Imaging has been vital in elucidating biological events in live cells and organisms. Driven by curiosity, scientists have always longed to “see” biomolecules to believe in their existence. With several decades of progress in imaging techniques and agents, recent advances and applications have enhanced our ability to directly visualize the physicochemical properties, chemical and biological reactivities, and molecular mechanisms of biomolecules. With sufficient spatiotemporal resolution, the application of these advancements has empowered new discoveries and deepened our understanding of a wide range of cellular processes.
This virtual special issue on Advances in Biological Imaging assembles a collection of thought-provoking Reviews and Articles from eminent researchers worldwide. Their contributions collectively represent the cutting-edge of research in several areas of biological imaging techniques. These areas include imaging biological activity or reactivity using activity-based reagents, chemical probes that provide insight into biological events and substructures, microscopy assays that dissect cellular pathways, and advanced techniques with enhanced resolution and multiplexing. Taken together, the Articles and Reviews in this issue not only present an overview of modern imaging technologies but also provide a perspective on future directions for this exciting field.
Activity-Based Imaging: A New Direction to Visualize Biological Activity
Chemical reactivity provides essential support to cellular viability and growth. As an emerging field, activity-based sensing (ABS) has enabled the real-time detection of biomolecular activity and chemical reactivity in live cells. This virtual special issue features a comprehensive Review by Messina, Quargnali, and Chang highlighting this emerging field, “Activity-Based Sensing for Chemistry-Enabled Biology...”, which elucidates the design and application of ABS reagents for a major type of reactive oxygen species (ROS), hydrogen peroxide (H2O2). The specific and selective recognition of H2O2 involves a series of chemical mechanisms based on its chemical reactivity. Thus, the system is capable of differentiating H2O2 from other ROS molecules. As a timely summary of current progress, this Review introduces an extensive family of small molecule H2O2 probes and their application in revealing biological processes related to H2O2.
Expanding on these principles, Chan et al. present an extensive Review of Activity-Based Photoacoustic Probes. Photoacoustic (PA) imaging is a powerful approach for in vivo imaging, with superior imaging depth and low background noise. In each tissue or disease type, specific biomarkers exhibit distinct chemical reactivities. PA probes have been developed to provide selective imaging strategies by leveraging these tissue- or disease-specific activities. This Review introduces a set of PA probes that target the activity of biomarkers in various organs (brain, liver, and stomach), physiological or pathological events (inflammatory and blood clotting), and pathogens, including bacteria and viruses. As a particular focus, the design principles for these probes are highlighted, thus guiding PA probe development in related areas.
Enzymatic activity is another important reactivity that chemical imaging reagents can target and visualize, generating valuable insight into biological processes related to enzymes of interest. For example, Edgington-Mitchell et al. systematically summarized “Chemical Tools to Image the Activity of PAR-Cleaving Proteases” in this virtual special issue. As a family of G protein-coupled receptors (GPCR) that require cleavage by proteases for activation, the activity of PAR-cleaving proteases is essential to understanding PAR-related biological events. This Review provides a thorough analysis on how protease activities are measured and imaged using various chemical probes, focusing on substrate-based probes, activity-based probes, serine protease probes, and cysteine protease probes.
Illuminating Biological Events and Substructures Using Chemical Probes
As we delve deeper, imaging probes often reveal previously unseen details of biological events. As an essential cellular process, cell death is related to many diseases. Tang and colleagues summarize fluorogenic imaging approaches to directly study this process in their Review, “Aggregation-Induced Emission (AIE) Luminogens for Cell Death Research”. AIE luminogens (AIEgens) harbor special features wherein their molecular aggregates exhibit stronger activated emission than individual molecules. This Review showcases AIEgens developed to monitor and induce various types of cell death, including apoptosis, necrosis, immunogenic cell death, pyroptosis, autophagy, lysosome-dependent cell death, and ferroptosis.
Imaging subcellular organelles continuously attracts research interest, with a view to improving selectivity and photophysical properties. In their Article, Lavis and colleagues describe the development of “2,7-Diaminobenzopyrylium Dyes Are Live-Cell Mitochondrial Stains”. While most of the existing mitochondrial dyes exhibit spectral features similar to those of standard blue, green-yellow, and red excitation windows, the DAB framework is demonstrated as a novel scaffold for mitochondrial staining with a violet excitation spectrum. The superior brightness and photostability make DAB-based stains a good mitochondrial stain, using a previously less explored spectral region.
Dissecting Cell Biology via Microscopic Assays
Combined with microscopy, fluorescence-based assays often provide mechanistic details that other experimental approaches cannot parallel. For example, metabolism is an important topic in both chemistry and cell biology. In particular, energy-related metabolic pathways, represented by glucose metabolism, have received attention in numerous studies. An et al.’s Article in this virtual special issue presents compelling research on “Size-Specific Modulation of a Multienzyme Glucosome Assembly during the Cell Cycle.” Using single-cell imaging strategies, the authors show that the size of glucosomes, cytoplasmic enzyme assemblies for glucose metabolism, is modulated in varying cell cycle stages to regulate glucose flux between glycolysis and building-block biosynthesis. This work highlights the power of quantitative imaging techniques to reveal behind-the-scenes mechanisms that could not be uncovered without rigorous quantification.
Continuing on this topic, West and colleagues developed “A Fluorescence-Based Assay to Probe Inhibitory Effect of Fructose Mimics on GLUT5 Transport in Breast Cancer Cells.” The design follows the uptake of a fluorescent substrate for the principal fructose transporters (GLUT5), 6-NBDF (6-NBD-D-fructose), whose entry into the intracellular space is dependent on the active form of GLUT5 and results in a quantifiable fluorescent signal. Using this assay, the researchers evaluated the capacity of a series of d-fructose mimics to bind with GLUT5 and inhibit the entry of 6-NBDF. Because GLUT5 is overexpressed in human breast cancer cells, these compounds could advance the development of potent probes targeting and modulating GLUT5-expressing cancerous cells.
This virtual special issue also touches upon another essential cellular process, protein misfolding and aggregation, which is discussed by Zhang et al. in their development of “A High-Fidelity Assay Based on Turn-off Fluorescence to Detect the Perturbations of Cellular Proteostasis.” Most previous methods rely on the activation of fluorescence emission from solvatochromic and viscosity-sensitive fluorophores to visualize protein misfolding and aggregation. When searching for conditions that inhibit protein aggregation, these sensors fall short due to inhibiting their fluorescence. In this work, the researchers developed a series of rhodamine-based probes whose fluorescence decreases with protein misfolding; this signal change is the opposite of that of traditional probes. This new development could be useful in evaluating the conditions that assist folding and prevent the aggregation of cellular proteins.
Developing Imaging Techniques to Deepen and Broaden Vision
Finally, we touch upon advanced imaging techniques and strategies. Whelan, Bell, and colleagues present the development of “Live-Cell SOFI Correlation with SMLM and AFM Imaging” to acquire super-resolution microscopic images of live cells. SOFI correlation refers to super-resolution optical fluctuation imaging, which adds to a previously reported correlative process using single-molecule localization microscopy (SMLM) and atomic force microscopy (AFM). Using reversibly switching fluorescent proteins, SOFI-SMLM-AFM can image microtubules in live cells via SOFI and then in fixed cells via SMLM and AFM. This correlative strategy can increase resolution by two to 3-fold without compromising the information about live cells possibly lost in the fixation process.
Multiplex protein imaging provides spatial information for multiple targets, potentiating the discovery of novel interactions in biological processes. To this end, Li and Xue et al. have developed “Multiplex Protein Imaging through PACIFIC: Photoactive Immunofluorescence with Iterative Cleavage”. Focusing on immunofluorescence with fixed cells, the PACIFIC approach realizes multiplexing imaging by implementing a photocleavable group in fluorophores for antibody labeling. After each round of imaging, the fluorophore can be cleaved by irradiation with 365 nm UV light. This allows for the next round of imaging for new targets using antibodies labeled with a distinct fluorophore. Such a process can be iterated, resulting in multiplex imaging for as many as six targets imaged in the same cell.
This virtual special issue also highlights the intersection between nanotechnology and imaging technology. In their thorough and timely Review, Calatayud, Pascu, and colleagues summarize “Functional Diversity in Radiolabeled Nanoceramics and Related Biomaterials for the Multimodal Imaging of Tumors.” To enable the preclinical investigation of tumors, several molecular imaging techniques have been employed, including magnetic resonance imaging, optical imaging, positron emission tomography imaging, and single photon emission computed tomography imaging. This Review introduces how multiple imaging agents can be incorporated into radio-nanoparticles to trace tumors in living organisms.
Overall, this virtual special issue weaves together a comprehensive narrative of contemporary biological imaging techniques, setting the stage for future directions in the field. We thank the authors for their contributions to this virtual special issue and the reviewers for their contributions to the papers. The Editors of ACS Bio & Med Chem Au look forward to publishing research from this exciting field in the coming years.
Biography
Xin Zhang is a Professor of Chemistry at Westlake University. He earned a doctoral degree at the California Institute of Technology and was a postdoctoral fellow at the Scripps Research Institute, California. His research is focused on the chemistry of biological aggregates formed by proteins and RNAs under physiological and pathological conditions. He has expertise in chemistry and molecular and cell biology and has developed chemical biology approaches to study biological aggregates. His independent work has been recognized by awards, including the NSF CAREER award, Pew Scholar in the Biomedical Sciences, Sloan Research Fellowship, and the Burroughs Wellcome Fund Career Award at the Scientific Interface.
Views expressed in this editorial are those of the authors and not necessarily the views of the ACS.
The authors declare no competing financial interest.