Researchers have developed a fluorescence microscope that uses structured illumination for fast, super-resolution imaging over a wide field of view. It can also be used for multi-color and high-speed shooting. Credit: Henning Ortkras, Bielefeld University

A microscope based on fiber-optic components designed to study the cellular effects of drug groups.

Researchers have developed a fluorescence microscope that uses structured illumination for fast, super-resolution imaging over a wide field of view. This advanced microscope is designed to capture high-resolution images of many living cells at once, making it easier to analyze how different drugs and their combinations affect the body.

“Polypharmacy — the effect of several combinations of medications that are typically prescribed to chronic or elderly patients — can lead to serious interactions and become a major problem,” said Henning Ortkrass of Bielefeld University in Germany. “We developed this microscope as part of the EIC Pathfinder OpenProject DeLIVEry, which aims to develop a platform that can investigate polypharmacy in individual patients.”

New microscopic liver cell

The researchers used their new microscopic setup to image fixed, multicolored liver cells. The image showed the cells’ small membranous structures, which are smaller than the diffraction limit. Credit: Henning Ortkras, Bielefeld University

In the journal Optica Publishing Group Optics Express, researchers describe their new microscope that uses fiber-optic delivery of excitation light to enable very high image quality over a very large field of view with multi-color, high-speed capability. They showed that the instrument can be used to image liver cells, achieving a field of view of up to 150 x 150 square micrometers and imaging rates up to 44 Hz while maintaining a spatiotemporal resolution of less than 100 nm.

“Using this new microscope, individual drug combinations can be tested on isolated cells and then imaged at ultra-high resolution to observe the dynamics of cell membrane features or organelles,” Ortkras said. “The large field of view can provide statistical information about cell response, which can be used to improve personalized healthcare. Thanks to the potentially small size of the system, it may also be useful for clinical applications where high accuracy is important.”

Set up a new fluorescence microscope

The new fluorescence microscope uses structured illumination for rapid, ultra-resolution imaging over a wide field of view. Multi-color imaging can also be performed, as shown in the video. Credit: Henning Ortkras, Bielefeld University

High resolution across a wide field of view

The new microscope is based on super-resolution structured illumination microscopy (SR-SIM), which uses a structured pattern of light to excite fluorescence in the sample and achieve spatial resolution beyond the diffraction limit. SR-SIM is particularly suitable for live cell imaging because it uses low-energy excitation that does not damage the sample while producing highly detailed images.

To achieve high resolution across a wide field of view, the new microscope reconstructs ultra-resolution images from a set of raw images. These raw images are acquired using an array of six optical fibers to illuminate the sample in a sinusoidal pattern that is shifted and rotated to obtain additional information. This improves resolution 2-fold while still achieving fast imaging and compatibility with live cell imaging.

Ultra-resolution image of multiple cells at once

Thanks to the microscope’s large field of view, it is possible to obtain ultra-resolution images of multiple cells simultaneously. Credit: Henning Ortkras, Bielefeld University

“Fiber selection and phase shift are performed using a newly designed fiber switch based on galvanometric and MEMS mirrors,” Ortkras said. “We also designed a custom hexagonal holder that collimates and refocuses the six fiber bundles in the microscope to illuminate a large field of view and allow fine-tuning of all beams. This allows the setup to be used for total internal reflection fluorescence (TIRF)-SIM excitation, which is used to limit fluorescence excitation and detection It is reported in a thin area of ​​the sample.

Liver cell imaging

Since the liver is the main organ involved in drug metabolism, the researchers tested the setup using samples of fixed, multicolored rat liver cells. Reconstructed images using the new microscope allowed visualization of small membrane structures that are smaller than the diffraction limit of light.

“This uniquely compact system combines a large field of view and fast pattern switching speed with multi-color, energy-efficient excitation,” Ortkras said. “In addition, the setup achieves very high image quality and can be set to perform as either 2D-SIM or TIRF-SIM.”

Next, the researchers plan to apply the microscopy setup to live cell studies of liver cells to observe the dynamics of cells treated with several drugs. They also plan to improve the image reconstruction process to accomplish direct reconstruction of the acquired raw data.

Reference: “High-speed TIRF and 2D super-resolution structured illumination microscopy with a large field of view based on fiber-optic components” by Henning Ortkras, Jasmine Schorsted, Gerd Weibusch, Karolina Szafranska, Peter McCourt, and Thomas Hauser, 16 August 2023, Optics Express.
doi: 10.1364/OE.495353

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