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Revolutionizing Cellular Imaging with soTILT3D

A pioneering team of scientists, led by Anna-Karin Gustavsson at Rice University, has introduced an advanced imaging platform known as soTILT3D, which holds the potential to transform our understanding of cellular structures at the nanoscale. This innovative system, which stands for single-objective tilted light sheet with 3D point spread functions (PSFs), enhances super-resolution microscopy, allowing for rapid and highly accurate 3D imaging of various cellular components while offering control over the extracellular environment. The findings were recently shared in Nature Communications.

Exploring cells at the nanoscale not only reveals intricate biological processes but also sheds light on the complex molecular interactions that underpin cellular functions. This knowledge is pivotal in the advancement of targeted therapies and in comprehending the mechanisms behind various diseases.

While traditional fluorescence microscopy has proven useful in cellular studies, it is hampered by the diffraction of light, making it challenging to resolve structures smaller than a few hundred nanometers. Although single-molecule super-resolution microscopy has gained traction for its insights into nanoscale structures, many techniques still grapple with issues like high background fluorescence and slow imaging speeds, particularly in thick or densely packed samples. Additionally, these methods often lack precise control over the sample environment.

The soTILT3D platform aims to rectify these limitations. By combining an angled light sheet, a nanoprinted microfluidic system, and sophisticated computational tools, soTILT3D greatly enhances both imaging resolution and speed, providing clearer insights into the interactions of cellular structures, even in challenging samples.

Innovative Features of soTILT3D

One of the core innovations of the soTILT3D platform is its use of a single-objective tilted light sheet, which selectively illuminates thin layers of a sample. This approach significantly boosts contrast levels by minimizing background fluorescence from out-of-focus regions, particularly in thick biological specimens like mammalian cells.

“The light sheet is created with the same objective lens used for imaging. It is fully steerable and dithered to eliminate common shadowing artifacts present in light sheet microscopy. This allows for comprehensive imaging of the sample from top to bottom with enhanced accuracy,” explained Gustavsson, who serves as an assistant professor of chemistry at Rice University and is the corresponding author of the study.

The system also features a uniquely designed microfluidic apparatus with an embedded customizable metalized micromirror, granting researchers precise control over the extracellular environment. This setup facilitates swift solution exchange, enabling sequential imaging of multiple targets without color distortion.

“The adaptable design and structure of the microfluidic chip and the integrated micromirror make it versatile for a broad array of samples and experimental frameworks,” noted Nahima Saliba, a co-first author of the research alongside her graduate colleague Gabriella Gagliano.

Additionally, the platform employs advanced computational tools, including deep learning algorithms, to analyze higher fluorophore concentrations, thus improving imaging speeds and providing real-time drift correction for prolonged, stable imaging sessions.

“The PSF engineering within the platform supports 3D imaging of single molecules, while deep learning capabilities address the challenges of dense emitter conditions, greatly enhancing acquisition speed,” Saliba added.

Furthermore, the microfluidic component of soTILT3D accommodates automated Exchange-PAINT imaging, allowing for the sequential visualization of various targets without the color offsets that can complicate multicolor imaging at the nanoscale.

Remarkable Improvements in Imaging

The soTILT3D platform has yielded substantial advancements in imaging precision and speed. Its angled light sheet can enhance the signal-to-background ratio by up to six times in cellular imaging compared to conventional epi-illumination techniques, leading to higher contrast and more accurate nanoscale localization.

“This unprecedented level of detail highlights complex aspects of 3D cell architecture that have previously posed challenges for traditional imaging methods,” Gagliano stated.

In terms of speed, soTILT3D can achieve a tenfold increase in imaging efficiency when used in conjunction with high emitter densities and deep learning techniques. This capability enables researchers to capture intricate images of cellular structures such as the nuclear lamina, mitochondria, and cell membrane proteins in a fraction of the time typically required. It also allows for precise whole-cell 3D multitarget imaging, revealing the distribution of multiple proteins and measuring nanoscale distances between them with exceptional accuracy.

Wide-Ranging Applications in Biology and Medicine

soTILT3D presents expansive opportunities for researchers across numerous scientific disciplines. Its ability to image intricate samples, including stem cell aggregates, extends its usefulness beyond individual cell studies. The microfluidic system’s compatibility with live-cell imaging enables scientists to monitor cellular responses to various stimuli in real-time while minimizing photo damage. Additionally, the controlled solution exchange feature makes it an excellent tool for real-time assessments of drug impacts on cellular behavior.

“With soTILT3D, we aimed to create a versatile imaging instrument that addresses the constraints of conventional super-resolution microscopy,” Gustavsson said. “We believe these innovations will significantly advance research across biology, biophysics, and biomedicine, where understanding nanoscale interactions is crucial for deciphering cellular functions in health and disease.”

This research received partial funding from the National Institute of General Medical Sciences of the National Institutes of Health through grants R00GM134187 and R35GM155365, as well as support from the Welch Foundation grant C-2064-20210327 and startup funds from the Cancer Prevention and Research Institute of Texas grant RR200025.

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