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Precision vibratome for high-speed ultrathin biotissue cutting and organ-wide imaging.

ABSTRACT: Cutting tissues into ultrathin slices is highly desired in sectioning-based organ-wide imaging. However, it is difficult to perform tissue cutting at a high speed with excellent quality. Here, we design a precision vibratome based on a paired double parallelogram flexure, which enables a vibrating blade to move strictly along a straight line. Meanwhile, we develop a high-speed cutting method that does not compromise cutting quality, which the vibratome operated at a high frequency mode. The characterized parasitic motion errors of a 180-Hz vibratome were less than 300 nm. It achieved a cutting speed six times that of an 85-Hz vibratome with acceptable quality. The capacity of the vibratome was investigated by organ-wide imaging, and the results revealed that it can be adapted in different tissues, such as the mouse brain and liver. This new vibratome shows great potential in speeding up organ-wide imaging applications especially for large volume biotissues.

SUBMITTER: Li Y

PROVIDER: S-EPMC8426277 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labelling or there is concern that labelling will disrupt function. Commonly used techniques for observing unlabelled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has a similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high-speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the optical quality of interference reflection microscopy falls within the range of other microscope techniques, being inferior to some and superior to others, depending on the metric used and, with minimal microscope modification, can be used to study the dynamics of unlabelled microtubules.Lay descriptionThe cytoskeleton gives a cell its shape and plays a major role in its movement and division. It's also helps organise the content of cells and is the base for intracellular transport. Important components of the cytoskeleton are microtubules, which are hollow cylindrical beams (25 nm in diameter) that assemble from protein building blocks called tubulin. Deficiencies in microtubules are related to many diseases including cancer and Alzheimer. Given their important role, microtubules are heavily investigated in many laboratories. One way to study microtubules is to isolate them from cells and image them using light microscopy. Over the years a number of imaging techniques have been used. These techniques have a number of drawbacks which are addressed by ongoing efforts which this work is a part of. Here, we present a method based on light interference that produce high quality images of microtubules. The technique is cheap and easy to implement making it accessible to a wide base of researchers.

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Precision vibratome for high-speed ultrathin biotissue cutting and organ-wide imaging.

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