Researchers at the Broad Institute of MIT and Harvard have developed a new way of mapping cell populations and visualizing how biomolecules, including different sequences of DNA and RNA, are organized spatially in cells and tissues. The approach, dubbed DNA microscopy, doesn’t require any optical or other specialized equipment, but instead uses nucleic acid barcodes to pinpoint molecules’ relative positions within a sample. Carried out using standard laboratory equipment, the process requires just the sample of cells, some reagents and pipettes, and allows large numbers of samples to be processed simultaneously.

“DNA microscopy is an entirely new way of visualizing cells that captures both spatial and genetic information simultaneously from a single specimen,” said Joshua Weinstein, PhD, a postdoctoral associate at the Broad Institute of MIT and Harvard. “It will allow us to see how genetically unique cells—those comprising the immune system, cancer, or the gut, for instance—interact with one another and give rise to complex multicellular life.”

Using DNA microscopy (left), scientists can accurately reconstruct an image of cells captured with a fluorescence microscope (right). Scale bar = 100 micrometers. [J. Weinstein et al./Cell 2019]
Weinstein, together with computational and systems biologist Aviv Regev, PhD, and molecular biologist Feng Zhang, PhD, reported on the new technology in Cell. “It’s not just a new technique, it’s a way of doing things that we haven’t ever considered doing before, Regev added. The team’s published paper describing the new approach and tests on human cell lines, is titled “DNA microscopy: Optics-free spatio-genetic imaging by stand-alone chemical reaction.”

Microscopy techniques today fall into two basic categories. One uses some form of optics, and includes basic light microscopes—which have been around since the 1600s—and more recent electron, fluorescence, and light-sheet microscopes that all rely on the fact that a sample emits photons or electrons, which can be detected. This type of microscopy can provide information on subcellular structure and function. The second category of microscopy is based on dissecting samples at defined locations, and harnessing computer programs to reassemble each piece into the whole. Unlike optical techniques, dissection-based microscopy techniques can provide genetic information.

What hasn’t yet been developed is a single technology that can do the job of both optical and dissection-based methods. “Although imaging of cells and tissues has been a cornerstone of biology ever since cells were discovered under the light microscope centuries ago, advances in microscopy have to date largely not incorporated the growing capability to make precise measurements of genomic sequences,” the authors stated. “While microscopy illuminates spatial detail, it does not capture genetic information unless it is performed in tandem with separate genetic assays.”

Ideally, then, we would be able to identify where cells with differential gene expression patterns are located within a specific tissue. “The spatial organization of cells with unique gene expression patterns within tissues is essential to their function and is at the foundation of differentiation, specialization, and physiology in higher organisms,” the team continued. This is true for tissues including the gut and nervous system, the immune system, and tumors, in which the organization of distinct cells that express genes with different mutations can influence tumorigenesis.

In contrast with existing microscopy approaches, DNA microscopy relies on image reconstruction from the relative physical proximity of individual molecules, the authors explained, and in this way can obtain precise genetic information at high spatial resolution. For their reported study the researchers used DNA microscopy to map the locations of individual human cancer cells in a sample.

This image shows a visualization of the data provided by DNA microscopy, which has a resolution comparable to optical imaging. [Weinstein et al. / Cell]
To visualize the spatially organized genetic landscape of a sample, the researchers first take the lab-grown cells and fix them into position in a reaction chamber. They then randomly tag individual DNA or RNA molecules in the cells using small synthetic DNA barcodes known as unique molecular identifiers (UMIs). These tags are then replicated, diffusing in “clouds” outwards from their original position. “… picture every single molecule as a radio tower broadcasting its own signal outward,” Weinstein said. As they diffuse out, the replicated tagged molecules eventually collide with other tagged molecules, and they combine into DNA pairs. Each collision effectively then represents a new DNA sequence product as a chemical reaction. Molecules that are close to one another are more likely to collide and generate DNA pairs. Conversely, fewer DNA pairs will be generated if the diffusing tags are relatively far apart from others.

The sample essentially becomes dotted with chemically discrete points. By tracking the collisions between clouds of UMI copies researchers can reduce the uncertainty of the original UMI positions. The labeled biomolecules are collected, sequenced, and computer algorithms are then used to decode the data and reconstruct the relative positions of the tags. The resulting image is a two- or three-dimensional genetically detailed plot of molecular positions in physical space.

The plot is based on hundreds of thousands of different dimensions, dictated by the number of molecules with which the tagged molecules can plausibly communicate. “A chemical reaction within the specimen encodes information into DNA from which an algorithm can decode the relative positions of molecules without needing to know in advance cell identity or the nature of genetic variation,” Weinstein summarized. “DNA microscopy gives us microscopic information without a microscope-defined coordinate system.”

flyover of scatterplot data from DNA microscopy
In this flyover of scatterplot data from DNA microscopy, scientists use an algorithm to determine the relative location of molecules (colored dots, classified by the molecules’ gene sequences) at each point in space. [J. Weinstein et al./Cell 2019]
DNA microscopy could in practice be used to map any groups of molecules that can interact with synthetic DNA tags, including genomic DNA, RNA, or proteins with DNA-labeled antibodies. “In the same way that light microscopy images molecules that interact with photons (either due to diffraction or scattering or because these molecules emit photons themselves) and encodes these images in the wavelengths and directions of these photons, DNA microscopy images molecules that interact with DNA (including DNA, RNA, or molecules that have been tagged with either DNA or RNA) and encodes these images,” the authors noted. “Because DNA microscopy does not rely on specialized equipment and can be performed in a multi-well format with normal lab pipettes, it is highly scalable, such that a large number of samples can be processed in parallel.”

“You’re basically able to reconstruct exactly what you see under a light microscope,” Weinstein stated “We’ve used DNA in a way that’s mathematically similar to photons in light microscopy. This allows us to visualize biology as cells see it and not as the human eye does.”

The technology isn’t perfect, the authors acknowledged, as it can’t resolve empty spaces, such as gaps between two cells. More work will be needed to address this issue. “…  one key weakness of DNA microscopy remains the resolution of empty space, and future work will be needed to eliminate this obstacle to produce high-quality reconstructions of samples over large lengths where there are gaps in molecular density,” the team commented. Nevertheless, “DNA microscopy is a compelling approach to study the tissue organization of cells such as lymphocytes, neurons, or mutated cancer cells, where somatic mutation, recombined gene segments, and other sources of nucleotide-level variation endow unique molecular identities with important physical consequences.”

Weinstein believes that the most exciting applications of the technology will be in areas of biology in which mutations, RNA editing, and other forms of nucleotide-level variation work in concert within the organism to either produce particular physiological outcomes, or cause disease. “By capturing information directly from the molecules being studied, DNA microscopy opens up a new way of connecting genotype to phenotype,” Zheng commented.

Potential applications include looking at immune system development, nervous system architecture, and mapping genetic mutations in different cells within the same tumor in relation to their interaction with other cells and with the immune system. “We hope that it sparks the imagination—that people will be inspired with great ideas that we’ve never thought about,” Regev said.

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