosensor and in a 96-well plate seeded with cell samples expressing varying concentrations of a FRETting and non-FRETting constructs.
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