Hyperspectral oblique plane microscopy provides Raman contrast images of biological specimens with unprecedented pixel throughput. Being a light-sheet technique it can capture hundreds of spectra in the time a point scanning system would take to capture a single spectrum. We will demonstrate the utility of this new system by monitoring wound healing in a zebrafish at five minute intervals, Followed by spontaneous Raman imaging of a beating zebrafish heart.
Structured Illumination Microscopy (SIM) is a widely used super-resolution microscopy method, capable of imaging at twice the diffraction limit of conventional widefield microscopes. We developed a new method to assess in silico the spatio-temporal resolution limits of SIM, and demonstrated that its capacity to reconstruct super-resolved information is substantially worse than the time required to acquire a full stack of raw frames. We also applied our method to gauge the efficacy of a reconstruction method termed “rolling SIM” which claimed to improve the temporal resolution of SIM, and we showed that this is not the case.
Temporal focusing is a technique for performing axially resolved widefield multiphoton microscopy with a large field of view. Despite significant advantages over conventional point-scanning multiphoton microscopy in terms of imaging speed, the need to collect the whole image simultaneously means that it is expected to achieve a lower penetration depth in common biological samples compared to point-scanning. We assess the penetration depth using a rigorous objective criterion based on the modulation transfer function, comparing it to point-scanning multiphoton microscopy. Measurements are performed in a variety of mouse organs in order to provide practical guidance as to the achievable penetration depth for both imaging techniques. It is found that two-photon scanning microscopy has approximately twice the penetration depth of temporal-focusing microscopy, and that penetration depth is organ-specific; the heart has the lowest penetration depth, followed by the liver, lungs, and kidneys, then the spleen, and finally white adipose tissue.
High resolution imaging in three dimension is important for biological research. RESOLFT (Reversible Saturable Optical Fluorescence Transitions) microscopy is one technique can achieve lateral super-resolution imaging. Twophoton microscopy naturally generate high resolution in the longitudinal direction with less background compared to single photon excitation. In this paper, we combine these two methods to realize three-dimensional high-resolution imaging. Spatial light modulator (SLM) is used as a flexible phase mask of the microscopy. Multiple super-resolution focuses as an array or in arbitrary positions could be generated by phase retrieval. This microscopy by SLM control could applied to parallel two-photon RESOLFT imaging or multiple spots tracking in high-resolution.
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