Fast Frame Scanning Camera System For Light-sheet Microscopy

Optical microscopy with visible light, which has an undisputed advantage of allowing non-contact and minimally invasive imaging, has played an important role in the study of modern cytology and biology. Fluorescence microscopy is a leader among many optical microscopic techniques because of its inherent high selectivity of the region of interest. This inherent high selectivity originates from immunofluorescence technique and fluorescence labeling probe technique. In Light-sheet fluorescence microscopy(LSFM), out-of-focus background is inherently not generated, so the familiar problem, when an object goes out of focus, its image becomes blurred but does not dis-appear, that had to be faced when we used a standard fluorescence microscope has been solved. Compared with the wide field, confocal, multiple-photon and other fluorescent microscopy, the photo bleaching and the phototoxicity of the LSFM are very low. LSFM is very suitable for imaging deep layer and transparent tissue or the whole organism and has a significant advantage in increasing the observation time, increasing the imaging speed and improving the number of multiple-view imaging per given volume. But regardless of adopting a widefield illumination strategy or a line-scanning illumination strategy, the imaging rate of LSFM is generally limited by the frame rate of the camera. In order to improve the situation, this thesis covers the following research works: 1.We have proposed the idea of the digital scanning high speed camera which was developed from a rotating mirror high speed camera and built a fast frame scanning camera system based on a homebuilt light-sheet fluorescence microscope system. The fast frame scanning camera system incorporated a variable mechanical slit and a galvanometer scanning mirror to the imaging path of the light-sheet microscope. In the imaging optical path, a detection objective lens and a tube lens imaged the illuminated area onto a mechanical slit whose width controlled the size of the field of view(FOV). The rotation of the scanning mirror positioned multiple sub-images on the CCD sensor in a single camera frame. So that multiple images could be acquired during one exposure period. The improvement factor of the frame rate was dependent on the number of sub-images that could be tiled on the sensor without overlapping each other and was thereby a trade-off of the image size. Then we can break the speed limit of the camera in the traditional LSFM fluorescence microscope system. 2.Written by G language of LabVIEW, a control program synchronized the scanning and image acquisition. NI USB-6361 was used to produce both the trigger signals and the driving waveform of the galvanometer via channels of analogue outputs. The voltage applied to the galvanometer during one exposure period was a step-shaped waveform, the level of each step corresponds to different angle of galvanometer’s deflection i.e. different sub-image’s position and the length of the step represents the dwell time of galvanometer. The incremental step size, the number of the steps and the width of the mechanical slit were carefully chosen to ensure the CCD sensor filled with sub-images without overlap. A trigger signal started each exposure of the CCD device and, at the same time, the scanning cycle of the galvanometer. The galvanometer flew back and got ready for the next cycle during the readout period. When the exposure and the scanning were synchronized well, we were able to acquire frames which comprise stable and clear sub-images. 3.The fast frame scanning camera system for light-sheet microscopy have acquired images that were comprised of 16 or 32 ribbons of sub-images, respectively. The flowing fluorescent beads, which was driven by a peristaltic pump, were able to be recorded clearly without motion blur. The system captures the clear fluorescent beads and their movement trajectories. The actual imaging frame rate was successfully made higher with 16 or 32 times over the frame rate of the CCD; as a result, we achieved 480 fps and 960 fps. However, the galvanometer can run at much higher speed and the imaging speed can be further improved for capturing extremely fast events happening within a very short time. The factor of improvement is theoretically limited by the size of sub-images and the speed of scanner, and ultimately by the number of detectable photons that give out reasonable contrasts of images.