Researchers have developed a fluorescence microscope that uses structured illu­mination for fast super-resolution imaging over a wide field of view. The new microscope was designed to image multiple living cells simul­taneously with a very high resolution to study the effects of various drugs and mixtures of drugs on the body. “Poly­pharmacy – the effect of the many combinations of drugs typically prescribed to the chronically sick or elderly – can lead to dangerous interactions and is becoming a major issue,” said Henning Ortkrass from Biele­feld University in Germany. “We developed this microscope to develop a platform that can investi­gate poly­pharmacy in individual patients.”

The new microscope uses optical fiber delivery of excitation light to enable very high image quality over a very large field of view with multicolor and high-speed capability. The researchers show that the instrument can be used to image liver cells, achieving a field of view up to 150 x 150 square micro­meters and imaging rates up to 44 Hertz while maintaining a spatio­temporal resolution of less than 100 nanometers. “With this new microscope, individual drug combina­tions can be tested on isolated cells and then imaged with super-resolution to observe dynamics of cell membrane features or organelles,” said Ortkrass. “The large field of view can provide statistical information about the cell response, which could be used to improve persona­lized healthcare. Thanks to the system’s potentially small size, it might also be useful for clinical applications where high resolution is important.”

The new microscope is based on super-resolved structured illumination micro­scopy (SR-SIM), which uses a structured pattern of light to excite fluorescence in a sample and achieve a spatial resolution beyond the diffraction limit of light. SR-SIM is particularly well suited for live cell imaging because it uses low-power excitation that doesn’t harm the sample while producing highly detailed images. To achieve high resolution across a wide field of view, the new micro­scope reconstructs super-resolved images from a set of raw images. These raw images are acquired by using a set of six optical fibers to illuminate the sample with a sinusoidal striped pattern that is shifted and rotated to gain extra information. This creates a two-fold resolution improve­ment while still achieving fast imaging and being compatible with live-cell imaging.

“The fiber selection and phase shift is performed using a newly designed fiber switch based on galvanometric mirrors and MEMS-mirrors,” said Ortkrass. “We also custom-designed a hexagonal holder that collimates and refocuses the beams of the six fibers into the micro­scope to illuminate a large FOV and allow precise adjustment of all beams. This allows the setup to be used for total internal reflection fluorescence excitation (TIRF)-SIM, which is used to restrict fluorescence excita­tion and detection to a thin region of the sample.”

Since the liver is the primary organ involved in drug metabolism, the researchers tested the setup using samples of fixed multicolor-stained rat liver cells. The reconstructed images produced with the new microscope allowed visuali­zation of the tiny membrane structures that are smaller than the diffraction limit of light. “This compact system uniquely combines a large field of view and fast pattern switching speed with multicolor, power-efficient excitation,” said Ortkrass. “In addition, the setup achieves very high image quality and can be tuned to perform 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 live reconstruc­tion of the acquired raw data. (Source: Optica)

Reference: H. Ortkrass et al.: High-speed TIRF and 2D super-resolution structured illumination microscopy with a large field of view based on fiber optic components, Opt. Exp. 31, 29156 (2023); DOI: 10.1364/OE.495353

Link: Biomolecular Photonics Research Group, Faculty of Physics, Bielefeld University, Bielefeld, Germany