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PAINT-ing the future of fluorescence imaging

DNA Exchange imaging as a multiplexed biomarker strategy to see inside cells and tissues

By Eriona Hysolli

(BOSTON) – Many different types of cells located within various tissues and organs work together to sustain life in an organism. Visualizing which proteins are found at any given time in each cell type, and how the cells are arranged in tissues, is vital to understanding how organs function and malfunction. One of the most popular imaging methods – immunofluorescence microscopy – visualizes specific proteins through a microscope using an antibody directed against the protein that is linked to a fluorescent chemical compound called a fluorophore. This approach, however, is currently limited to detecting about four different fluorophores simultaneously with conventional microscopy, which is not sufficient to characterize the full biomolecular complexity of cells and tissues. Now, a team of scientists at the Wyss Institute for Biological Engineering has found a new way to generate highly detailed, fluorescently labeled images of a wide range of proteins and cells easily and rapidly, using DNA Exchange Imaging (DEI).

DNA Exchange Imaging of fixed mouse hippocampal neurons stained sequentially with antibodies recognizing neuronal markers Synapsin I, vGAT, MAP2, pNFH, α-tubulin, acetyl-tubulin, GFAP and nuclear marker DAPI. Credit: Wyss Institute at Harvard University

Imaging of living material can be done through use of dyes that intercalate between biomolecules, but this method is generally non-specific as the whole cell or tissue sections will stain non-discriminately, making scientists favor immunofluorescence instead. In immunofluorescence microscopy, lasers are used to excite the fluorophore, which in turn emits light at a specific wavelength, illuminating the stained molecular components. Distinguishing fluorophores depends on isolating the specific wavelength of their emitted light. This makes it difficult to visualize multiple proteins, each with its own fluorophore-labeled antibody simultaneously because the emitted light waves produced by different fluorophores can interfere with each other resulting in mixed signals.

An alternative strategy to using many fluorophores simultaneously is to sequentially stain the same cells or tissue sections with different antibodies, image them separately, and generate a composite image of all the individual antibodies. In its current form, this approach is laborious, time-consuming and inefficient as it requires repeated cycles of overnight antibody incubations and harsh washing.  “Our DEI approach is basically a generalized platform that solves the problem of multiplexed immunostaining for both conventional and super-resolution microscopy,” says Yu Wang, a graduate student at Harvard University, and lead author of a recent article published in Nano Letters on adapting DEI in cells and tissues.

Super-resolution image stack of a human HeLa cell stained sequentially with antibodies for nuclear membrane (laminin b – green), mitochondria (TOM20 – red) and microtubules (α-tubulin – white). Credit: Wyss Institute at Harvard University

DEI is based on fluorescently-labeled oligonucleotides, short DNA fragments that transiently bind to a complementary DNA fragment conjugated to an antibody, and stain proteins in cells or tissues of choice. In their study, the team stained fixed cells and paraffin-embedded tissues with up to 8 different antibodies carrying docking sites for binding the fluorescent oligonucleotides. “Our protocol allows for repeated staining because the DNA binding and washing steps are simpler and less time-consuming than traditional sequential staining procedures. The oligonucleotides we use are designed to be short and orthogonal (non-interfering) to make the staining very specific, but multiplexable and transient,” continues Wang. The team leveraged this technology to generate a composite image of all the cell layers comprising the mouse retina in order to prove its feasibility in complex tissue as well as highlight the technique’s multiplexing capabilities.

DEI piggybacks on the use of  Exchange-PAINT technology pioneered in the laboratory of Peng Yin, Ph.D., Core faculty member at the Wyss Institute and Professor in the Department of Systems Biology at Harvard Medical School (HMS). In conventional fluorescence microscopy, two points that are 200 nm apart or closer are not distinguished as two spots, but rather as one due to their fluorescence halos overlapping (wavelength interference). In order to visualize cellular structures inside the cells that are less than 200 nm apart from each other, super-resolution microscopy was developed. To achieve the high resolution, fluorescence intensity is intentionally depleted to create narrower fluorescent points (STED microscopy) that result in a crisper image, or fluorescence is activated, then switched off in individual molecules that are far apart such that their halos don’t overlap, and a complete image is reconstructed in the end with the stored individual fluorescence points (PALM and STORM microscopy).

Two-color DNA Exchange Imaging of mouse retina revealing astrocytes and Müller cells (GFAP – blue) and white blood cells and microglia (Coronin1a – magenta). Credit: Wyss Institute at Harvard University

PAINT (Point Accumulation for Imaging in Nanoscale Topography) was initially developed as a dye-based alternative strategy to achieve super-resolution imaging via use of dyes that transiently bind and quickly dissociate from their targets. Because the binding is fast and transient, it results in a “blinking” fluorescence signal that can be captured at many time points. The center of a fluorescence peak can then be resolved much more accurately, turning a blurry image, a common problem with conventional microscopy, into a sharp one. In a Nature Methods publication in 2014, Dr. Yin and his research team combined the PAINT principles with DNA (DNA-PAINT) in an exchangeable manner (Exchange-PAINT). In this study, short DNA fragments (oligonucleotides) were linked to a wide variety of fluorophores and bound to a complementary docking sequence in a DNA origami, or via antibodies to microtubules and mitochondria in a human cell line.

With Exchange-PAINT, the team successfully accomplished repeated exchanges of different oligo-fluorophore combinations with 10-color exchange in DNA targets and 4-color exchange in human cells. This methodology enables multiple targets to be processed sequentially, and an overlay image of all cycles generates a multicolored image delineating different regions or components of the specimen.

In a follow up study, the Wyss scientists validated the use of 52 different orthogonal DNA docking sites that can be used to theoretically accommodate 52 sequential antibody bindings, and optimized conjugation of DNA to antibodies and fluorophores to find the more efficient combination for multiplex imaging. These findings were published in early 2017 in Chemical Science.

Prior to the DEI report, the Wyss team had optimized DNA- and Exchange-PAINT  in DNA origami nanostructures and cells utilizing solely super-resolution platforms that better complement the use of PAINT approaches. This study was published in Angewandte Chemie and Nano Letters. Now, Wang and collaborators have not only validated the DEI technique in primary neuronal culture as well as neuronal and tumor tissue, but also extended the DEI compatibility so that it can be used with standard confocal fluorescence microscopy in order to make the methodology more widely useful for academia and industry, where super-resolution imaging techniques are still not fully accessible.

The technology is powerful and has the potential to be a game-changer in the world of fluorescent imaging, but is limited by today’s library of antibodies.  Development of a better, cheaper, and wider selection of antibodies remains a bottleneck to be overcome in order to stretch the limit of sequential imaging in tissues and cells used in DEI.

Yin envisions the broad availability of complete DEI imaging packages for laboratories in academia and industry alike. “There is a critical need to visualize multiple biomarkers in the same specimen efficiently and with high accuracy. We believe DEI can illuminate our understanding of the organization of cellular structures and complex biological architecture,” says Yin. DEI has already been licensed by Ultivue, a start-up company launched out of the Wyss Institute to commercialize high-definition biological imaging.

Multiplexed DNA Exchange Imaging of mouse retina tissue with antibodies demarcating 6 distinct layers of cells. Credit: Wyss Institute at Harvard University

“It is remarkable to witness the translation of DNA-PAINT and Exchange-PAINT from approaches that color nanostructures and living materials in the lab to technologies in the commercial space with promising applications to visually resolve molecular components with brain and cancer tissues,” says Donald Ingber, M.D., Ph.D., who is the Founding Director of the Wyss Institute, the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital, as well as Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS).

Other authors contributing to DEI study are Ralf Jungmann, Ph.D.,  Group Leader at Max Planck Institute of Biochemistry; George M. Church, Ph.D., Professor at Harvard Medical School and MIT;  Sarit S. Agasti Ph.D., Assistant Professor in Jawaharlal Nehru Centre for Advanced Scientific Research; Edward S. Boyden Ph.D., Professor at MIT Media Lab and McGovern Institute; past and present members of the Yin lab at Wyss Institute Johannes B. Woehrstein, Noah Donoghue Ph.D., Mingjie Dai Ph.D., Maier S. Avendaño Ph.D., Sylvain W. Lapan Ph.D.; Harvard Medical School affiliates Ron C. J. Schackmann Ph.D., Jason J. Zoeller Ph.D., Shan Shan H. Wang Ph.D., Joan S. Brugge Ph.D., and Pascal S. Kaeser Ph.D.;  MIT affiliates Paul W. Tillberg Ph.D., and Demian Park Ph.D.

This research is supported by NIH grants (1U01MH106011, 1R01EB018659), NSF grant (CCF- 1317291), and ONR grants (N00014-13-1-0593 and N00014- 14-1-0610) to Peng Yin.

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