Light Sheet Microscopy
What ASI currently has available for iSPIM & diSPIM systems is all of the necessary hardware for generating the light sheet from a laser, and for scanning the sample. We have systems for iSPIM where we focus the light sheet onto the sample with one objective & image with the other, and diSPIM where we can illuminate & image from both objectives. The objectives both can be moved very rapidly & precisely with one of our piezo objective movers. The scan head utilizes Micro-Mirrors for fast reliable & economical positioning of the illumination beam.
The components that we have can be mounted onto the transmitted illumination pillar of an inverted microscope, or onto our RAMM system. In this configuration a third objective can be used to image the sample from below. We can provide systems with the lasers, objectives, and cameras for these systems.
here are three basic configurations of SPIM systems that we have built for various users.
Fixed Sheet systems: The light sheet is stationary, with only mechanical adjustment for the sheet position. The two objectives are manually adjusted to correctly focus on the sheet. The specimen is scanned through the sheet using the X and Z stages to generate volume images.
Advantages: Least expensive – not requiring either galvo scanners or a piezo objective positioner.
Disadvantages: difficulty in correctly overlapping the objective focal planes with fixed positioners and the relatively slower stage scanning at odd angles.
Standard single-sided system: Light sheet from one side, emission objective on the other. The light sheet can be scanned using galvos to sweep across the sample volume. There is an emission objective piezo so the viewing objective can be positioned to follow the light sheet as it is scanned through the sample.
Advantages: Rapid Scanning, straight-forward setup.
Disadvantages: Better XY resolution than Z resolution.
Double-side system: Light sheet excitation and emission on each side. Both sides have a piezo-objective positioner. You need light sheets on each side as well. Dr. Hari Shroff is currently getting very nice results from the dual system and will be publishing a paper on this in a few months.
Advantages: With proper post processing, XY & Z resolutions are all very good – yielding a combination of speed and resolution that is unsurpassed for live cell imaging.
Disadvantages: Complicated and expensive.