scholarly journals Optical Probing of Local Membrane Potential with Fluorescent Polystyrene Beads

2021 ◽  
Author(s):  
Zehavit Shapira ◽  
Nurit Degani-Katzav ◽  
Shimon Yudovich ◽  
Asaf Grupi ◽  
Shimon Weiss
Keyword(s):  
Membrane Potential ◽  
Electrical Activity ◽  
Single Particle ◽  
Imaging Techniques ◽  
Single Cells ◽  
Membrane Voltage ◽  
Functional Surface ◽  
Fluorescent Intensity ◽  
Voltage Imaging ◽  
Local Circuits

Studying the electrical activity in single cells and in local circuits of excitable cells, like neurons, requires an easy to use and high throughput methodology that enables the measurement of membrane potential. Studying the electrical properties in particular sub-compartments of neurons, or in a specific type of neurons produces additional complexity. An optical voltage-imaging technique that allows high spatial and temporal resolution could be an ideal solution. However, most of the valid voltage imaging techniques are nonspecific; The ones that are more site-directed require much pre-work and specific adaptations in addition to other disadvantages. Here, a new technique for membrane voltage imaging, based on FRET between fluorescent polystyrene (FPS) beads and Dipicrylamine (DPA) is explored. Not only fluorescent intensity is demonstrated to be correlated with membrane potential, but more importantly, single particle voltage detection is demonstrated. Among other advantages, FPS beads can be synthesized with functional surface groups, and be further targeted to specific proteins via conjugation of recognition molecules. Therefore, FPS beads, in the presence of DPA, constitute single-particle detectors for membrane voltage, with a potential to be localized to specific membrane compartments. This new and accessible platform for targeted optical voltage imaging may further elucidate the mechanisms of neuronal electrical activity.

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

Fluorescent potentiometric dyes for membrane-potential imaging

1994 ◽  
Vol 52 ◽  
pp. 166-167
Author(s):  
Leslie M. Loew
Keyword(s):  
Membrane Potential ◽  
Single Cells ◽  
Quantitative Imaging ◽  
Optical Recording ◽  
Biological Processes ◽  
Major Focus ◽  
The Past ◽  
Excitable Systems ◽  
Major Application ◽  
The Impact

A major application of potentiometric dyes has been the multisite optical recording of electrical activity in excitable systems. After being championed by L.B. Cohen and his colleagues for the past 20 years, the impact of this technology is rapidly being felt and is spreading to an increasing number of neuroscience laboratories. A second class of experiments involves using dyes to image membrane potential distributions in single cells by digital imaging microscopy - a major focus of this lab. These studies usually do not require the temporal resolution of multisite optical recording, being primarily focussed on slow cell biological processes, and therefore can achieve much higher spatial resolution. We have developed 2 methods for quantitative imaging of membrane potential. One method uses dual wavelength imaging of membrane-staining dyes and the other uses quantitative 3D imaging of a fluorescent lipophilic cation; the dyes used in each case were synthesized for this purpose in this laboratory.

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

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

<p>We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent V<u>olt</u>age 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.</p> <p> </p>

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

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

<p>We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent V<u>olt</u>age 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.</p> <p> </p>

scholarly journals Single-particle investigation of summertime and wintertime Antarctic sea spray aerosols using low-Z particle EPMA, Raman microspectrometry, and ATR-FTIR imaging techniques

2016 ◽  
Author(s):  
Hyo-Jin Eom ◽  
Dhrubajyoti Gupta ◽  
Hye-Rin Cho ◽  
HeeJin Hwang ◽  
SoonDo Hur ◽  
...  
Keyword(s):  
Chlorophyll A ◽  
Single Particle ◽  
Imaging Techniques ◽  
Sea Spray ◽  
Chemical Compositions ◽  
Research Station ◽  
Ftir Imaging ◽  
X Ray ◽  
Inorganic Species ◽  
Raman Microspectrometry

Abstract. Two aerosol samples collected at King Sejong Korean scientific research station, Antarctica on Dec. 9, 2011 in the austral summer (sample S1) and July 23, 2012 in the austral winter (sample S2), when the oceanic chlorophyll-a levels were quite different, by ~19 times (2.46 vs. 0.13 μg/L, respectively), were investigated on a single particle basis using quantitative energy-dispersive electron probe X-ray microanalysis (ED-EPMA), called low-Z particle EPMA, Raman microspectrometry (RMS), and attenuated total reflectance Fourier transform infrared (ATR-FTIR) imaging techniques to obtain their characteristics based on the elemental chemical compositions, molecular species, and mixing state. X-ray analysis showed that the supermicron summertime and wintertime Antarctic aerosol samples have different elemental chemical compositions, even though all the individual particles analyzed were sea spray aerosols (SSAs); i.e., the contents of C, O, Ca, S, and Si were more elevated, whereas Cl was more depleted, for sample S1 having a much higher chlorophyll-a level than for sample S2. Based on qualitative analysis of the chemical species present in individual SSAs by the combined application of RMS and ATR-FTIR imaging, different organic species were encountered in samples S1 and S2; i.e., Mg hydrate salts of alanine were predominant in samples S1 and S2, whereas Mg salts of fatty acids internally mixed with Mg hydrate salts of alanine were significant in sample S2. Although CaSO4 was encountered significantly in both samples S1 and S2, other inorganic species, such as Na2SO4, NaNO3, Mg(NO3)2, SiO2, and CH3SO3Mg, were encountered more significantly in sample S1, suggesting that those compounds may be related to the higher phytoplankton activity in summer.

scholarly journals Electron microscopy snapshots of single particles from single cells

2018 ◽  
Vol 294 (5) ◽  
pp. 1602-1608 ◽  
Author(s):  
Xiunan Yi ◽  
Eric J. Verbeke ◽  
Yiran Chang ◽  
Daniel J. Dickinson ◽  
David W. Taylor
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Protein Complexes ◽  
Signal To Noise Ratio ◽  
Single Cells ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particle Analysis ◽  
Whole Cells ◽  
Potential Applications

Cryo-electron microscopy (cryo-EM) has become an indispensable tool for structural studies of biological macromolecules. Two additional predominant methods are available for studying the architectures of multiprotein complexes: 1) single-particle analysis of purified samples and 2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, whereas the latter can capture proteins in their native state but has a low signal-to-noise ratio and yields lower-resolution structures. Here, we present a simple, adaptable method combining microfluidic single-cell extraction with single-particle analysis by EM to characterize protein complexes from individual Caenorhabditis elegans embryos. Using this approach, we uncover 3D structures of ribosomes directly from single embryo extracts. Moreover, we investigated structural dynamics during development by counting the number of ribosomes per polysome in early and late embryos. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary, and disease contexts.

scholarly journals Single-particle investigation of summertime and wintertime Antarctic sea spray aerosols using low-<i>Z</i> particle EPMA, Raman microspectrometry, and ATR-FTIR imaging techniques

2016 ◽  
Vol 16 (21) ◽  
pp. 13823-13836 ◽  
Author(s):  
Hyo-Jin Eom ◽  
Dhrubajyoti Gupta ◽  
Hye-Rin Cho ◽  
Hee Jin Hwang ◽  
Soon Do Hur ◽  
...  
Keyword(s):  
Single Particle ◽  
Imaging Techniques ◽  
Attenuated Total Reflection ◽  
Sea Spray ◽  
Chemical Compositions ◽  
Research Station ◽  
Ftir Imaging ◽  
X Ray ◽  
Inorganic Species ◽  
Raman Microspectrometry

Abstract. Two aerosol samples collected at King Sejong Korean scientific research station, Antarctica, on 9 December 2011 in the austral summer (sample S1) and 23 July 2012 in the austral winter (sample S2), when the oceanic chlorophyll a levels on the collection days of the samples were quite different, by  ∼  19 times (2.46 vs. 0.13 µg L−1, respectively), were investigated on a single-particle basis using quantitative energy-dispersive electron probe X-ray microanalysis (ED-EPMA), called low-Z particle EPMA, Raman microspectrometry (RMS), and attenuated total reflection Fourier transform infrared (ATR-FTIR) imaging techniques to obtain their characteristics based on the elemental chemical compositions, molecular species, and mixing state. X-ray analysis showed that the supermicron summertime and wintertime Antarctic aerosol samples have different elemental chemical compositions, even though all the individual particles analyzed were sea spray aerosols (SSAs); i.e., the contents of C, O, Ca, S, and Si were more elevated, whereas Cl was more depleted, for sample S1 than for sample S2. Based on qualitative analysis of the chemical species present in individual SSAs by the combined application of RMS and ATR-FTIR imaging, different organic species were observed in samples S1 and S2; i.e., Mg hydrate salts of alanine were predominant in samples S1 and S2, whereas Mg salts of fatty acids internally mixed with Mg hydrate salts of alanine were significant in sample S2. Although CaSO4 was observed significantly in both samples S1 and S2, other inorganic species, such as Na2SO4, NaNO3, Mg(NO3)2, SiO2, and CH3SO3Mg, were observed more significantly in sample S1, suggesting that those compounds may be related to the higher phytoplankton activity in summer.

Electrical activity of small intestinal smooth muscle and its temperature dependence

1975 ◽  
Vol 229 (5) ◽  
pp. 1268-1276 ◽  
Author(s):  
TY El-Sharkawy ◽  
EE Daniel
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Electrical Activity ◽  
Temperature Dependence ◽  
Intestinal Smooth Muscle ◽  
Plateau Phase ◽  
Control Activity ◽  
Electrical Control ◽  
Electrical Control Activity ◽  
Small Intestinal

Some important features of the intracellularly recorded electrical control activity of rabbit jejunal smooth muscle and its temperature dependence are reported in this study. This activity consisted of repetitive 18-mV depolarizations (control potentials (CP) or slow waves), which at 37degreesC lasted 2 s and had a frequency of 18/min and arose from a membrane potential of --55 mV. In some cells periods between CP's exhibited "diastolic" progressive depolarizations (intercontrol-potential depolarization), which may be the trigger of the CP in driving cells. While CP was usually monophasic, some cells persistently exhibited a notch early in the plateau phase. We suggest that CP consists of two components, an "initial depolarization" and a "secondary depolarization," which are usually fused together to give a monophasic potential. Cooling reduced CP frequency and prolonged its duration and caused more cells to show notching. While amplitude and rate of CP initial depolarization had low Q10's, duration and rates of onset and offset of the secondary depolarization had higher Q10's. Thus, the process responsible for secondary depolarization is more sensitive to temperature thant that underlying initial depolarization of the CP.

Role of HERG-like K+ currents in opossum esophageal circular smooth muscle

1999 ◽  
Vol 277 (6) ◽  
pp. C1284-C1290 ◽  
Author(s):  
Hamid I. Akbarali ◽  
Hemant Thatte ◽  
Xue Dao He ◽  
Wayne R. Giles ◽  
Raj K. Goyal
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Smooth Muscle Cells ◽  
Single Cells ◽  
Circular Muscle ◽  
Muscle Cells ◽  
Resting Membrane Potential ◽  
Channel Blockers ◽  
Circular Smooth Muscle ◽  
Inward Currents

An inwardly rectifying K+ conductance closely resembling the human ether-a-go-go-related gene (HERG) current was identified in single smooth muscle cells of opossum esophageal circular muscle. When cells were voltage clamped at 0 mV, in isotonic K+ solution (140 mM), step hyperpolarizations to −120 mV in 10-mV increments resulted in large inward currents that activated rapidly and then declined slowly (inactivated) during the test pulse in a time- and voltage- dependent fashion. The HERG K+ channel blockers E-4031 (1 μM), cisapride (1 μM), and La3+ (100 μM) strongly inhibited these currents as did millimolar concentrations of Ba2+. Immunoflourescence staining with anti-HERG antibody in single cells resulted in punctate staining at the sarcolemma. At membrane potentials near the resting membrane potential (−50 to −70 mV), this K+ conductance did not inactivate completely. In conventional microelectrode recordings, both E-4031 and cisapride depolarized tissue strips by 10 mV and also induced phasic contractions. In combination, these results provide direct experimental evidence for expression of HERG-like K+ currents in gastrointestinal smooth muscle cells and suggest that HERG plays an important role in modulating the resting membrane potential.

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