Unlocking the microscopic universe within living organisms through cutting-edge single-cell analysis technologies
Imagine being able to count and identify every person in a crowded stadium individually rather than just knowing the crowd's average height or weight.
This is the fundamental difference between traditional biology and the revolutionary field of single-cell analysis. For centuries, scientists studied cells in bulk, grinding up tissues and analyzing the average molecular content. This approach, while valuable, completely missed the incredible diversity and heterogeneity that exists between individual cells within the same tissue.
The Biophysical Society of Japan's 2019 "Single-cell PRESTO" session showcased cutting-edge technologies that are now making it possible to observe biological processes with unprecedented, single-cell resolution, opening new windows into the microscopic universe within living organisms 1 .
Averaging measurements across thousands or millions of cells, masking important differences between individual cells.
Examining each cell individually to uncover the true diversity and specialized functions within tissues.
At the heart of the single-cell revolution lies a simple but profound concept: every cell is unique. Even within the same tissue type, individual cells can exhibit striking differences in gene expression, protein content, and function. This cellular heterogeneity plays crucial roles in development, disease progression, and treatment response.
Single-cell RNA sequencing (scRNA-seq) allows researchers to classify and characterize each cell at the transcriptome level, identifying rare but functionally important cell populations that would otherwise be masked in bulk analyses 2 .
The year 2019 was declared "Method of the Year" for single-cell multimodal omics by Nature Methods, highlighting the growing ability to simultaneously measure multiple molecular aspects of individual cells 8 .
Beyond just identifying cell types, understanding their spatial organization and interactions within tissues provides critical insights into how organs function during health and disease.
Did you know? A single tissue sample can contain dozens of distinct cell types, each with specialized functions and gene expression profiles that bulk analysis methods completely average out.
One of the most significant technical challenges in single-cell biology has been how to visualize molecular components deep within intact tissues without having to slice them into thin sections. Traditional methods required labor-intensive serial sectioning, which often distorted tissue architecture and made it difficult to reconstruct three-dimensional structures accurately.
A team of researchers developed an ingenious solution called ACT-PRESTO (Active Clarity Technique-Pressure Related Efficient and Stable Transfer of Macromolecules into Organs), which enables rapid tissue clearing and labeling for detailed 3D imaging at single-cell resolution 9 . This method allows researchers to transform opaque biological tissues into transparent samples while preserving cellular structures and protein-based signals.
Samples are first fixed with paraformaldehyde and infused with acrylamide to create a tissue-hydrogel hybrid that preserves the structural and molecular integrity of the tissue.
The sample undergoes electrophoresis in a specially designed chamber that efficiently removes light-scattering lipid molecules while retaining proteins and nucleic acids. This process, which takes just 4-20 hours instead of weeks required by earlier methods, is what renders the tissue transparent.
The PRESTO component applies pressure to drive antibodies or nucleic acid probes deep into the thick, cleared tissues, allowing specific cellular components to be labeled with high efficiency.
The cleared and labeled samples can then be imaged using specialized microscopy techniques to visualize cellular structures and molecular distributions throughout the entire tissue volume.
The ACT-PRESTO method achieved what previously seemed impossible—rendering whole organs transparent within a single day while preserving the ability to label specific cellular components. When researchers injected trypan blue into the cleared brain, they could beautifully visualize the intricate architecture of the lateral ventricles 9 . Furthermore, they demonstrated that the method works with conventional immunolabeling, successfully testing 75 out of 82 commercially available antibodies against various cellular structures.
| Method | Processing Time | Tissue Size Change | Compatibility with Immunostaining |
|---|---|---|---|
| ACT-PRESTO | 1 day | Returns to original size | Excellent (91.5% of tested antibodies worked) |
| CLARITY | 1-2 weeks | Swelling during process, returns in solution | Good |
| iDISCO | Several days | Shrinks to ~50% of original size | Good |
| CUBIC | Several days | Minimal size change | Moderate |
| Reagent/Technology | Function | Application Examples |
|---|---|---|
| PrestoBlue Cell Viability Reagent | Measures cell proliferation and viability using resazurin-based chemistry | Quickly assessing cell health before single-cell RNA sequencing 5 |
| Unique Molecular Identifiers (UMIs) | Tags individual mRNA molecules to eliminate PCR amplification bias | Accurate quantification of transcript numbers in scRNA-seq 2 |
| 10x Genomics Chromium Platform | Microfluidic partitioning of single cells into droplets for barcoding | High-throughput single-cell transcriptome analysis of thousands of cells 7 |
| ACT-PRESTO Solutions | Tissue clearing and rapid macromolecule penetration | 3D imaging of cellular structures in intact organs 9 |
The toolkit for single-cell research has expanded dramatically, with several platform technologies enabling different types of analyses:
| Technology Type | Key Features | Best Suited For |
|---|---|---|
| Droplet-based scRNA-seq (10x Genomics) | High-throughput analysis of thousands of cells | Identifying cell types and states in complex tissues 7 |
| Single-nucleus RNA-seq | Sequences mRNA from cell nuclei only | Tissues difficult to dissociate (brain, muscle) or frozen samples 2 |
| Multimodal Omics | Simultaneously measures multiple molecular layers | Understanding connections between gene expression, surface proteins, and epigenetics 8 |
| Spatial Transcriptomics | Maps gene expression within tissue context | Understanding cellular organization and communication in tissues |
The technologies showcased in the BSJ 2019 "Single-cell PRESTO" session represent more than just incremental advances—they mark a fundamental shift in how we study biological systems.
By allowing scientists to observe molecular processes at the level of individual cells within their native tissue contexts, these methods are revealing a previously invisible world of cellular heterogeneity, organization, and interaction.
As these technologies continue to evolve and become more accessible, they hold tremendous promise for transforming medical diagnosis and treatment. From identifying rare cancer cells that drive tumor resistance to mapping the complex cellular interactions in autoimmune diseases, single-cell technologies powered by approaches like PRESTO are providing the resolution needed to understand biological complexity in health and disease. The session highlighted that we're not just perfecting existing methods but creating entirely new ways of seeing the biological world—one cell at a time 1 .
The future of single-cell biology lies in integrating these powerful technologies to build comprehensive cellular atlases of entire organisms, providing foundational resources that will drive biological discovery and medical innovation for decades to come 2 . As we continue to develop tools that minimize the technical artifacts and maximize the biological insights, we move closer to understanding life at its most fundamental level—one remarkable cell at a time.