Achieving Low Photobleaching Using LSFM

Researchers in biological imaging strive for 3D, high-speed, and high-resolution imaging with minimal photobleaching and phototoxicity. The light-sheet fluorescence microscope (LSFM) contributes to this goal. The LSFM can image live specimens with high spatiotemporal resolution and low photobleaching thanks to a novel excitation and detection scheme. It has demonstrated great promise for the 3D imaging of biological samples. The basic idea behind LSFM technology is to illuminate the sample with a thin light-sheet and then collect the emitted fluorescence along an axis perpendicular to the light-sheet’s transmission.

A team of researchers recently developed a photon-efficient method for increasing the FoV. The researchers used image scanning microscopy (ISM), a high-resolution imaging technique, to create ISM-enhanced laterally swept light-sheet microscopy (iLSLM).

Both iLSLM and axially swept light-sheet microscopy (ASLM) outperform conventional swept focus light-sheet microscopy (SFLM) in axial resolution and optical sectioning, and iLSLM outperforms ASLM when >55% photon efficiency is required. However, iLSLM’s current work is based on digital pixel reassignment, which significantly slows imaging speed. The researchers will investigate optical pixel reassignment in the future to achieve the same imaging speed as ASLM. Meanwhile, iLSLM will be suitable for applications where photobleaching is a serious issue, or the specimen is phototoxic.

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