Abstract
The in-vivo investigation of highly dynamic biological samples, for example the beating zebrafish heart, requires high-speed volume imaging techniques. Light-sheet microscopy is ideal for such samples as it records high-contrast images of entire planes within large samples at once. However, in order to obtain images of different planes, it has been necessary to move the sample relative to the fixed focal plane of the detection objective lens. This mechanical movement limits speed, precision and may be harmful to the sample. We have built a light-sheet microscope that uses remote focusing with an electrically tunable lens (ETL). Without moving specimen or objective we have thereby achieved flexible volume imaging at much higher speeds than previously reported. Our high-speed microscope delivers 3D snapshots of sensitive biological samples. As an example, we imaged 17 planes within a beating zebrafish heart at 510 frames per second, equivalent to 30 volume scans per second. Movements, shape changes and signals across the entire volume can be followed which has been impossible with existing reconstruction techniques.
Highlights
The analysis of many interesting biological phenomena such as neuronal activity in the brain or blood flow in the heart requires the rapid recording of extended three-dimensional volumes
We found that sinusoidal driving signals were best for rapid volume imaging in a light sheet microscope with our electrically tunable lens (ETL)
As the image quality depends on the focal length of the ETL and deteriorates towards the borders of the image, the effectively usable field of view (FOV) and scan range depend on the specific image quality requirements
Summary
The analysis of many interesting biological phenomena such as neuronal activity in the brain or blood flow in the heart requires the rapid recording of extended three-dimensional volumes. Images of different planes within the sample are captured sequentially, usually by moving the sample relative to the detection lens. This mechanical movement of the specimen or the lens limits acquisition speed, precision and may even be harmful to the sample. In order to capture the dynamic state of the sample and avoid distortions in the recorded data, volume imaging has to be performed by imaging multiple planes either in parallel or sequentially at very high speed, ideally without any sample movement. The most straightforward approach consists in using a set of cameras, each camera focused on a different plane [1] This approach is only practicable for a low number of planes due to the low light efficiency and high complexity of such a multi-camera setup. For large deviations from the ideal working distance the image quality deteriorates, especially due to spherical aberration
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