New microscope images entire living organisms
Researchers at the Howard Hughes Medical Institute's Janelia Research Campus have built the first light microscope capable of imaging large, non-transparent specimens at sub-second temporal resolution and sub-cellular spatial resolution in all dimensions.
Rather than collecting a single image of a sample with a single objective, the new microscope simultaneously collects light and creates images from multiple angles. Each image still suffers from poor resolution along one axis, but the most useful data from each image can be combined to generate a final image with good resolution in all dimensions.
The researchers designed a microscope with four objective lenses positioned around the sample at right angles to one another, like traffic lights at an intersection. Each objective sends light into the sample to illuminate it, and also collects fluorescent light emitted by the sample.
From each side, an objective produces a thin beam of light that sweeps the sample from top to bottom so quickly that the detection camera across from it sees a continuous sheet of light. The beams from each of the four objectives are staggered so that they do not interfere or intersect with one another, and a rolling shutter in each camera keeps pace with the beam, so that the camera's detector remains focused on the narrow slit along which it can pick up high-resolution information. These features allow the microscope to collect four different images of the sample from different angles simultaneously, without any crosstalk between these multiple views.
Each of the four cameras in the microscope collects data for an image that, by itself, still lacks good spatial resolution in one dimension. An image-processing software transformed those images into a final high-resolution image.
A typical experiment with this microscope might run for one hour. During that time, the four sCMOS cameras in the microscope can stream data from the experiment at rates up to 3.2 gigabytes per second. This results in about 10 terabytes of multi-view image data from one hour of imaging.
The researchers have used the IsoView to visualize cell-by-cell activity throughout the nervous system of an entire living fruit fly larvae, an organism that has more than 10,000 neurons and is about 50 times larger than the roundworm C. elegans, the only animal whose complete nervous system has previously been imaged at the single-cell level. Because the IsoView can produce images as the larvae moves freely in a loose gel, this opens up the possibility of functional imaging in an entire, behaving animal.
The scientists also performed high-resolution functional imaging of activity in the entire brain of a larval zebrafish, demonstrating that neurons in the deepest, least accessible regions of the brain could be seen clearly, separate from their neighbors.