Improved Sensitivity in a Modified Berkeley Red Sensor of Transmembrane Potential - PubMed (original) (raw)

Improved Sensitivity in a Modified Berkeley Red Sensor of Transmembrane Potential

Marisol X Navarro et al. ACS Chem Biol. 2024.

Abstract

Voltage imaging is an important complement to traditional methods for probing cellular physiology, such as electrode-based patch clamp techniques. Unlike the related Ca2+ imaging, voltage imaging provides a direct visualization of bioelectricity changes. We have been exploring the use of sulfonated silicon rhodamine dyes (Berkeley Red Sensor of Transmembrane potential, BeRST) for voltage imaging. In this study, we explore the effect of converting BeRST to diEt BeRST, by replacing the dimethyl aniline of BeRST with a diethyl aniline group. The new dye, diEt BeRST, has a voltage sensitivity of 40% Δ_F_/F per 100 mV, a 33% increase compared to the original BeRST dye, which has a sensitivity of 30% Δ_F_/F per 100 mV. In neurons, the cellular brightness of diEt BeRST is about 20% as bright as that of BeRST, which may be due to the lower solubility of diEt BeRST (300 μM) compared to that of BeRST (800 μM). Despite this lower cellular brightness, diEt BeRST is able to record spontaneous and evoked action potentials from multiple neurons simultaneously and in single trials. Far-red excitation and emission profiles enable diEt BeRST to be used alongside existing fluorescent indicators of cellular physiology, like Ca2+-sensitive Oregon Green BAPTA. In hippocampal neurons, simultaneous voltage and Ca2+ imaging reveals neuronal spiking patterns and frequencies that cannot be resolved with traditional Ca2+ imaging methods. This study represents a first step toward describing the structural features that define voltage sensitivity and brightness in silicon rhodamine-based BeRST indicators.

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Figures

Figure 1.

Characterization of diEt BeRST. a) Plot of relative absorbance (dotted black line) or emission intensity (solid purple line) for diEt BeRST. Spectra were acquired in pH = 7.5 buffer (50 mM TBS, 150 mM NaCl, 0.1% w/w SDS). b) Plot of fractional change in fluorescence (ΔF/F) over time in HEK cells stained with 1 μM of diEt BeRST under voltage clamp conditions. Cells were held at −60 mV and stepped to hyper- or depolarizing potentials (±100 mV) in 20 mV increments. c) Plot of %ΔF/F vs final membrane potential for diEt BeRST. Data are the mean ΔF/F. Error bars are S.E.M. for n = 7 cells, across 4 different experiments. If error bars are not visible, they are smaller than the marker. Dotted line is the linear fit through the data (R2 = 0.995). Plots of mean d) voltage sensitivity or e) signal-to-noise ratio (SNR) per 100 mV in voltage-clamped HEK cells for diEt BeRST and BeRST. ** p < 0.002; *** p < 0.0002. Circles are the voltage sensitivity for each measured cell, n = 7 (diEt BeRST), n = 6 (BeRST). Error bars are S.E.M.

Figure 2.

Fluorescence imaging of diEt BeRST in HEK 293T cells and rat hippocampal neurons. a) Transmitted light and b) wide field epifluorescence image of HEK293T cells stained with diEt BeRST (1 μM). Scale bar is 20 μm. c) Plot of fluorescence intensity of either diEt BeRST or BeRST staining in HEK293T cells. d) Transmitted light and e) wide field epifluorescence image of rat hippocampal neurons stained with diEt BeRST (1 μM). Scale bar is 20 μm. f) Plot of fluorescence intensity of either diEt BeRST or BeRST staining in neurons. Bars are the mean value. Error bars are standard error of the mean (S.E.M.) for n = 73 (diEt BeRST, HEK293T), 69 (diEt BeRST, neurons), 67 (BeRST, HEK293T), or 49 (BeRST, neurons) cells. Each point represents the fluorescence intensity for a single cell. **** indicates a p-value of <0.0001 (two-tailed t-test).

Figure 3.

Optical recordings of spontaneous neuronal activity in rat hippocampal neurons using diEt BeRST. a) Widefield epifluorescence and b) transmitted light image of rat hippocampal neurons stained with 1 μM diEt BeRST. c) Plots of ΔF/F vs time for the indicated neurons in panels a/b. Optical recordings are acquired at 500 Hz. Scale bar is 20 μm.

Figure 4.

Multi-color imaging in rat hippocampal neurons with diEt BeRST. Neurons were stained with diEt BeRST, Oregon Green BAPTA (OGB), and Hoechst 33342 (1 μM for all dyes). Widefield epifluorescence a) diEt BeRST, b) diEt BeRST (grey) and OGB (green), c) diEt BeRST (grey) and Hoechst (cyan), and d) transmitted light image of rat hippocampal neurons. Scale bar is 20 μm. Plots of ΔF/F vs time for e) diEt BeRST or f) OGB. Colors indicate the same neuron, observed first under voltage imaging and then under Ca2+ imaging.

Scheme 1.

Diethyl BeRST (diEt BeRST) and BeRST 1

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Improved Sensitivity in a Modified Berkeley Red Sensor of Transmembrane Potential - PubMed (original) (raw)