Using Fluorescence Microscopy Applications for DNA-PAINT
Whether by confocal microscopy or FISH, producing attractive and useful images of cellular phenomena requires a special touch that most scientists never get the chance to develop. With the advent of a new technique for super resolution microscopy, now everyone in the lab has a fresh chance to get their hands on a 5 nm scale imaging technique, should they desire. In a new paper published in Nature Protocols, researchers outline the technique which they call DNA-PAINT for its use of dye labeled nucleotide bonding as a resolution enhancing element.1 DNA-PAINT allows for molecule counting, particle averaging, and super resolution image reconstruction, making for a formidable technique even among advanced microscopy methods.2 Importantly, the researchers behind DNA-PAINT have opted to include introductory programs intended for novice microscopists so as to make the technique accessible to everyone in the laboratory environment, rather than just those who already have advanced imaging skills. If researchers want to start taking nanometer scale images of their cellular and molecular subjects, they’ll need to understand how to use the data output of DNA-PAINT by using powerful software to transform it into publication-grade material.
How Can DNA Be Useful in Super Resolution Microscopy?
While DNA-PAINT is a form of fluorescence microscopy, it’s nothing like the other form of fluorescent microscopy that researchers will be familiar with, FISH. FISH uses fluorescent probes to bind to DNA, and then researchers subsequently excite the fluorescent probes using light and detect the resulting fluorescent excitation.3 This means that FISH is used largely to image chromosomes and certain sequences of DNA, rather than to replace the traditional “making small things look large enough to examine” role of a microscope.
In contrast, DNA-PAINT uses fluorescent probes to bind to arbitrary cellular features tagged with complementary labels, creating a “blinking” effect.4 The blinking prevents background from accumulating too much at any one instant of time, and also allows for getting around the refraction limits of light microscopes.5 6 The complementary labels are bound to antibodies against the cellular features. By using software to measure the repeated blinking of each oligonucleotide probe, researchers can image surfaces with confidence, because each probe is specific to the feature it’s binding to, thanks to being attached to an antibody. This means that unlike in FISH, DNA-PAINT actually produces an image rather than making the desired subject appear in isolation against the dark background which presumably contains other structures.
This workflow can be a bit confusing, but it’s easier when hashed out into steps:
- Produce antibodies against cellular features to be imaged
- Create complementary probes
- Conjugate complementary probes to the respective antibody
- Make note of which complementary probe sequences are conjugated to which antibody of which specificity
- Combine wet components of experimental apparatus and start imaging
- Detect transient binding fluorescent events using the fluorescent microscope
- Process the blinking transient binding events with software
- Develop model of imaged surface using software
Astute researchers will quickly note that unlike other fluorescence-based imaging techniques intended for imaging cellular surfaces, DNA-PAINT is capable of intense background fluorescence control via the selection of antibody binding specificities.7 Researchers can calibrate their experimental apparatus to deliver the precise answer to their experimental question without fear of noisy data coming from harder questions, as is commonly the case. It’s easy to imagine one researcher using DNA-PAINT to image a small handful of characterized features to understand their relationship to each other using highly specific antibodies.
It’s equally easy to imagine another researcher using DNA-PAINT with very weakly specific or multispecific antibodies to image an entire cellular area to detect previously unknown features for further investigation. The flexibility of DNA-PAINT means that researchers need to get their heads around a lot of different possibilities and a lot of unfamiliar methods and data, however. Easing into this new imaging landscape will be a lot easier if researchers team up.
Passing the PAINT
If teaming up to execute a high-resolution microscopy experiment seems like it still won’t be any more complicated than doing a quick sample preparation and passing it off to the mysterious imaging expert, let’s go over the skills required to use DNA-PAINT:
- Fluorescent microscope operation
- DNA probe generation
- Antibody generation
- Antibody-probe conjugation
- High-resolution imaging experimental planning
- DNA-PAINT protocol execution
- High-resolution imaging execution
- Fluorescent microscopy interpretation
- Nanometer scale imagery interpretation
It looks like there will still be a role for the imaging expert as DNA-PAINT becomes more common, but it’s assured that imaging experts are no longer enough to implement a successful imaging experiment with DNA-PAINT. For all its strengths, DNA-PAINT requires more resources, more expertise, more pieces of data to track, and ultimately, more personnel to execute successfully.
To take advantage of DNA-PAINT’s high-resolution microscopy potential, researchers will need to team up with a handful of others who can provide the missing pieces from the experimental setup and data analysis. Molecular biologists will need to team up with immunologists and microscopists before a DNA-PAINT picture can be made—without probe generation, antibody preparation, and high-resolution microscopy expertise under the same roof, running a complex experiment isn’t possible. This kind of high-bandwidth collaboration requires powerful collaboration enabling software designed specifically for the purpose.
Collaborative Science Solutions is the collaboration, data tracking, experiment planning, personnel organizing, and imaging research promoting software that the researchers of today will use to develop the high-resolution images produced by DNA-PAINT of tomorrow. Using Science Solutions, your team will be able to pass data between sections of the imaging pipeline and rest assured that the vast quantity of specific data remains linked to the relevant experimental pieces. Contact us today to find out how you can use Science Solutions to start seeing cellular structures using DNA-PAINT.
- “Super-Resolution Microscopy With DNA-PAINT.” May 2017, https://www.nature.com/nprot/journal/v12/n6/full/nprot.2017.024.html?foxtrotcallback=true ↩
- “Single-Molecule Kinetics And Super-Resolution Microscopy By Fluorescence Imaging Of Transient Binding On DNA Origami.” October 2010, http://pubs.acs.org/doi/abs/10.1021/nl103427w ↩
- “Immunological Method For Mapping Genes on Drosophila Polytene Chromosomes.” 1982, https://www.ncbi.nlm.nih.gov/pubmed/6812046 ↩
- “Wide-field Subdiffraction Imaging By Accumulated Binding Of Diffusing Probes.” October 2006, http://www.pnas.org/content/103/50/18911 ↩
- “Nonlinear Structured-illumination Microscopy: Wide-Field Fluorescence Imaging With Theoretically Unlimited Resolution.” July 2005, http://www.pnas.org/content/102/37/13081 ↩
- “Nonblinking and Long-Lasting Single-Molecule Fluorescence Imaging.” October 2006, https://www.nature.com/nmeth/journal/v3/n11/full/nmeth934.html ↩
- “DNA-barcoded Labeling Probes For Highly Multiplexed Exchange-PAINT Imaging.” January 2017, http://pubs.rsc.org/en/Content/ArticleLanding/2017/SC/C6SC05420J#!divAbstract ↩