The Relationship between Light Sheet Microscopes and Precision Motion Components

The flamingo conjures up pink feathers, an elongated beak, and the infamous silhouette that constantly stands on one leg while sleeping. It is a celebrated member of the genus ‘Phoenicopteridae’, and it sure does know how to stand out.

These exotic birds caught the imagination of Professor Dr. Jan Huisken, Research Group Leader and Director of the Medical Engineering Department at Morgridge Institute for Research in Madison, WI, as the name-giving property for the special light-sheet microscopes developed by him and his team. The form and silhouette of these microscopes are reminiscent of the flamboyant wading bird’s sleeping habits.

Supported by several sponsors, the objective of the Flamingo project is that these modular microscopes would be freely available to researchers in their laboratories without charge.

This illustration of a compact Flamingo light sheet microscope (right) with L-SPIM configuration (see text) is reminiscent of the one-legged sleeping bird. (Image: Image: Morgridge Institute for Research, Madison, WI)

Light Sheet Microscopy: Technology and Advantages

Sketch of the Light Sheet Microscopy principles (Image: Morgridge Institute for Research, Madison, WI)

Illumination and detection are two distinctive optical systems that operate perpendicular to each other throughout Light Sheet Fluorescence Microscopy (LSFM). The laser beam that is utilized in the illumination of the sample is concentrated in one direction and constructs a light sheet that enhances a thin layer of the sample. An objective lens detects the fluorescent light that is emitted in this plane for subsequent capture.

One of the key features on the microscope is that it can virtually be constructed ‘around the experiment’ as separating the illumination from the detection is possible. Rather than mounting samples in between microscope slide and coverslip, they are saturated in a hydrogel and then placed into a cylindrical sample chamber inside the microscope. Furthermore, recordings from living specimens are possible on a continual basis as the environment is comparable to physiological conditions

Sample Positioning is Particularly Important

A special role is played by the positioning of the sample since linear or rotational movement is carried out as required. For this task, PI (Physik Instrumente) offers a multitude of subsystems.

We are proud to be among the seed supporters and sponsors of Jan Huisken’s groundbreaking Flamingo project .

For the Flamingo project, reliability and precision of modules are key factors. This means, high precision, fast movements, and a compact size of the drives together with high-quality production standards and services from PI make the difference.

Dr. Thomas Bocher, Head of Segment Marketing 'Microscopy & Life Sciences', PI

Low Phototoxicity

"LSFM is a very powerful and flexible platform for gentle in vivo imaging offering low phototoxicity and fast image acquisition," says Jan Huisken regarding the particular benefits provided by the technology. Due to the of the careful "treatment" of the sample, LSFM is preordained for the study of living organisms. A major research goal of the technology is to obtain an improved understanding of energetic and complex processes such as embryonic morphogenesis (i.e., the growth cycles of organs, organisms, and organelles).

Light sheet microscopy gives amazing images of living organisms and of time sequences of growth processes. Zebrafish recorded by Alyssa Graves with Flamingo #1. (Image: Morgridge Institute for Research, Madison, WI)

Modular Design

So, for the success of integration and implementation, certain conditions were necessary to the process: The system needed to be easy to set-up, extremely robust, and straightforward to operate. The solution: A simple, accessible design with components proven to be of high-standard in conjunction with a minimal footprint as well as being cost-effective. This enables varied production for application-specific designs: In the T-SPIM design, the process involves illuminating the sample from two opposing optics for more uniform illumination. The inclusion of a second detection path results in the X-SPIM design.

Modular conceptualization of FLAMINGO: PI’s linear and rotation stage modules are shown on the right (Image: Morgridge Institute for Research, Madison, WI)

Sample Positioning within the Microscope

L-505 compact linear stage with folded drivetrain (left) Learn more

The creation of Z stacks is one of the most widely used applications in Light Sheet Microcopy which is when the sample can be fully depicted across three dimensions. For this task, the sample is moved in the Z direction (that is, along the optical axis of the objective lens). For this purpose, PI provides compact linear stages of the L-505 type. These significantly compacted linear stages have a folded drivetrain, linear encoders, DC motors with gearhead, and provide 26 mm travel range at bidirectional repeatability of ±250 nm. The stage is unsurpassed due to robust stiffness and precision guiding attributable to ball guides.

Ultrasonic piezo motors offer high-speed, precision motion in a compact package. Piezo motors can self-clamp at rest, which provides high stability. PI has decades years of experience designing piezo motors from the bottom up, relying on expertise from its piezo ceramic manufacturing division PI Ceramic. Learn more

Rotating the sample or specimen can be of great use in many different scenarios. Typically, researchers desire the ability to image capture their sample from the perfect angle (side view, top view etc.) or want to image an organ that can only be viewed from certain angles (like the heart).

The other major purpose for effective 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 requisite parts of each dataset are merged to produce one singular 3D image that is representative of the entire sample. This can be of significant use when imaging larger samples as the sample itself scatters, refracts, and absorbs the light, which in turn affects the illumination and detection quality of structures deep inside the sample. Therefore, multiple angles are required to ensure precision capture of all the details.

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

Motivation and Philosophy Behind the Flamingo Project

Co-inventor of Light Sheet Microscopy, Jan Huisken, who recently started this project when working on his PhD at the EMBL Heidelberg (Germany), stated, "We aim to democratize high-end light microscopy, bringing it to campuses and labs for free. We think this will be especially useful for reproducing scientific results, something increasingly important to science." Michael Weber, the Field Application Specialist for the US East Coast in the Flamingo project adds, "We’re turning the idea of a research imaging facility upside down. We want to bring the microscope as close to the sample as possible, rather than biologists bringing their delicate samples to us." Thus, in bringing the microscope to the biologist, one major advantage is offered: there is no requisite travel for either the biologist or the sample. This is advantageous because most samples are extremely sensitive to variances in their environmental conditions (e.g., gravity, humidity, temperature), this approach helps researchers to focus on their work rather than worrying about any logistical issues.

The project also seeks to intensify the dialogue between system developers and users to further the evolution of LSFM technology.

Outlook

On the East Coast of the United States, the second Flamingo system has already been assembled. What’s more, over the next few months, and in the years to come, continual assembly of over several dozens of Flamingos will be completed to meet the enormous demand for ultra-modernized light-sheet microscopes for research around the world. Thomas Bocher states, "PI will continue to support this philanthropic endeavor."

This information has been sourced, reviewed and adapted from materials provided by PI (Physik Instrumente) LP.

For more information on this source, please visit PI (Physik Instrumente) LP.