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Article ; Online: Expanding the family of genetically encoded voltage indicators with a candidate Heliorhodopsin exhibiting near-infrared fluorescence.

Ganapathy, Srividya / Meng, Xin / Mossel, Delizzia / Jagt, Mels / Brinks, Daan

2023 Volume 299, Issue 6, Page(s) 104771

Abstract: Genetically encoded voltage indicators, particularly those based on microbial rhodopsins, are gaining traction in neuroscience as fluorescent sensors for imaging voltage dynamics with high-spatiotemporal precision. Here we establish a novel genetically ... ...

Abstract	Genetically encoded voltage indicators, particularly those based on microbial rhodopsins, are gaining traction in neuroscience as fluorescent sensors for imaging voltage dynamics with high-spatiotemporal precision. Here we establish a novel genetically encoded voltage indicator candidate based on the recently discovered subfamily of the microbial rhodopsin clade, termed heliorhodopsins. We discovered that upon excitation at 530 to 560 nm, wildtype heliorhodopsin exhibits near-infrared fluorescence, which is sensitive to membrane voltage. We characterized the fluorescence brightness, photostability, voltage sensitivity, and kinetics of wildtype heliorhodopsin in HEK293T cells and further examined the impact of mutating key residues near the retinal chromophore. The S237A mutation significantly improved the fluorescence response of heliorhodopsin by 76% providing a highly promising starting point for further protein evolution.
MeSH term(s)	Humans ; Fluorescence ; HEK293 Cells ; Rhodopsins, Microbial/chemistry
Chemical Substances	heliorhodopsin ; Rhodopsins, Microbial
Language	English
Publishing date	2023-04-29
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1016/j.jbc.2023.104771
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Article ; Online: Voltage Imaging with Engineered Proton-Pumping Rhodopsins: Insights from the Proton Transfer Pathway.

Meng, Xin / Ganapathy, Srividya / van Roemburg, Lars / Post, Marco / Brinks, Daan

ACS physical chemistry Au

2023 Volume 3, Issue 4, Page(s) 320–333

Abstract: Voltage imaging using genetically encoded voltage indicators (GEVIs) has taken the field of neuroscience by storm in the past decade. Its ability to create subcellular and network level readouts of electrical dynamics depends critically on the kinetics ... ...

Abstract	Voltage imaging using genetically encoded voltage indicators (GEVIs) has taken the field of neuroscience by storm in the past decade. Its ability to create subcellular and network level readouts of electrical dynamics depends critically on the kinetics of the response to voltage of the indicator used. Engineered microbial rhodopsins form a GEVI subclass known for their high voltage sensitivity and fast response kinetics. Here we review the essential aspects of microbial rhodopsin photocycles that are critical to understanding the mechanisms of voltage sensitivity in these proteins and link them to insights from efforts to create faster, brighter and more sensitive microbial rhodopsin-based GEVIs.
Language	English
Publishing date	2023-05-03
Publishing country	United States
Document type	Journal Article ; Review
ISSN	2694-2445
ISSN (online)	2694-2445
DOI	10.1021/acsphyschemau.3c00003
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Article ; Online: Linearly polarized excitation enhances signals from fluorescent voltage indicators.

Bloxham, Blox / Brinks, Daan / Kheifets, Simon / Cohen, Adam E

Biophysical journal

2021 Volume 120, Issue 23, Page(s) 5333–5342

Abstract: Voltage imaging in cells requires high-speed recording of small fluorescent signals, often leading to low signal/noise ratios. Because voltage indicators are membrane bound, their orientations are partially constrained by the plane of the membrane. We ... ...

Abstract	Voltage imaging in cells requires high-speed recording of small fluorescent signals, often leading to low signal/noise ratios. Because voltage indicators are membrane bound, their orientations are partially constrained by the plane of the membrane. We explored whether tuning the linear polarization of excitation light could enhance voltage indicator fluorescence. We tested a panel of dye- and protein-based voltage indicators in mammalian cells. The dye BeRST1 showed a 73% increase in brightness between the least and most favorable polarizations. The protein-based reporter ASAP1 showed a 22% increase in brightness, and QuasAr3 showed a 14% increase in brightness. In very thin neurites expressing QuasAr3, improvements were anomalously large, with a 170% increase in brightness between polarization parallel versus perpendicular to the dendrite. Signal/noise ratios of optically recorded action potentials were increased by up to 50% in neurites expressing QuasAr3. These results demonstrate that polarization control can be a facile means to enhance signals from fluorescent voltage indicators, particularly in thin neurites or in high-background environments.
MeSH term(s)	Action Potentials ; Animals ; Coloring Agents ; Fluorescent Dyes ; Indicators and Reagents
Chemical Substances	Coloring Agents ; Fluorescent Dyes ; Indicators and Reagents
Language	English
Publishing date	2021-10-26
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2021.10.028
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Article ; Online: Waveguide-based total internal reflection fluorescence microscope enabling cellular imaging under cryogenic conditions.

Li, Qingru / Hulleman, Christiaan N / Moerland, Robert J / Mailvaganam, Elil / Ganapathy, Srividya / Brinks, Daan / Stallinga, Sjoerd / Rieger, Bernd

Optics express

2021 Volume 29, Issue 21, Page(s) 34097–34108

Abstract: Total internal reflection fluorescence (TIRF) microscopy is an important imaging tool for the investigation of biological structures, especially the study on cellular events near the plasma membrane. Imaging at cryogenic temperatures not only enables ... ...

Abstract	Total internal reflection fluorescence (TIRF) microscopy is an important imaging tool for the investigation of biological structures, especially the study on cellular events near the plasma membrane. Imaging at cryogenic temperatures not only enables observing structures in a near-native and fixed state but also suppresses irreversible photo-bleaching rates, resulting in increased photo-stability of fluorophores. Traditional TIRF microscopes produce an evanescent field based on high numerical aperture immersion objective lenses with high magnification, which results in a limited field of view and is incompatible with cryogenic conditions. Here, we present a waveguide-based TIRF microscope, which is able to generate a uniform evanescent field using high refractive index waveguides on photonic chips and to obtain cellular observation at cryogenic temperatures. Our method provides an inexpensive way to achieve total-internal-reflection fluorescence imaging under cryogenic conditions.
MeSH term(s)	Cell Membrane ; Equipment Design ; Fluorescent Dyes ; Freezing ; HEK293 Cells ; Humans ; Lenses ; Lighting ; Microscopy, Fluorescence/instrumentation ; Microscopy, Fluorescence/methods ; Photons ; Refractometry
Chemical Substances	Fluorescent Dyes
Language	English
Publishing date	2021-11-22
Publishing country	United States
Document type	Journal Article
ZDB-ID	1491859-6
ISSN	1094-4087 ; 1094-4087
ISSN (online)	1094-4087
ISSN	1094-4087
DOI	10.1364/OE.433945
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Article ; Online: Photoactivated voltage imaging in tissue with an archaerhodopsin-derived reporter.

Chien, Miao-Ping / Brinks, Daan / Testa-Silva, Guilherme / Tian, He / Phil Brooks, F / Adam, Yoav / Bloxham, Blox / Gmeiner, Benjamin / Kheifets, Simon / Cohen, Adam E

Science advances

2021 Volume 7, Issue 19

Abstract: Photoactivated genetically encoded voltage indicators (GEVIs) have the potential to enable optically sectioned voltage imaging at the intersection of a photoactivation beam and an imaging beam. We developed a pooled high-throughput screen to identify ... ...

Abstract	Photoactivated genetically encoded voltage indicators (GEVIs) have the potential to enable optically sectioned voltage imaging at the intersection of a photoactivation beam and an imaging beam. We developed a pooled high-throughput screen to identify archaerhodopsin mutants with enhanced photoactivation. After screening ~10
Language	English
Publishing date	2021-05-05
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ZDB-ID	2810933-8
ISSN	2375-2548 ; 2375-2548
ISSN (online)	2375-2548
ISSN	2375-2548
DOI	10.1126/sciadv.abe3216
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Article ; Online: Two-photon imaging of a magneto-fluorescent indicator for 3D optical magnetometry.

Lee, Hohjai / Brinks, Daan / Cohen, Adam E

Optics express

2015 Volume 23, Issue 21, Page(s) 28022–28030

Abstract: We developed an optical method to visualize the three-dimensional distribution of magnetic field strength around magnetic microstructures. We show that the two-photon-excited fluorescence of a chained donor-bridge-acceptor compound, phenanthrene-(CH2)12- ... ...

Abstract	We developed an optical method to visualize the three-dimensional distribution of magnetic field strength around magnetic microstructures. We show that the two-photon-excited fluorescence of a chained donor-bridge-acceptor compound, phenanthrene-(CH2)12-O-(CH2)2-N,N-dimethylaniline, is sensitive to ambient magnetic field strength. A test structure is immersed in a solution of the magneto-fluorescent indicator and a custom two-photon microscope maps the fluorescence of this compound. The decay kinetics of the electronic excited state provide a measure of magnetic field that is insensitive to photobleaching, indicator concentration, or local variations in optical excitation or collection efficiency.
Language	English
Publishing date	2015-10-19
Publishing country	United States
Document type	Journal Article
ZDB-ID	1491859-6
ISSN	1094-4087 ; 1094-4087
ISSN (online)	1094-4087
ISSN	1094-4087
DOI	10.1364/OE.23.028022
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Article ; Online: Two-Photon Lifetime Imaging of Voltage Indicating Proteins as a Probe of Absolute Membrane Voltage.

Brinks, Daan / Klein, Aaron J / Cohen, Adam E

Biophysical journal

2015 Volume 109, Issue 5, Page(s) 914–921

Abstract: Genetically encoded voltage indicators (GEVIs) can report cellular electrophysiology with high resolution in space and time. Two-photon (2P) fluorescence has been explored as a means to image voltage in tissue. Here, we used the 2P electronic excited- ... ...

Abstract	Genetically encoded voltage indicators (GEVIs) can report cellular electrophysiology with high resolution in space and time. Two-photon (2P) fluorescence has been explored as a means to image voltage in tissue. Here, we used the 2P electronic excited-state lifetime to probe absolute membrane voltage in a manner that is insensitive to the protein expression level, illumination intensity, or photon detection efficiency. First, we tested several GEVIs for 2P brightness, response speed, and voltage sensitivity. ASAP1 and a previously described citrine-Arch electrochromic Förster resonance energy transfer sensor (dubbed CAESR) showed the best characteristics. We then characterized the voltage-dependent lifetime of ASAP1, CAESR, and ArcLight under voltage-clamp conditions. ASAP1 and CAESR showed voltage-dependent lifetimes, whereas ArcLight did not. These results establish 2P fluorescence lifetime imaging as a viable means of measuring absolute membrane voltage. We discuss the prospects and improvements necessary for applications in tissue.
MeSH term(s)	Action Potentials ; HEK293 Cells ; Humans ; Membrane Potentials ; Membrane Proteins/metabolism ; Optical Imaging ; Photons
Chemical Substances	Membrane Proteins
Language	English
Publishing date	2015-09-01
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2015.07.038
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Zs.A 235: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Linking the genotypes and phenotypes of cancer cells in heterogenous populations via real-time optical tagging and image analysis.

You, Li / Su, Pin-Rui / Betjes, Max / Rad, Reza Ghadiri / Chou, Ting-Chun / Beerens, Cecile / van Oosten, Eva / Leufkens, Felix / Gasecka, Paulina / Muraro, Mauro / van Tol, Ruud / van Steenderen, Debby / Farooq, Shazia / Hardillo, Jose Angelito U / de Jong, Robert Baatenburg / Brinks, Daan / Chien, Miao-Ping

Nature biomedical engineering

2022 Volume 6, Issue 5, Page(s) 667–675

Abstract: Linking single-cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, could help unravel molecular mechanisms driving the growth of tumour-cell subpopulations. Here we show that a ... ...

Abstract	Linking single-cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, could help unravel molecular mechanisms driving the growth of tumour-cell subpopulations. Here we show that a custom-built optical microscope with an ultrawide field of view, fast automated image analysis and a dye activatable by visible light enables the screening and selective photolabelling of cells of interest in large heterogeneous cell populations on the basis of specific functional cellular dynamics, such as fast migration, morphological variation, small-molecule uptake or cell division. Combining such functional single-cell selection with single-cell RNA sequencing allowed us to (1) functionally annotate the transcriptomic profiles of fast-migrating and spindle-shaped MCF10A cells, of fast-migrating MDA-MB-231 cells and of patient-derived head-and-neck squamous carcinoma cells, and (2) identify critical genes and pathways driving aggressive migration and mesenchymal-like morphology in these cells. Functional single-cell selection upstream of single-cell sequencing does not depend on molecular biomarkers, allows for the enrichment of sparse subpopulations of cells, and can facilitate the identification and understanding of the molecular mechanisms underlying functional phenotypes.
MeSH term(s)	Genotype ; Humans ; Neoplasms ; Phenotype ; Transcriptome
Language	English
Publishing date	2022-03-17
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2157-846X
ISSN (online)	2157-846X
DOI	10.1038/s41551-022-00853-x
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Article ; Online: Painting with Rainbows: Patterning Light in Space, Time, and Wavelength for Multiphoton Optogenetic Sensing and Control.

Brinks, Daan / Adam, Yoav / Kheifets, Simon / Cohen, Adam E

Accounts of chemical research

2016 Volume 49, Issue 11, Page(s) 2518–2526

Abstract: Photons are a fascinating reagent, flowing and reacting quite differently compared to more massive and less ephemeral particles of matter. The optogenetic palette comprises an ever growing set of light-responsive proteins, which open the possibility of ... ...

Abstract	Photons are a fascinating reagent, flowing and reacting quite differently compared to more massive and less ephemeral particles of matter. The optogenetic palette comprises an ever growing set of light-responsive proteins, which open the possibility of using light to perturb and to measure biological processes with great precision in space and time. Yet there are limits on what light can achieve. Diffraction limits the smallest features, and scattering in tissue limits the largest. Photobleaching, diffusion of photogenerated products, and optical crosstalk between overlapping absorption spectra further muddy the optogenetic picture, particularly when one wants to use multiple optogenetic tools simultaneously. But these obstacles are surmountable. Most light-responsive proteins and small molecules undergo more than one light-driven transition, often with different action spectra and kinetics. By overlapping multiple laser beams, carefully patterned in space, time, and wavelength, one can steer molecules into fluorescent or nonfluorescent, active or inactive conformations. By doing so, one can often circumvent the limitations of simple one-photon excitation and achieve new imaging and stimulation capabilities. These include subdiffraction spatial resolution, optical sectioning, robustness to light scattering, and multiplexing of more channels than can be achieved with simple one-photon excitation. The microbial rhodopsins are a particularly rich substrate for this type of multiphoton optical control. The natural diversity of these proteins presents a huge range of starting materials. The spectroscopy and photocycles of microbial rhodopsins are relatively well understood, providing states with absorption maxima across the visible spectrum, which can be accessed on experimentally convenient time scales. A long history of mutational studies in microbial rhodopsins allows semirational protein engineering. Mutants of Archaerhodopsin 3 (Arch) come in all the colors of the rainbow. In a solution of purified Arch-eGFP, a focused green laser excites eGFP fluorescence throughout the laser path, while a focused red laser excites fluorescence of Arch only near the focus, indicative of multiphoton fluorescence. This nonlinearity occurs at a laser intensity ∼10
MeSH term(s)	Animals ; Fluorescence ; Optogenetics/methods ; Photons ; Rhodopsins, Microbial/chemistry ; Rhodopsins, Microbial/radiation effects
Chemical Substances	Rhodopsins, Microbial
Language	English
Publishing date	2016--15
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ZDB-ID	1483291-4
ISSN	1520-4898 ; 0001-4842
ISSN (online)	1520-4898
ISSN	0001-4842
DOI	10.1021/acs.accounts.6b00415
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Article: Coherent control of single molecules at room temperature.

Brinks, Daan / Hildner, Richard / Stefani, Fernando D / van Hulst, Niek F

Faraday discussions

2012 Volume 153, Page(s) 51–60; discussion 73–91

Abstract: The detection of individual molecules allows to unwrap the inhomogeneously broadened ensemble and reveal the spatial disorder and temporal dynamics of single entities. During 20 years of increasing sophistication this approach has provided valuable ... ...

Abstract	The detection of individual molecules allows to unwrap the inhomogeneously broadened ensemble and reveal the spatial disorder and temporal dynamics of single entities. During 20 years of increasing sophistication this approach has provided valuable insights into biomolecular interactions, cellular processes, polymer dynamics, etc. Unfortunately the detection of fluorescence, i.e. incoherent spontaneous emission, has essentially kept the time resolution of the single molecule approach out of the range of ultrafast coherent processes. In parallel coherent control of quantum interferences has developed as a powerful method to study and actively steer ultrafast molecular interactions and energy conversion processes. However the degree of coherent control that can be reached in ensembles is restricted, due to the intrinsic inhomogeneity of the synchronized subset. Clearly the only way to overcome spatio-temporal disorder and achieve key control is by addressing individual units: coherent control of single molecules. Here we report the observation and manipulation of vibrational wave-packet interference in individual molecules at ambient conditions. We show that adapting the time and phase distribution of the optical excitation field to the dynamics of each molecule results in a superior degree of control compared to the ensemble approach. Phase reversal does invert the molecular response, confirming the control of quantum coherence. Time-phase maps show a rich diversity in excited state dynamics between different, yet chemically identical, molecules. The presented approach is promising for single-unit coherent control in multichromophoric systems. Especially the role of coherence in the energy transfer of single antenna complexes under physiological conditions is subject of great attention. Now the role of energy disorder and variation in coupling strength can be explored, beyond the inhomogeneously broadened ensemble.
MeSH term(s)	Organic Chemicals/chemistry ; Quantum Theory ; Temperature ; Vibration
Chemical Substances	Organic Chemicals
Language	English
Publishing date	2012-01-24
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1359-6640
ISSN	1359-6640
DOI	10.1039/c1fd00087j
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