Rice University improves views of nanoscale cellular structures

03 Dec 2024

New super-resolution platform combines tilted light-sheet, microfluidics and deep learning.

A project at Rice University has developed a novel approach to nanoscale cellular imaging, one that could lead to improved knowledge of subcellular structures.

Reported in Nature Communications, the new platform is intended to tackle some of the specific current challenges involved in single-molecule super-resolution imaging of whole cells.

Studying mammalian cells at the nanoscale provides insights into the intricate mechanisms that drive cellular behavior, commented the project, enabling researchers to uncover details that are essential for understanding health and disease.

These details can reveal how molecular interactions contribute to cellular functions, which is critical for advancing targeted therapies and understanding disease development, or pathogenesis.

However, the imaging operation is often hampered by high fluorescence background and slow acquisition speeds, especially when imaging multiple targets in 3D.

"While conventional fluorescence microscopy has been useful for studying cellular structures, it has been limited by the diffraction of light, restricting its ability to resolve features smaller than a few hundred nanometers," commented Rice University.

"Moreover, while single-molecule super-resolution microscopy has provided groundbreaking insights into biological structures at the nanoscale, existing techniques often suffer from high background fluorescence and slow imaging speeds, particularly when dealing with thick samples or complex cell aggregates."

The Rice solution is called soTILT3D, for "single-objective tilted light-sheet with 3D point spread functions." By integrating an angled light sheet, a nanoprinted microfluidic sample system and advanced computational tools, soTILT3D allows clearer visualization of how different cellular structures interact at the nanoscale, even in conventionally challenging samples.

Overcoming the limitations of traditional cell microscopy

Using a single-objective tilted light sheet as its illumination method lets the platform selectively illuminate thin slices of a sample, reducing background fluorescence from the out-of-focus areas and effectively enhancing the contrast.

"The light sheet is formed using the same objective lens used in the microscope for imaging and is fully steerable, dithered to remove shadowing artifacts that are common in light-sheet microscopy, and angled to enable imaging all the way down to the coverslip," commented Anna-Karin Gustavsson from Rice University. "This allows us to image entire samples from top to bottom with improved precision."

Alongside its slanted illumination, soTILT3D incorporates a specially designed microfluidic system with an embedded customizable metalized micromirror, giving precise control over the extracellular environment and allowing rapid solution exchange.

This is ideal for sequential multi-target imaging without color offsets while also allowing for reflection of the light sheet into the sample, said the project.

In trials using the U2OS cell line, one commonly used in cell research, the new platform successfully achieved "whole-cell multi-target 3D single-molecule super-resolution imaging with improved precision and imaging speed." The spatial arrangement of closely situated proteins could be imaged, offering new insights into protein organizations.

"Our goal was to create a flexible imaging tool that overcomes limitations of traditional super-resolution microscopy," commented Gustavsson. "We hope these advancements will enhance studies in biology, biophysics and biomedicine, where intricate interactions at the nanoscale are key to understanding cellular function in health and pathogenesis."