According to a study published in Nature Communications, a team of Rice University researchers led by Anna-Karin Gustavsson created a novel imaging technology that promises to increase the understanding of nanoscale cellular structures.
From left to right, Anna-Karin Gustavsson, Gabriella Gagliano and Nahima Saliba. Image Credit: Jeff Fitlow/Rice University
This platform, known as soTILT3D (single-objective tilted light sheet with 3D point spread functions), represents significant advances in super-resolution microscopy. It allows for quick and precise 3D imaging of multiple cellular structures while the extracellular environment can be controlled and adjusted flexibly.
Nanoscale cell studies provide insights into the subtle mechanisms that regulate cellular function, allowing researchers to unearth details critical to understanding health and disease. These insights can disclose how molecular interactions influence cellular processes, which is important for developing targeted therapeutics and understanding disease pathogenesis.
While conventional fluorescence microscopy has been effective in investigating cellular structures, it has been hampered by light diffraction, making it unable to resolve features smaller than a few hundred nanometers.
While single-molecule super-resolution microscopy has offered innovative insights into biological structures at the nanoscale, current techniques frequently suffer from high background fluorescence and slow imaging speeds, especially when dealing with thick samples or complex cell aggregates. They also generally lack precise, customizable control over the sample environment.
The soTILT3D platform immediately addresses these issues. An angled light sheet, a nanoprinted microfluidic system, and sophisticated computational tools work in concert to greatly increase imaging speed and accuracy with soTILT3D. This makes it possible to see more clearly how various cellular structures interact at the nanoscale, even in samples that are usually difficult to analyze.
Key Innovations
The soTILT3D platform employs a single-objective tilted light sheet to selectively illuminate thin slices of a sample, significantly increasing contrast by decreasing background fluorescence from out-of-focus areas, particularly in dense biological samples like mammalian cells.
The light sheet is formed using the same objective lens as used in the microscope for imaging, and it 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. This allows us to image entire samples from top to bottom with improved precision.
Anna-Karin Gustavsson, Assistant Professor, Rice University
The platform also includes a specially designed microfluidic system with an embedded metalized micromirror that can be customized. This system allows for rapid solution exchange and precise control over the extracellular environment, making it perfect for sequential multitarget imaging without color offsets. It also allows for the reflection of the light sheet into the sample.
The design and geometry of the microfluidic chip and nanoprinted insert with the micromirror can be easily adapted for various samples and length scales, providing versatility in different experimental setups.
Nahima Saliba, Study Co-First Author and Graduate Student, Rice University
Furthermore, soTILT3D uses computational tools, including real-time drift correction algorithms and deep learning, to analyze larger fluorophore concentrations for faster imaging, allowing for stable, high-precision imaging over long periods of time.
Saliba added, “The platform’s PSF engineering enables 3D imaging of single molecules, while deep learning handles dense emitter conditions which conventional algorithms have trouble with, which significantly improves the acquisition speed.”
When imaging in-depth at the nanoscale, SoTILT3D’s microfluidic device also provides automated Exchange-PAINT imaging, enabling sequential visualization of various targets without the color offsets typical of multicolor techniques.
Groundbreaking Results
The soTILT3D platform has shown impressive gains in imaging speed and accuracy. The platform’s tilted light sheet enhances contrast and permits accurate nanoscale localization by increasing the signal-to-background ratio for cellular imaging by up to six times compared to conventional epi-illumination techniques.
This level of detail reveals intricate aspects of 3D cell architecture that have been traditionally difficult to observe with conventional approaches.
Gabriella Gagliano, Graduate Student, Rice University
When combined with high emitter density and deep learning analysis, soTILT3D achieves a tenfold increase in speed, enabling researchers to capture detailed images of complex structures such as the nuclear lamina, mitochondria, and cell membrane proteins across entire cells in a fraction of the time.
Furthermore, the platform enables precise whole-cell 3D multitarget imaging, which captures the distributions of numerous proteins within a single cell and measures nanoscale distances between them. Researchers can now visualize the spatial arrangement of closely spaced proteins with remarkable precision and accuracy, such as nuclear lamina proteins lamin B1 and lamin A/C and lamina-associated protein 2, providing new insights into protein organization and its role in regulating cellular function.
Broad Applications in Biology and Medicine
The soTILT3D platform opens up new options for researchers from various sectors. Its capacity to image complex samples, including stem cell aggregation, broadens its application beyond single cells. The microfluidic system's biocompatibility makes it suited for live-cell imaging, allowing scientists to analyze cellular responses to various stimuli in real time with minimal photodamage. SoTILT3D’s finely controlled solution exchange capability is also an excellent tool for assessing how drugs influence cells in real time.
Gustavsson added, “Our goal with soTILT3D was to create a flexible imaging tool that overcomes limitations of traditional super-resolution microscopy. 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.”
The study was supported by partial financial support from the National Institute of General Medical Sciences of the National Institutes of Health grant R00GM134187 and grant R35GM155365, the Welch Foundation grant C-2064-20210327, and startup funds from the Cancer Prevention and Research Institute of Texas grant RR200025.
Journal Reference:
Saliba, N. et. al. (2024) Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet. Nature Communications. doi.org/10.1038/s41467-024-54609-z