When single-molecule super-resolution microscopes were first commercialized some 15 years ago, they made headlines for their ability to resolve individual molecules and structures at the nanometer scale, well beyond the limits of traditional light microscopy. They quickly became virtually indispensable in biopharmaceutical research. Now, as their capabilities continue to evolve, their applications are expanding commensurately to help scientists determine mechanisms of action, track protein folding dynamics in real time, monitor vector delivery and gene expression, and, for biopharmaceutical manufacturing, monitor cell cultures, protein aggregation, and contamination risks.
Disrupting science-as-usual
The next-generation super-resolution microscopes are coming to the market now, with even greater advances anticipated in the near future. Some of these devices boast significantly wider fields of view without the tradeoffs in resolution that characterized many of their predecessors. Others minimize phototoxicity, while still others can handle thick samples (up to 1mm) that cause some microscopy technologies to struggle, such as stimulated emission depletion (STED) and single molecule localization microscopy (SMLM). Artificial intelligence is becoming a critical component for many.
These and other advances in super-resolution microscopy are disrupting science-as-usual. High-throughput single-molecule tracking is but one example.
“The randomness of chemical reactions poses a challenge to the function of living things,” Abraham Kohrman, applications scientist for biological microscopy at Bruker, points out, setting the scene. “Cells solve this problem through organization.”
Conventional microscopy, however, is insufficient to reveal the details. It “measures biochemical behaviors in aggregate, rather than at the individual interaction level. Directly measuring the motion of particles involved (in various organization tasks, such as transporting organelles and actively amplifying or propagating signals) provides researchers the opportunity to better understand these mechanisms,” Kohrman says. Applications include life sciences, production chemistry, industrial processes, and material sciences, he says, as well as medicine, “where it can be used to measure the uptake of medications by cells and organisms, allowing for the development of new drug delivery strategies.”
The ability to combine data from super-resolution microscopes with genomics or proteomics datasets precipitates additional advances. “Traditional proteomics—mass spec and protein microarrays—tell us what is expressed in a population of cells, but super resolution microscopes tell us precisely where in a specific cell something is,” Kohrman explains.
For example, he continues, “Combining imaging of multiple types of biomolecules could allow researchers to show, in a given cell, that a particular DNA topology leads to a specific number of mRNA transcripts (being) produced, and a resulting amount of protein. This allows for a more complete understanding of the effects of perturbations from experimental conditions or disease.”
“Single molecule localization microscopy (like Bruker’s Vutara VXL, a subset of super-resolution microscopy)…allows the location and interaction of proteins to be directly measured and the precise mechanisms of drug candidates elucidated,” he says. As examples, Kohrman cites measuring the kinetics of particles and drug uptake from engineered nanoparticles, and tracking the secretion of signaling factors. The high degree of localization that’s possible today can even determine the cargo of lipid nanoparticles and the structure and degree of infectivity of viruses.
Future view
The role of super-resolution microscopy is expanding, encompassing drug development from early discovery and target validation to cell-based assays and biomarker analysis.
“These projects often shift direction as new data emerges, so flexibility in imaging systems is essential,” Evi Menelaou, PhD, software product manager at Nikon Instruments, points out. She calls it “a cornerstone of future microscopy platforms.” In this context, flexibility includes “pivoting quickly to scale-up for high-content screening, integrating with robotic automation, or modifying imaging protocols to accommodate new assay formats.”
For maximum flexibility, the ability to communicate and integrate with other software systems, devices, or workflows is vital, and transforms microscopes from standalone instruments into networked, programmable tools in data-rich environments, like the biopharma industry. Such connectivity and modularity “will be key to ensuring imaging workflows remain agile and compatible across diverse research environments,” Menelaou says.
The imaging pipeline itself is a growing area of interest for regulators as well as instrument makers. That interest is linked to biopharma companies’ growing focus on translational medicine, Menelaou says. As data from these instruments becomes more consequential, “Future systems will likely place greater emphasis on traceability, auditability, and standardized data handling to meet the rigorous demands of clinical validation and regulatory review.”
When instrument makers think about the current and near-future uses for super-resolution microscopes, there are “two major directions…in biopharma applications: live-cell imaging and 3D cell models,” according to Shohei Imamura, director, product management, life science, high-end imaging systems at Evident Scientific. Currently, “Most super-resolution technologies involve tradeoffs between resolution, throughput, and phototoxicity. Prioritizing one often limits the others.”
That’s about to change.
One imaging kit developed for super-resolution microscopes—still in development—offers views down to 1nm using resolution enhancement by sequential imaging (RESI) technology. In contrast, resolutions for other super-resolution microscopes are about 10 to 20 nm for single-molecule localization microscopes, 30 to 80 nm for stimulated emission depletion (STED), and 100 to 120 nm for structured illumination microscopes (SIM).
The 110 µm x 110 µm field-of-view of another microscope offers three-fold greater resolution than many microscopes from the previous generation, many of which range from about 5 to 30 µm, and peak at about 100 µm for SIM microscopes. The increased field of view helps scientists study heterogeneous populations and rare phenotypes, “validating observations in super molecule localization microscope data as true biological variability, and the representation of underlying mechanisms,” ONI spokesperson Anna Cabelle, PhD, elaborates.
Additionally, super-resolution microscopes are evolving to simplify sample preparation and support real-time imaging to maximize throughput and minimize phototoxicity. (Imamura cites Evident’s IXplore™ IX85 SpinSR, which launched in May, as an example.)
“3D cell models such as spheroids and organoids are increasingly recognized as physiologically relevant, as they better mimic the in vivo environment,” Imamura says. “These samples typically are thick and large, often requiring primary screening followed by super-resolution imaging for detailed analysis.” New technologies are streamlining that workflow with high-content analysis platforms integrated into the microscope. Additional advances include a lens that supports supper-resolution imaging of samples as thick as 1 mm.
Another example of today’s advances is ONI’s sub-20 nm resolution Aplo Scope, which is both compact and transitions from capturing dynamic events to capturing subcellular details without reconfiguration, Cabelle says. Also, “It [lets] samples be characterized for different metrics in a single assay, offering higher throughput.” Integrated advanced image analysis and reporting tools further streamline the workflow, making the microscope accessible to “researchers of all skill levels.”
AI: a critical enabler
AI is becoming increasingly important, too. In fact, Imamura calls it “a critical enabler.”
For super-resolution microscopes, AI helps by restoring images from even noisy, low-fluorescence data. That capability helps minimize phototoxicity by reducing exposure time and illumination power that, in turn, decreases noise and signal loss. Reducing phototoxicity helps maintain cell viability during live-cell imaging, Imamura explains.
“In addition, AI greatly improves the robustness and efficiency of complex analysis, such as phenotypic screening—a growing trend where detecting subtle cellular differences can be challenging,” Imamura adds. He cites technology as an example. Imamura says its “segmentation technology can identify and classify phenotypic variations with minimal training data, enabling one-step quantification of phenotype ratios.”
[Nikon Instruments]
As Nikon’s Menelaou adds, “AI is becoming an integral part of intelligent imaging workflows, where it helps microscopes adaptively focus on regions of interest, optimize imaging parameters in real time, and reduce phototoxicity and acquisition time. AI also simplifies complex imaging protocols, making super-resolution (microscopy) more accessible to non-specialist users and expanding its utility across multidisciplinary teams.”
AI’s role in analysis is equally pivotal, Menelaou adds. For example, “Classifiers can be trained on specific datasets and imaging conditions, enabling reproducible segmentation, classification, and quantification of biological structures. This is particularly valuable in biopharma, where consistency and scalability are essential for drug discovery and development.
“Perhaps most compelling,” she continues, “is AI’s ability to uncover subtle patterns and correlations within the vast datasets generated by super-resolution imaging. These insights are often missed by manual analysis and can truly accelerate hypothesis generation and experimental design, helping researchers move from image to insight more quickly and confidently.”
Other tools affected
With super-resolution microscopes becoming crucial for biopharmaceutical labs, ancillary tools also are evolving, and new ones are being developed to leverage their power.
Overall, these tools are becoming increasingly automated, “particularly for microfluidic exchange and sample preparation, image acquisition, and data analysis,” ONI’s Cabelle says.
For example, “Commercially available dye families…continue to expand to offer multi-color flexibility,” Cabelle says. “Newer dyes include self-blinking dyes or those for the far-red and near-infrared spectrum, which offer far deeper tissue penetration and reduced cellular background.”
Additionally, more precise labeling methods are being developed, she says, “that minimize the physical distance (localization or linkage error) between the fluorophore and the target biomolecule. This is critical for achieving sub-10 nm resolutions.” Examples include single-domain antibodies (including nanobodies), self-labeling tags, and chemical methodologies that can label molecules in living systems.
Notably, ONI and Massive Photonics, Cabelle says, are developing a two-plex DNA-Paint imaging kit engineered for elevated temperatures (above 32°C). The already commercialized DNA-Paint kit achieves resolutions down to 1 nm using resolution enhancement by sequential imaging technology.
Entry barriers falling
Super-resolution microscopes historically have required dedicated dark rooms and optical tables, which limited their adoption. Usage was further constrained by complex setups, difficulty of operation, need for specialist training, complicated data analysis tools, and, often, low experimental throughput.
New entries into the field, however, are overcoming many of those barriers, reducing the complexity associated with traditional hardware and analysis. “Super resolution (microscopes are) becoming faster, more powerful, and intuitive to use,” Cabelle notes. As they continue to evolve, they are becoming important tools not only for research, but for manufacturing and other applications.
