Linearly polarized excitation enhances signals from fluorescent voltage indicators

Genetically encoded voltage indicators (GEVIs) hold great promise for monitoring neuronal population activity, but GEVI imaging in dense neuronal populations remains difficult due to a lack of contrast and/or speed. To address this challenge, we developed a novel confocal microscope that allows simultaneous multiplane imaging with high-contrast at near-kHz rates. This approach enables high signal-to-noise ratio voltage imaging in densely labeled populations and minimizes optical crosstalk during concurrent optogenetic photostimulation.

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Review on Complete Mueller Matrix Optical Scanning Microscopy Imaging

Applied Sciences ◽

10.3390/app11041632 ◽

2021 ◽

Vol 11 (4) ◽

pp. 1632

Author(s):

Aymeric Le Gratiet ◽

Ali Mohebi ◽

Fabio Callegari ◽

Paolo Bianchini ◽

Alberto Diaspro

Keyword(s):

High Speed ◽

Polarized Light ◽

Mueller Matrix ◽

Scanning Microscopy ◽

Label Free ◽

Microscopy Imaging ◽

Excitation Light ◽

Optical Scanning ◽

Polarization Control ◽

Main Challenge

Optical scanning microscopy techniques based on the polarization control of the light have the capability of providing non invasive label-free contrast. By comparing the polarization states of the excitation light with its transformation after interaction with the sample, the full optical properties can be summarized in a single 4×4 Mueller matrix. The main challenge of such a technique is to encode and decode the polarized light in an optimal way pixel-by-pixel and take into account the polarimetric artifacts from the optical devices composing the instrument in a rigorous calibration step. In this review, we describe the different approaches for implementing such a technique into an optical scanning microscope, that requires a high speed rate polarization control. Thus, we explore the recent advances in term of technology from the industrial to the medical applications.

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Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163800 ◽

1996 ◽

Vol 54 ◽

pp. 268-269

Author(s):

W.F. Marshall ◽

K. Oegema ◽

J. Nunnari ◽

A.F. Straight ◽

D.A. Agard ◽

...

Keyword(s):

Confocal Microscopy ◽

High Speed ◽

Laser Scanning ◽

Ccd Camera ◽

Image Plane ◽

Time Lapse ◽

Laser Scanning Confocal Microscopy ◽

Three Dimensions ◽

Excitation Light ◽

Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

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A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

10.26434/chemrxiv.13426412.v1 ◽

2020 ◽

Author(s):

Brittany Benlian ◽

Pavel Klier ◽

Kayli Martinez ◽

Marie Schwinn ◽

Thomas Kirkland ◽

...

Keyword(s):

Energy Transfer ◽

Membrane Potential ◽

Small Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Potential Oscillations ◽

Excitation Light ◽

Voltage Imaging ◽

Induced Pluripotent

We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This Quenching Bioluminescent Voltage Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.

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A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

10.26434/chemrxiv.13426412 ◽

2020 ◽

Author(s):

Brittany Benlian ◽

Pavel Klier ◽

Kayli Martinez ◽

Marie Schwinn ◽

Thomas Kirkland ◽

...

Keyword(s):

Energy Transfer ◽

Membrane Potential ◽

Small Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Live Cells ◽

Potential Oscillations ◽

Excitation Light ◽

Voltage Imaging ◽

Induced Pluripotent

We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This Quenching Bioluminescent Voltage Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.

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Laser Silicon Interaction Study on Ring Oscillators to Establish Temperature Voltage Dependency Trends on 14 nm and Future FinFET Technologies

ISTFA 2017: Conference Proceedings from the 43rd International Symposium for Testing and Failure Analysis ◽

10.31399/asm.cp.istfa2017p0214 ◽

2017 ◽

Author(s):

Gaurav Mattey ◽

Lava Ranganathan

Keyword(s):

High Speed ◽

Laser Frequency ◽

Ring Oscillator ◽

Switching Speed ◽

Dual Nature ◽

Voltage Imaging ◽

Multiple Cores ◽

Speed Performance ◽

Self Test ◽

Frequency Changes

Abstract Critical speed path analysis using Dynamic Laser Stimulation (DLS) technique has been an indispensable technology used in the Semiconductor IC industry for identifying process defects, design and layout issues that limit product speed performance. Primarily by injecting heat or injecting photocurrent in the active diffusion of the transistors, the laser either slows down or speeds up the switching speed of transistors, thereby affecting the overall speed performance of the chip and revealing the speed limiting/enhancing circuits. However, recently on Qualcomm Technologies’ 14nm FinFET technology SOC product, the 1340nm laser’s heating characteristic revealed a Vt (threshold voltage) improvement behavior at low operating voltages which helped identify process issues on multiple memory array blocks across multiple cores failing for MBIST (Memory Built-in Self-test). In this paper, we explore the innovative approach of using the laser to study Vt shifts in transistors due to process issues. We also study the laser silicon interactions through scanning the 1340nm thermal laser on silicon and observing frequency shifts in a high-speed Ring Oscillator (RO) on 16nm FinFET technology. This revealed the normal and reverse Temperature Dependency Gate voltages for 16nm FinFET, thereby illustrating the dual nature of stimulation (reducing mobility and improving Vt) from a thermal laser. Frequency mapping through Laser Voltage Imaging (LVI) was performed on the Ring Oscillator (RO) using the 1340nm thermal laser, while concurrently stimulating the transistors of the RO. Spatial distribution of stimulation was studied by observing the frequency changes on LVI.

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All-optical electrophysiology with improved genetically encoded voltage indicators reveals interneuron network dynamics in vivo

10.1101/2021.11.22.469481 ◽

2021 ◽

Author(s):

He Tian ◽

Hunter C. Davis ◽

J. David Wong-Campos ◽

Linlin Z. Fan ◽

Benjamin Gmeiner ◽

...

Keyword(s):

Gap Junction ◽

Signal To Noise Ratio ◽

Voltage Imaging ◽

All Optical ◽

Precision Voltage ◽

Coupling Strengths ◽

Junction Coupling ◽

Multiple Cells ◽

Voltage Indicator

All-optical electrophysiology can be a powerful tool for studying neural dynamics in vivo, as it offers the ability to image and perturb membrane voltage in multiple cells simultaneously. The "Optopatch" constructs combine a red-shifted archaerhodopsin (Arch)-derived genetically encoded voltage indicator (GEVI) with a blue-shifted channelrhodopsin actuator (ChR). We used a video-based pooled screen to evolve Arch-derived GEVIs with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). By combining optogenetic stimulation of individual cells with high-precision voltage imaging in neighboring cells, we mapped inhibitory and gap junction-mediated connections, in vivo. Optogenetic activation of a single NDNF-expressing neuron in visual cortex Layer 1 significantly suppressed the spike rate in some neighboring NDNF interneurons. Hippocampal PV cells showed near-synchronous spikes across multiple cells at a frequency significantly above what one would expect from independent spiking, suggesting that collective inhibitory spikes may play an important signaling role in vivo. By stimulating individual cells and recording from neighbors, we quantified gap junction coupling strengths. Together, these results demonstrate powerful new tools for all-optical microcircuit dissection in live mice.

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Effect of glycosylation on the extracellular domain of the Ag43 bacterial autotransporter: enhanced stability and reduced cellular aggregation

Biochemical Journal ◽

10.1042/bj20071497 ◽

2008 ◽

Vol 412 (3) ◽

pp. 563-577 ◽

Cited By ~ 28

Author(s):

Stine K. Knudsen ◽

Allan Stensballe ◽

Magnus Franzmann ◽

Uffe B. Westergaard ◽

Daniel E. Otzen

Keyword(s):

Mammalian Cells ◽

Bacterial Attachment ◽

Passenger Domain ◽

Glycosylation Sites ◽

Structural And Functional Properties ◽

Cellular Aggregation ◽

Membrane Bound ◽

Extracellular Environment ◽

Resistant Structure ◽

Different Levels

Autotransporters constitute the biggest group of secreted proteins in Gram-negative bacteria and contain a membrane-bound β-domain and a passenger domain secreted to the extracellular environment via an unusually long N-terminal sequence. Several passenger domains are known to be glycosylated by cytosolic glycosyl transferases, promoting bacterial attachment to mammalian cells. In the present study we describe the effect of glycosylation on the extracellular passenger domain of the Escherichia coli autotransporter Ag43α, which induces frizzy colony morphology and cell settling. We identify 16 glycosylation sites and suggest two possible glycosylation motifs for serine and threonine residues. Glycosylation stabilizes against thermal and chemical denaturation and increases refolding kinetics. Unexpectedly, glycosylation also reduces the stabilizing effect of Ca2+ ions, removes the ability of Ca2+ to promote cell adhesion, reduces the ability of Ag43α-containing cells to form bacterial amyloid and increases the susceptibility of the resulting amyloid to proteolysis. In addition, our results indicate that Ag43α folds without a stable intermediate, unlike pertactin, indicating that autotransporters may arrive at the native state by a variety of different mechanisms despite a common overall structure. A small but significant fraction of Ag43α can survive intact in the periplasm if expressed without the β-domain, suggesting that it is able to adopt a protease-resistant structure prior to translocation across the membrane. The present study demonstrates that glycosylation may play significant roles in structural and functional properties of bacterial autotransporters at many different levels.

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