Flamingo Lightsheet Fluorescence Microscopy

Modular design with compact modules for sample movement

Lightsheet Microscopy: Technology and Advantages

In Light Sheet Fluorescence Microscopy (LSFM), also called Single Plane Illumination Microscopy (SPIM ), illumination and detection are two separate optical systems that are perpendicular to each other. The laser beam used to illuminate the sample is focused in one direction and forms a light sheet that illuminates a thin layer of the sample. The fluorescent light that is emitted in this plane is captured and detected by an objective lens.

The separation of illumination and observation brings two decisive advantages:

  • It makes it possible to build up the microscope virtually "around the experiment. Instead of mounting samples in between microscope slide and coverslip, they are immersed in a hydrogel and placed in a cylindrical sample chamber inside the microscope. There, the conditions are close to physiological and so even continuous recordings from living specimens are possible.
  • With LSFM, the light exposure of the samples is very low, which considerably reduces phototoxic effects. Only then long-term experiments are possible.

Sample Movement in the Microscope

One of the most common application in Light Sheet Microcopy is the creation of Z stacks with which the sample can be wholly depicted in three dimensions. For this purpose, the sample is moved in the Z direction (that is, along the optical axis of the objective lens). For this task, PI contributes compact linear stages of the L-505 type to the Flamingo Lightsheet microscopes of the >> Huisken Lab at the Morgridge Institute for Research. These very compact linear stages have a folded drivetrain, DC motors with gearhead, linear encoders, and offer 26 mm travel range at a bidirectional repeatability of ±250 nm. The stage also excels by a high stiffness and guiding accuracy due to ball guides.

Rotating the specimen can be useful in several scenarios. Often, researchers want to be able to image their sample from just the right angle (side view, top view etc.) or want to image an organ that’s only visible from certain angles (like the heart). The other big application for rotation is so-called multi-view imaging, where z-stacks are recorded from multiple angles, e.g. 6 angles covering 360 degrees, and then the useful parts of each dataset are fused to generate one 3D image that covers the entire sample. This is particularly useful for larger samples as the sample itself scatters, refracts and absorbs the light, affecting the illumination and detection quality of structures deep inside the sample. Therefore several angles are needed to capture all the details.

The rotation of the sample is supported by the U-628 ultrasonic piezo-motor-driven rotation stage from PI. It offers fast movement of up to 720°/s at a minimum step size of 51 µrad and a bidirectional repeatability of ±102 µrad.