Volumetric imaging in a snapshot

Microscopes that render tissue-volume images from single snapshots are making their way into biology.

Three-dimensional (3D) fluorescence images of biological samples are normally assembled from a focal stack of 2D images acquired from multiple planes in a sequential manner, such as in confocal or light-sheet microscopy. This process is time consuming and might not be ideally suited to record certain types of fast biological events or light-sensitive samples. Moreover, these techniques often require complicated microscopy setups.

Scanless volumetric imaging approaches instead record an entire 3D focal stack from a single snapshot of the camera. Conventional epifluorescence microscopes can be turned into fast volumetric imaging devices in a seemingly uncomplicated manner.

One such approach that was introduced years ago is the light-field microscope, developed by Levoy and his collaborators at Stanford (http://graphics.stanford.edu/projects/lfmicroscope/). A light-field microscope can be built by inserting a microlens array between the main lens and the sensor plane of a conventional optical microscope. Rays that would normally come to a focus in the intermediate plane instead pass through the microlenses and are recorded separately at a light-field plane, where different points in the specimen are recorded in different images. With the aid of deconvolution algorithms, this imaging setup produces perspective flyarounds and 3D volumes from each photograph. Although diffraction forces a trade-off between axial and lateral resolution, the resulting 3D videos contain enough information to allow, for example, scanless imaging of genetically encoded calcium indicators across the entire brain of a larval zebrafish.

Microlens arrays can be introduced into the illuminating path of the microscope as well, offering easy ways of generating 3D illumination patterns.

A portion of the entire light-field image of a zebrafish eye as seen before image reconstruction. Credit: Marina Corral

Another approach for scanless volumetric imaging is reported in this issue by Abrahamsson, Dahan and their colleagues. The researchers adapted multifocus fluorescence microscopy methods to collect all the focal stacks simultaneously on a single camera. The microscopy setup relies on the use of diffractive grating to form aberration-corrected, multifocus-shifted images of the specimen (Nat. Methods 10, 60–63, 2013). It achieves resolutions equal to that of a wide-field microscope with about half of its light sensitivity and can be applied for fast acquisition of 3D data at the level of single molecules and small organisms.

Both the light-field and multifocus microscopes are promising methodologies that could make fast volumetric imaging accessible to many.

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Pastrana, E. Volumetric imaging in a snapshot. Nat Methods 10, 37 (2013). https://doi.org/10.1038/nmeth.2297

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