Time-deterministic cryo-optical microscopy

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Paper Summary

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Freezing Cells Mid-Action: New Microscopy Technique Snaps High-Res Images of Cellular Dynamics

Researchers developed a technique to rapidly freeze cells during live observation with optical microscopy, allowing for high-resolution snapshots of dynamic processes. This method, coupled with time-deterministic freezing, enables researchers to observe fast reactions such as calcium waves and combine super resolution microscopy with Raman microscopy. Changes in the fluorescence intensity or spectrum shapes of some indicators under cryogenic conditions compared to those under room temperature, requiring careful analysis and interpretation.

Possible Conflicts of Interest

K.F. and Y.K. are inventors on a patent related to this work, and PCT national phase applications have been filed. K.F., M.Y., Y.K., and K.T. have pending patent applications. K.F. is also a co-founder and CTO of Millde Co., Ltd.

Identified Weaknesses

The difference of fluorescence intensity increases between the nucleus and cytoplasm regions after cryofixation

The changes in fluorescence intensity under cryogenic conditions might be caused by the changes in surrounding environment of the fluorescent indicator. The intensity changes need careful consideration when analyzing quantitative data.

The technique requires specialized equipment for cryogenic observations and manipulation, which can be complex and costly

This limits the widespread application of this technique.

The properties of some fluorescent probes may differ under cryogenic conditions compared to those in room temperature

Although many fluorescent probes work effectively under cryogenic condition, not all fluorescent probes are compatible with cryogenic conditions, especially those whose fluorescence intensity or spectrum shape is susceptible to temperature or its surrounding environment. This limits the choice of fluorescent probes used to label and visualize target structures or molecules in cells or tissues.

Rating Explanation

This paper presents a novel and valuable technique for studying cellular dynamics with improved spatial and temporal resolution under cryogenic conditions. The ability to freeze cells rapidly during optical microscopy observation offers unique advantages for visualizing and quantifying fast biological events, which is a significant advancement in the field. Despite the specialized equipment requirements, the limitations related to fluorescent probe compatibility and potential misinterpretations of intensity changes are manageable with careful experimental design and data analysis. The disclosed patent and the authors' affiliation with a company related to this technology raise a potential conflict of interest, which was considered when assigning the rating.

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