Winfried Denk

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Winfried Denk
Born (1957-11-12) November 12, 1957 (age 66)
Munich, Germany
Nationality German
OccupationPhysicist
Known for Serial block-face scanning electron microscopy
Two-photon excitation microscopy
Awards Michael and Kate Bárány Award (1998)
Leibniz Prize (2003)
W. Alden Spencer Award (2006)
Kavli Prize (2012)
Rosenstiel Award (2013)
The Brain Prize (2015)
Academic background
Academic advisors Watt W. Webb

Winfried Denk (born November 12, 1957, in Munich) is a German physicist. He built the first two-photon microscope while he was a graduate student (and briefly a postdoc) in Watt W. Webb's lab at Cornell University, in 1989.

Contents

Early life and education

Denk was born in Munich, Germany. As a child he spent most of his playtime learning to use the tools and building materials in his father's workshop. In school it became apparent that Denk’s ‘talents were unevenly spread across subjects, math and physics being favored’. [1] Fixing and constructing electronic devices was his main hobby throughout high school.

After high school, Denk completed the mandatory 15-month stint in the German army and spent the next 3 years at the Ludwig Maximilian University of Munich. In 1981 he moved to Zurich to study at the ETH. During this time, he also worked in the lab of Dieter Pohl, at the IBM laboratory. There he built one of the first super-resolution microscopes and developed a passion for scanning microscopy. He did his master's thesis in the lab of Kurt Wüthrich, under the direct guidance of Gerhard Wagner. But he felt that NMR spectroscopy was not for him because it did not involve enough opportunities to create new experimental gadgets.

In 1984 Denk joined the lab of Watt W. Webb at Cornell. While Webb himself was extremely interested in methods – both fluorescence-correlation and photo bleaching-recovery spectroscopy had been invented in his lab – he gave students and postdocs a lot of freedom. Denk enjoyed his time at Cornell but was almost fired after he went to Greece for six weeks to study monk seals. Given a second chance, he started a project aimed at measuring the motion of sensory hair-bundles in the inner ear. One of the attractions of this endeavor was that it required a stay in San Francisco, in order to learn from Jim Hudspeth and his group about hair-cells in general and specifically how to prepare them for the planned measurements.

Denk returned to Cornell and invented a method sensitive enough to measure the thermal movement of hair-bundles. He went on to show that hair cells can sense their own Brownian motion. [2]

Central to Denk's early career was his intuition that two-photon (2p) imaging might damage the sample less than one-photon confocal imaging. [3] He predicted this in spite of the fact that peak light intensity for 2p is almost one million times higher than for the confocal microscope. Equally important was his insight that infrared 2p excitation would allow scattered fluorescence to contribute to images even deep in turbid samples, improving the optical access and resolution of 2p imaging over what was possible using confocal imaging. [4]

Nowhere has this proven more valuable than when imaging neurons in living brain tissue. Two-photon microscopy remains the only technique that allows the recording of activity in living brains with high spatial resolution. 2p excitation can also be used to map cells' receptor distributions by releasing substances from their chemical "cages". [4]

Denk later demonstrated that 2p can be utilized to record activity in the visually stimulated retina. [5] He also showed that it can be combined with adaptive optics to improve resolution, and with amplified pulses to push the depth limit to 1mm in brain tissue. [6] [7] Today, two-photon excitation microscopy is also used in the fields of physiology, embryology and tissue engineering, as well as in cancer research.

The sparsity of data on connectivity between neurons had been a major limitation in circuit neuroscience. Denk’s 2004 paper [8] describing automated serial blockface microscopy rekindled the dormant science of comprehensive neural circuit mapping (connectomics), pioneered by Sydney Brenner. [9]

Denk, now Director at the Max Planck Institute for Biological Intelligence (formerly Max Planck Institute of Neurobiology [10] ), continues working to improve techniques for circuit-mapping in the rodent brain. [11] His most recent work involves precisely determining the positions, orientations, and identities of proteins and bound ligands in cryo-preserved cells. [12] [13]

Notable papers

Denk, Stricker & Webb1990, Science. Two-photon laser scanning fluorescence microscopy [3]

Denk 1994, Proc Natl Acad Sci USA. Two-photon scanning photochemical microscopy: mapping ligand-gated ion channel distribution [4]

Yuste & Denk 1995, Nature. Dendritic spines as basic functional units of neuronal integration [14]

Svoboda, Tank & Denk 1996, Science. Direct measurement of coupling between dendritic spines and shafts. [15]

Euler, Detwiler & Denk 2002, Nature. Directionally selective calcium signals in dendrites of starburst amacrine cells. [16]

Denk & Horstmann 2004, PLoS Biology. Serial Block-Face Scanning Electron Microscopy to Reconstruct Three-Dimensional Tissue Nanostructure [8]

Helmchen & Denk 2005, Nature Methods. Deep tissue two-photon microscopy [7]

Briggman et al. 2011, Nature. Wiring specificity in the direction-selectivity circuit of the retina [17]

Helmstaedter et al. 2013, Nature. Connectomic reconstruction of the inner plexiform layer in the mouse retina [18]

Recognition

Service

Related Research Articles

<span class="mw-page-title-main">Microscopy</span> Viewing of objects which are too small to be seen with the naked eye

Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.

<span class="mw-page-title-main">Dendritic spine</span> Small protrusion on a dendrite that receives input from a single axon

A dendritic spine is a small membranous protrusion from a neuron's dendrite that typically receives input from a single axon at the synapse. Dendritic spines serve as a storage site for synaptic strength and help transmit electrical signals to the neuron's cell body. Most spines have a bulbous head, and a thin neck that connects the head of the spine to the shaft of the dendrite. The dendrites of a single neuron can contain hundreds to thousands of spines. In addition to spines providing an anatomical substrate for memory storage and synaptic transmission, they may also serve to increase the number of possible contacts between neurons. It has also been suggested that changes in the activity of neurons have a positive effect on spine morphology.

<span class="mw-page-title-main">Motion perception</span> Inferring the speed and direction of objects

Motion perception is the process of inferring the speed and direction of elements in a scene based on visual, vestibular and proprioceptive inputs. Although this process appears straightforward to most observers, it has proven to be a difficult problem from a computational perspective, and difficult to explain in terms of neural processing.

<span class="mw-page-title-main">Two-photon excitation microscopy</span> Fluorescence imaging technique

Two-photon excitation microscopy is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness. Unlike traditional fluorescence microscopy, where the excitation wavelength is shorter than the emission wavelength, two-photon excitation requires simultaneous excitation by two photons with longer wavelength than the emitted light. The laser is focused onto a specific location in the tissue and scanned across the sample to sequentially produce the image. Due to the non-linearity of two-photon excitation, mainly fluorophores in the micrometer-sized focus of the laser beam are excited, which results in the spatial resolution of the image. This contrasts with confocal microscopy, where the spatial resolution is produced by the interaction of excitation focus and the confined detection with a pinhole.

Brain mapping is a set of neuroscience techniques predicated on the mapping of (biological) quantities or properties onto spatial representations of the brain resulting in maps.

<span class="mw-page-title-main">Pasko Rakic</span> Yugoslav-born American neuroscientist (born 1933)

Pasko Rakic is a Yugoslav-born American neuroscientist, who presently works in the Yale School of Medicine Department of Neuroscience in New Haven, Connecticut. His main research interest is in the development and evolution of the human brain. He was the founder and served as Chairman of the Department of Neurobiology at Yale, and was founder and Director of the Kavli Institute for Neuroscience. He is best known for elucidating the mechanisms involved in development and evolution of the cerebral cortex. In 2008, Rakic shared the inaugural Kavli Prize in Neuroscience. He is currently the Dorys McConell Duberg Professor of Neuroscience, leads an active research laboratory, and serves on Advisory Boards and Scientific Councils of a number of Institutions and Research Foundations.


Watt Wetmore Webb was an American biophysicist, known for his co-invention of multiphoton microscopy in 1990.

<span class="mw-page-title-main">Müller glia</span> Glial cell type in the retina

Müller glia, or Müller cells, are a type of retinal glial cells, first recognized and described by Heinrich Müller. They are found in the vertebrate retina, where they serve as support cells for the neurons, as all glial cells do. They are the most common type of glial cell found in the retina. While their cell bodies are located in the inner nuclear layer of the retina, they span across the entire retina.

Neurophysics is the branch of biophysics dealing with the development and use of physical methods to gain information about the nervous system. Neurophysics is an interdisciplinary science using physics and combining it with other neurosciences to better understand neural processes. The methods used include the techniques of experimental biophysics and other physical measurements such as EEG mostly to study electrical, mechanical or fluidic properties, as well as theoretical and computational approaches. The term "neurophysics" is a portmanteau of "neuron" and "physics".

<span class="mw-page-title-main">Connectome</span> Comprehensive map of neural connections in the brain

A connectome is a comprehensive map of neural connections in the brain, and may be thought of as its "wiring diagram". An organism's nervous system is made up of neurons which communicate through synapses. A connectome is constructed by tracing the neuron in a nervous system and mapping where neurons are connected through synapses.

Connectomics is the production and study of connectomes: comprehensive maps of connections within an organism's nervous system. More generally, it can be thought of as the study of neuronal wiring diagrams with a focus on how structural connectivity, individual synapses, cellular morphology, and cellular ultrastructure contribute to the make up of a network. The nervous system is a network made of billions of connections and these connections are responsible for our thoughts, emotions, actions, memories, function and dysfunction. Therefore, the study of connectomics aims to advance our understanding of mental health and cognition by understanding how cells in the nervous system are connected and communicate. Because these structures are extremely complex, methods within this field use a high-throughput application of functional and structural neural imaging, most commonly magnetic resonance imaging (MRI), electron microscopy, and histological techniques in order to increase the speed, efficiency, and resolution of these nervous system maps. To date, tens of large scale datasets have been collected spanning the nervous system including the various areas of cortex, cerebellum, the retina, the peripheral nervous system and neuromuscular junctions.

Serial block-face scanning electron microscopy is a method to generate high resolution three-dimensional images from small samples. The technique was developed for brain tissue, but it is widely applicable for any biological samples. A serial block-face scanning electron microscope consists of an ultramicrotome mounted inside the vacuum chamber of a scanning electron microscope. Samples are prepared by methods similar to that in transmission electron microscopy (TEM), typically by fixing the sample with aldehyde, staining with heavy metals such as osmium and uranium then embedding in an epoxy resin. The surface of the block of resin-embedded sample is imaged by detection of back-scattered electrons. Following imaging the ultramicrotome is used to cut a thin section from the face of the block. After the section is cut, the sample block is raised back to the focal plane and imaged again. This sequence of sample imaging, section cutting and block raising can acquire many thousands of images in perfect alignment in an automated fashion. Practical serial block-face scanning electron microscopy was invented in 2004 by Winfried Denk at the Max-Planck-Institute in Heidelberg and is commercially available from Gatan Inc., Thermo Fisher Scientific (VolumeScope) and ConnectomX.

<i>Eyewire</i> Human-based computation game

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Neuronal tracing, or neuron reconstruction is a technique used in neuroscience to determine the pathway of the neurites or neuronal processes, the axons and dendrites, of a neuron. From a sample preparation point of view, it may refer to some of the following as well as other genetic neuron labeling techniques,

Calcium imaging is a microscopy technique to optically measure the calcium (Ca2+) status of an isolated cell, tissue or medium. Calcium imaging takes advantage of calcium indicators, fluorescent molecules that respond to the binding of Ca2+ ions by fluorescence properties. Two main classes of calcium indicators exist: chemical indicators and genetically encoded calcium indicators (GECI). This technique has allowed studies of calcium signalling in a wide variety of cell types. In neurons, action potential generation is always accompanied by rapid influx of Ca2+ ions. Thus, calcium imaging can be used to monitor the electrical activity in hundreds of neurons in cell culture or in living animals, which has made it possible observe the activity of neuronal circuits during ongoing behavior.

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Three-photon microscopy (3PEF) is a high-resolution fluorescence microscopy based on nonlinear excitation effect. Different from two-photon excitation microscopy, it uses three exciting photons. It typically uses 1300 nm or longer wavelength lasers to excite the fluorescent dyes with three simultaneously absorbed photons. The fluorescent dyes then emit one photon whose energy is three times the energy of each incident photon. Compared to two-photon microscopy, three-photon microscopy reduces the fluorescence away from the focal plane by , which is much faster than that of two-photon microscopy by . In addition, three-photon microscopy employs near-infrared light with less tissue scattering effect. This causes three-photon microscopy to have higher resolution than conventional microscopy.

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Marla Beth Feller is the Paul Licht Distinguished Professor in Biological Sciences and Member of the Helen Wills Neuroscience Institute at the University of California, Berkeley. She studies the mechanisms that underpin the assembly of neural circuits during development. Feller is a Fellow of the American Association for the Advancement of Science, member of the American Academy of Arts and Sciences and member of the National Academy of Sciences.

References

  1. ↑ "Kavli biosketch". The Kavli Prize. 20 March 2014. Retrieved 2020-06-27.
  2. ↑ Denk, W.; Webb, W. W.; Hudspeth, A. J. (1989-07-01). "Mechanical properties of sensory hair bundles are reflected in their Brownian motion measured with a laser differential interferometer". Proceedings of the National Academy of Sciences. 86 (14): 5371–5375. Bibcode:1989PNAS...86.5371D. doi: 10.1073/pnas.86.14.5371 . ISSN   0027-8424. PMC   297624 . PMID   2787510.
  3. 1 2 Denk, W; Strickler, J.; Webb, W. (1990-04-06). "Two-photon laser scanning fluorescence microscopy". Science. 248 (4951): 73–76. Bibcode:1990Sci...248...73D. doi:10.1126/science.2321027. ISSN   0036-8075. PMID   2321027.
  4. 1 2 3 Denk, W. (1994-07-05). "Two-photon scanning photochemical microscopy: mapping ligand-gated ion channel distributions". Proceedings of the National Academy of Sciences. 91 (14): 6629–6633. Bibcode:1994PNAS...91.6629D. doi: 10.1073/pnas.91.14.6629 . ISSN   0027-8424. PMC   44256 . PMID   7517555.
  5. ↑ Denk, W.; Detwiler, P. B. (1999-06-08). "Optical recording of light-evoked calcium signals in the functionally intact retina". Proceedings of the National Academy of Sciences. 96 (12): 7035–7040. Bibcode:1999PNAS...96.7035D. doi: 10.1073/pnas.96.12.7035 . ISSN   0027-8424. PMC   22046 . PMID   10359834.
  6. ↑ Rueckel, M.; Mack-Bucher, J. A.; Denk, W. (2006-11-14). "Adaptive wavefront correction in two-photon microscopy using coherence-gated wavefront sensing". Proceedings of the National Academy of Sciences. 103 (46): 17137–17142. Bibcode:2006PNAS..10317137R. doi: 10.1073/pnas.0604791103 . ISSN   0027-8424. PMC   1634839 . PMID   17088565.
  7. 1 2 Helmchen, Fritjof; Denk, Winfried (2005). "Deep tissue two-photon microscopy". Nature Methods. 2 (12): 932–940. doi:10.1038/nmeth818. ISSN   1548-7091. PMID   16299478. S2CID   3339971.
  8. 1 2 Denk, Winfried; Horstmann, Heinz (2004-10-19). "Serial Block-Face Scanning Electron Microscopy to Reconstruct Three-Dimensional Tissue Nanostructure". PLOS Biology. 2 (11): e329. doi: 10.1371/journal.pbio.0020329 . ISSN   1545-7885. PMC   524270 . PMID   15514700.
  9. ↑ White, J. G.; Southgate, E.; Thomson, J. N.; Brenner, S. (1986-11-12). "The structure of the nervous system of the nematode Caenorhabditis elegans". Philosophical Transactions of the Royal Society of London. B, Biological Sciences. 314 (1165): 1–340. Bibcode:1986RSPTB.314....1W. doi:10.1098/rstb.1986.0056. ISSN   0080-4622. PMID   22462104.
  10. ↑ "New Max Planck Institute for Biological Intelligence, in foundation" . Retrieved 2022-04-20.
  11. ↑ Mikula, Shawn; Denk, Winfried (2015). "High-resolution whole-brain staining for electron microscopic circuit reconstruction". Nature Methods. 12 (6): 541–546. doi:10.1038/nmeth.3361. ISSN   1548-7091. PMID   25867849. S2CID   8524835.
  12. ↑ Rickgauer, J. Peter; Choi, Heejun; Lippincott-Schwartz, Jennifer; Denk, Winfried (2020-04-24). "Label-free single-instance protein detection in vitrified cells". doi:10.1101/2020.04.22.053868. S2CID   218467026 . Retrieved 2020-10-10.{{cite journal}}: Cite journal requires |journal= (help)
  13. ↑ Rickgauer, J Peter; Grigorieff, Nikolaus; Denk, Winfried (2017-05-03). "Single-protein detection in crowded molecular environments in cryo-EM images". eLife. 6: e25648. doi: 10.7554/eLife.25648 . ISSN   2050-084X. PMC   5453696 . PMID   28467302.
  14. ↑ Yuste, Rafael; Denk, Winfried (1995). "Dendritic spines as basic functional units of neuronal integration". Nature. 375 (6533): 682–684. Bibcode:1995Natur.375..682Y. doi:10.1038/375682a0. ISSN   0028-0836. PMID   7791901. S2CID   4271356.
  15. ↑ Svoboda, K.; Tank, D. W.; Denk, W. (1996-05-03). "Direct Measurement of Coupling Between Dendritic Spines and Shafts". Science. 272 (5262): 716–719. Bibcode:1996Sci...272..716S. doi:10.1126/science.272.5262.716. ISSN   0036-8075. PMID   8614831. S2CID   23080041.
  16. ↑ Euler, Thomas; Detwiler, Peter B.; Denk, Winfried (2002). "Directionally selective calcium signals in dendrites of starburst amacrine cells". Nature. 418 (6900): 845–852. Bibcode:2002Natur.418..845E. doi:10.1038/nature00931. ISSN   0028-0836. PMID   12192402. S2CID   1879454.
  17. ↑ Briggman, Kevin L.; Helmstaedter, Moritz; Denk, Winfried (2011). "Wiring specificity in the direction-selectivity circuit of the retina". Nature. 471 (7337): 183–188. Bibcode:2011Natur.471..183B. doi:10.1038/nature09818. ISSN   0028-0836. PMID   21390125. S2CID   4425160.
  18. ↑ Helmstaedter, Moritz; Briggman, Kevin L.; Turaga, Srinivas C.; Jain, Viren; Seung, H. Sebastian; Denk, Winfried (2013-08-07). "Connectomic reconstruction of the inner plexiform layer in the mouse retina". Nature. 500 (7461): 168–174. Bibcode:2013Natur.500..168H. doi:10.1038/nature12346. ISSN   0028-0836. PMID   23925239. S2CID   3119909.
  19. ↑ "Prizes awarded by the Optoelectronics Fund". The Rank Prize Funds. Retrieved 2020-06-27.
  20. ↑ "2012 Kavli Prize Laureates in Neuroscience". The Kavli Foundation. 2012-05-31. Retrieved 2017-07-21.
  21. ↑ "Winfried Denk".
  22. ↑ "Rosenstiel Award Past Winners". Rosenstiel Basic Medical Sciences Research Center. Retrieved 2020-06-27.
  23. ↑ "The Brain Prize – Prize Winners 2015". The Lundbeck Foundation, The Brain Prize. Retrieved 2017-07-21.
  24. ↑ "International Prize for Translational Neuroscience". Gertrud Reemtsma Foundation. Retrieved 2017-07-21.
  25. ↑ "List of Members: Winfried Denk". Leopoldina. Retrieved 2020-06-27.
  26. ↑ "Prof. Dr. Winfried Denk". Bayerische Akademie der Wissenschaften. Retrieved 2020-06-27.
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