Menu

Close

DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction

Joshua A. Weinstein, Aviv Regev, Feng Zhang

Preprint posted on November 19, 2018 https://www.biorxiv.org/content/early/2018/11/19/471219

DNA microscopy: imaging biology through the lens of DNA sequencing

Selected by Theo Sanderson

Background
The word “microscope” was coined in 1625, to describe a device with a glass lens made by Galileo. The centuries since have seen considerable improvements, with dramatic increases in the resolution at which cellular structures can be resolved. Nevertheless, the fundamentals are relatively unchanged: these techniques still involve bombarding a sample with particles (photons or electrons) and observing the result of their interactions using a series of lenses. By reconstructing the positions from which photons have emerged, we can understand spatial organisation within the sample.

Can DNA sequencing provide an alternative technology? In recent years DNA has begun to be used for far more than simply understanding the natural genetic code of living systems. DNA barcodes are now used in chemistry laboratories to uniquely tag billions of chemical compounds, and as a deterrant marker for forensics. In biology, such genetic barcodes have been used in high-throughput screens, and even as a ticker tape to record the developmental trajectories of cells.

In this preprint, the authors develop a new method to encode data on the spatial relationship between molecules into sequences of nucleotides, and use this information to do away with the need for an optical microscope, creating images with a DNA sequencing machine alone.

Key findings:

To be able to record spatial information within sequences of DNA the authors employ an innovative approach based on overlap-extension PCR. They first label molecules of interest (in this case RNA transcripts inside fixed cells) with short sequences of DNA. These DNA labels have a degenerate section which serves as a unique identifier for each labelled molecule in downstream analysis.

The labelled DNA is then amplified, producing many copies of these molecular identifiers in the local area of the original label. But these amplified molecules are not static – they drift around by diffusion, and it is this process that allows spatial information to be recorded. As the molecules drift, they encounter amplicons from other nearby labelled molecules, and complementary 5′ sequences on their primers cause the amplification of a concatenated version of the two initial sequences. These concatamer sequences record in a single piece of DNA the molecular identifiers from the two molecules. The researchers create a hydrogel in the sample which slows down the diffusion process so that concatameters will predominantly form between amplicons from nearby molecules. Each concatenated sequence also gets its own unique molecular identifier, created from degenerate sequences in the amplification primers. As molecules drift they create concatamers with molecules further and further from their initial location until the cycles of the PCR reaction are complete, and sequencing libraries are prepared from the concatenated molecules.

These sequencing libraries are now read using an Illumina sequencing machine. Inspecting this data allows a long list to be made of the molecular identifiers of all the molecules that were labelled, and the type of probe from which they originated, and next the number of contamers formed between each pair of molecules can be counted. In practise the number of such potential pairs is so vast that the researchers adopt sophisticated approaches to streamline their analyses, but their data essentially takes the form of a vast matrix recording which molecules are close to each other, rather like a table of driving times between cities.

 

 

From such a table, one could try to reconstruct a map of a country, and similarly the researchers analyse their data to reconstruct a 3D image of the positions of molecules in the cells they are studying.  In this experiment they looked at a co-culture of cells expressing RFP and GFP, and by visualising the position of RFP transcripts in red and GFP transcripts in green they form an image reminiscent of microscopy, but created entirely from DNA data. The mutually exclusive positions of GFP and RFP seen across cells demonstrates the success of their technique, and shows that this first demonstration has resolution sufficient to clearly resolve cells. They also directly compare their data to a light microscopy image and see similar cellular arrangements.

 

Figure 5E from the preprint, showing a DNA-microscopy image with GFP transcripts labelled in green and RFP transcripts labelled in red.

 

What I like about this preprint
This work opens up a range of opportunities for investigating spatial properties of biological systems in new ways. Light microscopy is fundamentally limited in how many channels can be imaged at the same time. Although multiple fluorophores can be used simultaneously, spectral overlap limits experimenters to using a handful in each experiment. By contrast, a 10 base pair sequence of DNA could potentially encode a million “channels”, in an approach like this. The experimenters demonstrate this scalability by resolving 20 different transcripts in parallel.

The preprint also does a good job of acknowledging current limitations of this technology. Whereas optical microscopy finds it easiest to resolve sources of material which are sparse and well-separated, DNA microscopy can actually struggle to resolve empty space, since this can only be inferred by reference to labelled molecules. The authors also highlight that the technique need not be limited to RNA transcripts. Almost anything can be tagged with a piece of DNA, whether using DNA-tagged antibodies or aptamers.

 

Future directions
Many of the images obtained in this work could have been acquired with a light microscope using fluorescent in-situ hybridisation, likely with higher resolution. The preprint presents strong proof of a concept and it will be exciting to see what refinements can be made to the technology, and what new biological questions can be asked using these approaches to look at a wider range of probe molecules.

Interestingly, in the same week that this work was posted on bioRxiv, two similar preprints were also posted. “From space to sequence and back again” uses ligation rather than overlap-extension and recovers patterns from 2D surfaces, and  “A Computational Framework for DNA Sequencing-Based Microscopy” presents a computational method, also described in a 2D setting, which uses a similar chemistry as the amplification reaction in Illumina sequencing. With a wide range of exciting work being carried out, this nascent field looks like one to watch.

 

Questions for the authors

  1. In traditional light microscopy there is a fundamental limit to resolution associated with the wavelength of light used. Do you have a sense of what would drive the analogous limits for DNA microscopy, and what their order of magnitude might be?
  2. In what areas of biology do you think DNA microscopy will first have tangible practical applications?

 

Posted on: 28th November 2018

Read preprint (3 votes)




  • 2 comments

    3 months

    Carmen Adriaens

    Hi Theo! Awesome highlight. I look forward to see higher resolution examples of this technique in the future, and I’m generally excited about non-genetic applications for DNA techniques!

    As relating to the third paper you mention, you can take a look at this one, too: https://www.biorxiv.org/content/early/2018/08/02/383943

    1

    3 months

    Theo Sanderson

    Thanks Carmen! The preprint you point to is fascinating. (It may be especially impactful when combined with the deep-learning approaches that are being developed to detect subtle morphological phenotypes.)

    Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the bioengineering category:

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    Defining the design requirements for an assistive powered hand exoskeleton

    Quinn A Boser, Michael R Dawson, Jonathon S Schofield, et al.



    Selected by Joanna Cross

    Imaging mechanical properties of sub-micron ECM in live zebrafish using Brillouin microscopy

    Carlo Bevilacqua, Héctor Sánchez Iranzo, Dmitry Richter, et al.



    Selected by Stephan Daetwyler

    1

    Polyacrylamide Bead Sensors for in vivo Quantification of Cell-Scale Stress in Zebrafish Development

    Nicole Traeber, Klemens Uhlmann, Salvatore Girardo, et al.



    Selected by Jacky G. Goetz

    SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues

    Jocelyn Y. Kishi, Brian J. Beliveau, Sylvain W. Lapan, et al.



    Selected by Yen-Chung Chen

    Live-cell imaging of marked chromosome regions reveals dynamics of mitotic chromosome resolution and compaction

    John K Eykelenboom, Marek Gierlinski, Zuojun Yue, et al.

    AND

    Quantitative imaging of chromatin decompaction in living cells

    Elisa Dultz, Roberta Mancini, Guido Polles, et al.



    Selected by Carmen Adriaens, Gautam Dey

    HIF1-alpha expressing cells induce a hypoxic-like response in neighbouring cancer cells

    Hannah Harrison, Henry J Pegg, Jamie Thompson, et al.



    Selected by Anh Hoang Le

    Zebrafish as a model to investigate the effects of exercise in cancer

    Alexandra Yin, Nathaniel R. Campbell, Lee W. Jones, et al.



    Selected by Jacky G. Goetz

    A 3D model of human skeletal muscle innervated with stem cell-derived motor neurons enables epsilon-subunit targeted myasthenic syndrome studies

    Mohsen Afshar Bakooshli, Ethan S Lippmann, Ben Mulcahy, et al.



    Selected by Chris Demers

    Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

    Tsung-li Liu, Srigokul Upadhyayula, Daniel E Milkie, et al.



    Selected by Arnaud Monnard, Gautam Dey

    Also in the biophysics category:

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

    Che-Hang Yu, Stefanie Redemann, Hai-Yin Wu, et al.



    Selected by Federico Pelisch

    1

    Mechanical Stretch Kills Transformed Cancer Cells

    Ajay Tijore, Mingxi Yao, Yu-Hsiu Wang, et al.



    Selected by Joseph Jose Thottacherry

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Force inference predicts local and tissue-scale stress patterns in epithelia

    Weiyuan Kong, Olivier Loison, Pruthvi Chavadimane Shivakumar, et al.



    Selected by Sundar Naganathan

    Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision

    Simao Coelho, Jongho Baek, Matthew S Graus, et al.



    Selected by Lars Hubatsch

    Phase transition and amyloid formation by a viral protein as an additional molecular mechanism of virus-induced cell toxicity

    Edoardo Salladini, Claire Debarnot, Vincent Delauzun, et al.



    Selected by Tessa Sinnige

    Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision

    Simao Coelho, Jongho baek, Matthew S Graus, et al.



    Selected by Sam Barnett

    Applications, Promises, and Pitfalls of Deep Learning for Fluorescence Image Reconstruction

    Chinmay Belthangady , Loic A. Royer



    Selected by Romain F. Laine

    Imaging mechanical properties of sub-micron ECM in live zebrafish using Brillouin microscopy

    Carlo Bevilacqua, Héctor Sánchez Iranzo, Dmitry Richter, et al.



    Selected by Stephan Daetwyler

    1

    Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex

    Diego Gauto, Leandro Estrozi, Charles Schwieters, et al.



    Selected by Reid Alderson

    1

    Structure of a cytochrome-based bacterial nanowire

    David J Filman, Stephen F Marino, Joy E Ward, et al.



    Selected by Amberley Stephens

    Strong preference for autaptic self-connectivity of neocortical PV interneurons entrains them to γ-oscillations

    Charlotte Deleuze, Gary S Bhumbra, Antonio Pazienti, et al.



    Selected by Mahesh Karnani

    Retrieving High-Resolution Information from Disordered 2D Crystals by Single Particle Cryo-EM

    Ricardo Righetto, Nikhil Biyani, Julia Kowal, et al.



    Selected by David Wright

    Microtubules Gate Tau Condensation to Spatially Regulate Microtubule Functions

    Ruensern Tan, Aileen J. Lam, Tracy Tan, et al.

    AND

    Kinetically distinct phases of tau on microtubules regulate kinesin motors and severing enzymes

    Valerie Siahaan, Jochen Krattenmacher, Amayra Hernandez-Vega, et al.



    Selected by Satish Bodakuntla

    2

    The structural basis for release factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

    Ziao Fu, Gabriele Indrisiunaite, Sandip Kaledhonkar, et al.



    Selected by David Wright

    Also in the cell biology category:

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

    Che-Hang Yu, Stefanie Redemann, Hai-Yin Wu, et al.



    Selected by Federico Pelisch

    1

    Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

    Evgeny Zatulovskiy, Daniel F. Berenson, Benjamin R. Topacio, et al.



    Selected by Zaki Ahmad

    1

    Minimal membrane interactions conferred by Rheb C-terminal farnesylation are essential for mTORC1 activation

    Shawn M Ferguson, Brittany Angarola



    Selected by Sandra Malmgren Hill

    Mechanical Stretch Kills Transformed Cancer Cells

    Ajay Tijore, Mingxi Yao, Yu-Hsiu Wang, et al.



    Selected by Vibha SINGH

    EHD2-mediated restriction of caveolar dynamics regulates cellular lipid uptake

    Claudia Matthaeus, Ines Lahmann, Severine Kunz, et al.



    Selected by Andreas Müller

    1

    Mechanical Stretch Kills Transformed Cancer Cells

    Ajay Tijore, Mingxi Yao, Yu-Hsiu Wang, et al.



    Selected by Joseph Jose Thottacherry

    Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

    Jayashree Chadchankar, Victoria Korboukh, Peter Doig, et al.



    Selected by Mila Basic

    A metabolic switch from OXPHOS to glycolysis is essential for cardiomyocyte proliferation in the regenerating heart

    Hessel Honkoop, Dennis de Bakker, Alla Aharonov, et al.



    Selected by Andreas van Impel

    1

    S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate desmosome assembly and cell adhesion.

    Keith T Woodley, Mark O Collins



    Selected by Abagael Lasseigne

    3

    A complex containing lysine-acetylated actin inhibits the formin INF2

    Mu A, Tak Shun Fung, Arminja N. Kettenbach, et al.



    Selected by Laura McCormick

    1

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

    Mark E. Lush, Daniel C. Diaz, Nina Koenecke, et al.

    AND

    Distinct progenitor populations mediate regeneration in the zebrafish lateral line.

    Eric D Thomas, David Raible



    Selected by Rudra Nayan Das

    1

    Actomyosin-II facilitates long-range retrograde transport of large cargoes by controlling axonal radial contractility

    Tong Wang, Wei Li, Sally Martin, et al.



    Selected by Ivana Viktorinová

    Atlas of Subcellular RNA Localization Revealed by APEX-seq

    Furqan M Fazal, Shuo Han, Pornchai Kaewsapsak, et al.

    AND

    Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

    Alejandro Padron, Shintaro Iwasaki, Nicholas Ingolia



    Selected by Christian Bates

    Applications, Promises, and Pitfalls of Deep Learning for Fluorescence Image Reconstruction

    Chinmay Belthangady , Loic A. Royer



    Selected by Romain F. Laine

    Also in the molecular biology category:

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    Structures of the Otopetrin Proton Channels Otop1 and Otop3

    Kei Saotome, Bochuan Teng, Che Chun (Alex) Tsui, et al.



    Selected by David Wright

    Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

    Che-Hang Yu, Stefanie Redemann, Hai-Yin Wu, et al.



    Selected by Federico Pelisch

    1

    Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

    Evgeny Zatulovskiy, Daniel F. Berenson, Benjamin R. Topacio, et al.



    Selected by Zaki Ahmad

    1

    Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

    Philipp Denninger, Anna Reichelt, Vanessa Aphaia Fiona Schmidt, et al.



    Selected by Marc Somssich

    Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

    Jayashree Chadchankar, Victoria Korboukh, Peter Doig, et al.



    Selected by Mila Basic

    Bacteriophage resistance alters antibiotic mediated intestinal expansion of enterococci

    Anushila Chatterjee, Cydney N Johnson, Phat Luong, et al.



    Selected by Yasmin Lau

    On-site ribosome remodeling by locally synthesized ribosomal proteins in axons

    Toshiaki Shigeoka, Max Koppers, Hovy Ho-Wai Wong, et al.



    Selected by Srivats Venkataramanan

    MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

    Emily Yun-Chia Chang, James P Wells, Shu-Huei Tsai, et al.



    Selected by Katie Weiner

    1

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Atlas of Subcellular RNA Localization Revealed by APEX-seq

    Furqan M Fazal, Shuo Han, Pornchai Kaewsapsak, et al.

    AND

    Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

    Alejandro Padron, Shintaro Iwasaki, Nicholas Ingolia



    Selected by Christian Bates

    Unlimited genetic switches for cell-type specific manipulation

    Jorge Garcia-Marques, Ching-Po Yang, Isabel Espinosa-Medina, et al.



    Selected by Rafael Almeida

    1

    Stable knockout and complementation of receptor expression using in vitro cell line derived reticulocytes for dissection of host malaria invasion requirements

    Timothy J Satchwell, Katherine E Wright, Katy L Haydn-Smith, et al.



    Selected by Alyson Smith

    The coordination of terminal differentiation and cell cycle exit is mediated through the regulation of chromatin accessibility

    Yiqin Ma, Daniel J McKay, Laura Buttitta



    Selected by Gabriel Aughey

    1

    Disrupting Transcriptional Feedback Yields an Escape-Resistant Antiviral

    Sonali Chaturvedi, Marie Wolf, Noam Vardi, et al.



    Selected by Pavithran Ravindran

    1

    Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding

    Sanne Boersma, Deepak Khuperkar, Bram M.P. Verhagen, et al.



    Selected by Lorenzo Lafranchi

    Also in the systems biology category:

    Lineage tracing on transcriptional landscapes links state to fate during differentiation

    Caleb Weinreb, Alejo E Rodriguez-Fraticelli, Fernando D Camargo, et al.



    Selected by Yen-Chung Chen

    1

    Short-range interactions govern cellular dynamics in microbial multi-genotype systems

    Alma Dal Co, Simon van Vliet, Daniel Johannes Kiviet, et al.

    AND

    Rapid microbial interaction network inference in microfluidic droplets

    Ryan H Hsu, Ryan L Clark, Jin Wei Tan, et al.



    Selected by Connor Rosen

    High-throughput functional analysis of lncRNA core promoters elucidates rules governing tissue-specificity

    Kaia Mattioli, Pieter-Jan Volders, Chiara Gerhardinger, et al.



    Selected by Clarice Hong

    Variability of bacterial behavior in the mammalian gut captured using a growth-linked single-cell synthetic gene oscillator

    David T Riglar, David L Richmond, Laurent Potvin-Trottier, et al.



    Selected by Meng Zhu

    Charting a tissue from single-cell transcriptomes

    Mor Nitzan, Nikos Karaiskos, Nir Friedman, et al.



    Selected by Irepan Salvador-Martinez

    Large-scale analyses of human microbiomes reveal thousands of small, novel genes and their predicted functions

    Hila Sberro, Nicholas Greenfield, Georgios Pavlopoulos, et al.



    Selected by Ganesh Kadamur

    Symmetry breaking in the embryonic skin triggers a directional and sequential front of competence during plumage patterning

    Richard Bailleul, Carole Desmarquet-Trin Dinh, Magdalena Hidalgo, et al.



    Selected by Alexa Sadier

    RNase L reprograms translation by widespread mRNA turnover escaped by antiviral mRNAs

    James M Burke, Stephanie L Moon, Evan T Lester, et al.



    Selected by Connor Rosen

    Acquired interbacterial defense systems protect against interspecies antagonism in the human gut microbiome

    Benjamin D. Ross, Adrian J. Verster, Matthew C. Radey, et al.



    Selected by Connor Rosen

    DNA microscopy: Optics-free spatio-genetic imaging by a stand-alone chemical reaction

    Joshua A. Weinstein, Aviv Regev, Feng Zhang



    Selected by Theo Sanderson

    2

    The Toll pathway inhibits tissue growth and regulates cell fitness in an infection-dependent manner

    Federico Germani, Daniel Hain, Denise Sternlicht, et al.



    Selected by Rohan Khadilkar

    LCM-seq reveals unique transcriptional adaption mechanisms of resistant neurons in spinal muscular atrophy

    Susanne Nichterwitz, Helena Storvall, Jik Nijssen, et al.

    AND

    Axon-seq decodes the motor axon transcriptome and its modulation in response to ALS

    Jik Nijssen, Julio Cesar Aguila Benitez, Rein Hoogstraaten, et al.



    Selected by Yen-Chung Chen

    Memory sequencing reveals heritable single cell gene expression programs associated with distinct cellular behaviors

    Sydney M Shaffer, Benjamin L Emert, Ann E. Sizemore, et al.



    Selected by Leighton Daigh

    2

    Conserved phosphorylation hotspots in eukaryotic protein domain families

    Marta J Strumillo, Michaela Oplova, Cristina Vieitez, et al.



    Selected by Gautam Dey

    LADL: Light-activated dynamic looping for endogenous gene expression control

    Mayuri Rege, Ji Hun Kim, Jacqueline Valeri, et al.



    Selected by Ivan Candido-Ferreira

    A minimal "push-pull" bistability model explains oscillations between quiescent and proliferative cell states.

    Sandeep Krishna, Sunil Laxman



    Selected by Lauren Neves

    1

    Close