Winfried Denk - Academia.edu (original) (raw)

Papers by Winfried Denk

eLife

We present an approach to study macromolecular assemblies by detecting component proteins’ charac... more We present an approach to study macromolecular assemblies by detecting component proteins’ characteristic high-resolution projection patterns, calculated from their known 3D structures, in single electron cryo-micrographs. Our method detects single apoferritin molecules in vitreous ice with high specificity and determines their orientation and location precisely. Simulations show that high spatial-frequency information and—in the presence of protein background—a whitening filter are essential for optimal detection, in particular for images taken far from focus. Experimentally, we could detect small viral RNA polymerase molecules, distributed randomly among binding locations, inside rotavirus particles. Based on the currently attainable image quality, we estimate a threshold for detection that is 150 kDa in ice and 300 kDa in 100 nm thick samples of dense biological material.

Nature Reviews Neuroscience, 2016

Optics Photonics News, Sep 1, 1997

Journal of Applied Physics, Aug 14, 1999

Appl Phys Lett, 1984

Optical stethoscopy: Image recording with resolution Л/20 DW Pohl, W. Denk,a) and M. Lanz IBM Zur... more Optical stethoscopy: Image recording with resolution Л/20 DW Pohl, W. Denk,a) and M. Lanz IBM Zurich Research Laboratory, 8803 Riischlikon, Switzerland (Received 28 November 1983; accepted for publication 12 January 1984) Subwave length-resolution optical ...

Proceedings of the National Academy of Sciences of the United States of America, Feb 16, 1999

Photochemical release (uncaging) of bioactive messengers with three-dimensional spatial resolutio... more Photochemical release (uncaging) of bioactive messengers with three-dimensional spatial resolution in light-scattering media would be greatly facilitated if the photolysis could be powered by pairs of IR photons rather than the customary single UV photons. The quadratic dependence on light intensity would confine the photolysis to the focus point of the laser, and the longer wavelengths would be much less affected by scattering. However, previous caged messengers have had very small cross sections for two-photon excitation in the IR region. We now show that brominated 7-hydroxycoumarin-4-ylmethyl esters and carbamates efficiently release carboxylates and amines on photolysis, with one- and two-photon cross sections up to one or two orders of magnitude better than previously available. These advantages are demonstrated on neurons in brain slices from rat cortex and hippocampus excited by glutamate uncaged from N-(6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl)-l-glutamate (Bhc-glu). Conventional UV photolysis of Bhc-glu requires less than one-fifth the intensities needed by one of the best previous caged glutamates, γ-(α-carboxy-2-nitrobenzyl)-l-glutamate (CNB-glu). Two-photon photolysis with raster-scanned femtosecond IR pulses gives the first three-dimensionally resolved maps of the glutamate sensitivity of neurons in intact slices. Bhc-glu and analogs should allow more efficient and three-dimensionally localized uncaging and photocleavage, not only in cell biology and neurobiology but also in many technological applications.

Spie Milestone Series, 2003

Characterization of the diffusional and electrotonic coupling of spines to the dendritic shaft is... more Characterization of the diffusional and electrotonic coupling of spines to the dendritic shaft is crucial to understanding neuronal integration and synaptic plasticity. Two-photon photobleaching and photorelease of fluorescein dextran were used to generate concentration gradients between spines and shafts in rat CA1 pyramidal neurons. Diffusional reequilibration was monitored with two-photon fluorescence imaging. The time course of reequilibration was exponential, with time constants in the range of 20 to 100 milliseconds, demonstrating chemical compartmentalization on such time scales. These values imply that electrical spine neck resistances are unlikely to exceed 150 megohms and more likely range from 4 to 50 megohms.

Journal of Neuroscience Methods, Mar 31, 1997

It is shown that the light intensity from a mercury short arc light bulb can be boosted to about ... more It is shown that the light intensity from a mercury short arc light bulb can be boosted to about 100 times its steady-state value for a period of about 1 ms by superimposing a current pulse of up to 100 amperes in magnitude and 1-2 ms in duration on a simmer current of 3 amperes. The output spectrum changes in a remarkable way from a line to a broadband distribution. The radiance delivered during a 1-ms pulse is comparable to what can be obtained from a Xe flash bulb. The lack of a high voltage ignition pulse accounts for an extreme reduction of the electrical artifact seen for the pulsed Hg bulb as compared to a Xe flash lamp. Possible applications include the release of caged compounds, high speed imaging, and wavelength ratio-imaging without filter switching.

A laser was designed and constructed with the goal of producing ultra-short pulses at a high repe... more A laser was designed and constructed with the goal of producing ultra-short pulses at a high repetition rate as needed for certain applications of multi-photon microscopy. With pure prism-based dispersion compensation repetition rates of up to 270MHz were achieved. The laser operates with hard-and soft-aperturing at the third (diverging output) and the first (parallel output) stability limits, respectively. At the third stability limit we found a pulse width of 27fs (FWHM) at 800nm central wavelength. At the first stability limit pulse widths of 23-40fs with tunability from 780nm to 920nm were reached.

Nonlinear Optics 98 Materials Fundamentals and Applications Topical Meeting, 1998

Spie Milestone Series, 2003

eLife

We present an approach to study macromolecular assemblies by detecting component proteins’ charac... more We present an approach to study macromolecular assemblies by detecting component proteins’ characteristic high-resolution projection patterns, calculated from their known 3D structures, in single electron cryo-micrographs. Our method detects single apoferritin molecules in vitreous ice with high specificity and determines their orientation and location precisely. Simulations show that high spatial-frequency information and—in the presence of protein background—a whitening filter are essential for optimal detection, in particular for images taken far from focus. Experimentally, we could detect small viral RNA polymerase molecules, distributed randomly among binding locations, inside rotavirus particles. Based on the currently attainable image quality, we estimate a threshold for detection that is 150 kDa in ice and 300 kDa in 100 nm thick samples of dense biological material.

Nature Reviews Neuroscience, 2016

Optics Photonics News, Sep 1, 1997

Journal of Applied Physics, Aug 14, 1999

Appl Phys Lett, 1984

Optical stethoscopy: Image recording with resolution Л/20 DW Pohl, W. Denk,a) and M. Lanz IBM Zur... more Optical stethoscopy: Image recording with resolution Л/20 DW Pohl, W. Denk,a) and M. Lanz IBM Zurich Research Laboratory, 8803 Riischlikon, Switzerland (Received 28 November 1983; accepted for publication 12 January 1984) Subwave length-resolution optical ...

Proceedings of the National Academy of Sciences of the United States of America, Feb 16, 1999

Photochemical release (uncaging) of bioactive messengers with three-dimensional spatial resolutio... more Photochemical release (uncaging) of bioactive messengers with three-dimensional spatial resolution in light-scattering media would be greatly facilitated if the photolysis could be powered by pairs of IR photons rather than the customary single UV photons. The quadratic dependence on light intensity would confine the photolysis to the focus point of the laser, and the longer wavelengths would be much less affected by scattering. However, previous caged messengers have had very small cross sections for two-photon excitation in the IR region. We now show that brominated 7-hydroxycoumarin-4-ylmethyl esters and carbamates efficiently release carboxylates and amines on photolysis, with one- and two-photon cross sections up to one or two orders of magnitude better than previously available. These advantages are demonstrated on neurons in brain slices from rat cortex and hippocampus excited by glutamate uncaged from N-(6-bromo-7-hydroxycoumarin-4-ylmethoxycarbonyl)-l-glutamate (Bhc-glu). Conventional UV photolysis of Bhc-glu requires less than one-fifth the intensities needed by one of the best previous caged glutamates, γ-(α-carboxy-2-nitrobenzyl)-l-glutamate (CNB-glu). Two-photon photolysis with raster-scanned femtosecond IR pulses gives the first three-dimensionally resolved maps of the glutamate sensitivity of neurons in intact slices. Bhc-glu and analogs should allow more efficient and three-dimensionally localized uncaging and photocleavage, not only in cell biology and neurobiology but also in many technological applications.

Spie Milestone Series, 2003

Characterization of the diffusional and electrotonic coupling of spines to the dendritic shaft is... more Characterization of the diffusional and electrotonic coupling of spines to the dendritic shaft is crucial to understanding neuronal integration and synaptic plasticity. Two-photon photobleaching and photorelease of fluorescein dextran were used to generate concentration gradients between spines and shafts in rat CA1 pyramidal neurons. Diffusional reequilibration was monitored with two-photon fluorescence imaging. The time course of reequilibration was exponential, with time constants in the range of 20 to 100 milliseconds, demonstrating chemical compartmentalization on such time scales. These values imply that electrical spine neck resistances are unlikely to exceed 150 megohms and more likely range from 4 to 50 megohms.

Journal of Neuroscience Methods, Mar 31, 1997

It is shown that the light intensity from a mercury short arc light bulb can be boosted to about ... more It is shown that the light intensity from a mercury short arc light bulb can be boosted to about 100 times its steady-state value for a period of about 1 ms by superimposing a current pulse of up to 100 amperes in magnitude and 1-2 ms in duration on a simmer current of 3 amperes. The output spectrum changes in a remarkable way from a line to a broadband distribution. The radiance delivered during a 1-ms pulse is comparable to what can be obtained from a Xe flash bulb. The lack of a high voltage ignition pulse accounts for an extreme reduction of the electrical artifact seen for the pulsed Hg bulb as compared to a Xe flash lamp. Possible applications include the release of caged compounds, high speed imaging, and wavelength ratio-imaging without filter switching.

A laser was designed and constructed with the goal of producing ultra-short pulses at a high repe... more A laser was designed and constructed with the goal of producing ultra-short pulses at a high repetition rate as needed for certain applications of multi-photon microscopy. With pure prism-based dispersion compensation repetition rates of up to 270MHz were achieved. The laser operates with hard-and soft-aperturing at the third (diverging output) and the first (parallel output) stability limits, respectively. At the third stability limit we found a pulse width of 27fs (FWHM) at 800nm central wavelength. At the first stability limit pulse widths of 23-40fs with tunability from 780nm to 920nm were reached.

Nonlinear Optics 98 Materials Fundamentals and Applications Topical Meeting, 1998

Spie Milestone Series, 2003