Estimation of the Mitochondrial Membrane Potential Using Fluorescence Lifetime Imaging Microscopy.
Okkelman Irina A,Papkovsky Dmitri B,Dmitriev Ruslan I
Cytometry. Part A : the journal of the International Society for Analytical Cytology
Monitoring of cell metabolism represents an important application area for fluorescence lifetime imaging microscopy (FLIM). In particular, assessment of mitochondrial membrane potential (MMP) in complex three-dimensional multicellular in vitro, ex vivo, and in vivo models would enable improved segmentation and functional discrimination of cell types, directly report on the mitochondrial function and complement the quenched-phosphorescence detection of cellular O and two-photon excited FLIM of endogenous NAD(P)H. Here, we report the green and orange-emitting fluorescent dyes SYTO and tetramethylrhodamine methyl ester (TMRM) as potential FLIM probes for MMP. In addition to nuclear, SYTO 16 and 24 dyes also display mitochondrial accumulation. FLIM with the culture of human colon cancer HCT116 cells allowed observation of the heterogeneity of mitochondrial polarization during the cell cycle progression. The dyes also demonstrated good performance with 3D cultures of Lgr5-GFP mouse intestinal organoids, providing efficient and quick cell staining and compatibility with two-photon excitation. Multiplexed imaging of Lgr5-GFP, proliferating cells (Hoechst 33342-aided FLIM), and TMRM-FLIM allowed us to identify the population of metabolically active cells in stem cell niche. TMRM-FLIM enabled to visualize the differences in membrane potential between Lgr5-positive and other proliferating and differentiated cell types. Altogether, SYTO 24 and TMRM dyes represent promising markers for advanced FLIM-based studies of cell bioenergetics with complex 3D and in vivo models. © 2019 International Society for Advancement of Cytometry.
10.1002/cyto.a.23886
Fluorescent indicators for imaging membrane potential of organelles.
Current opinion in chemical biology
Plasma membrane potential is a key driver of the physiology of excitable cells like neurons and cardiomyocytes. Voltage-sensitive fluorescent indicators offer a powerful complement to traditional electrode-based approaches to measuring and monitoring membrane potential. Intracellular organelles can also generate membrane potential, yet the electrode- and fluorescent indicator-based approaches used for plasma membrane potential imaging are difficult to implement on intact organelles in their native environment. Here, we survey recent advances in developing and deploying voltage-sensitive fluorescent indicators to interrogate organelle membrane potential in intact cells.
10.1016/j.cbpa.2022.102203
Picture perfect: Imaging mitochondrial membrane potential changes in retina slices with minimal stray fluorescence.
Haider Syeda Zehra,Mohanraj Nivedha,Markandeya Yogananda S,Joshi Preeti G,Mehta Bhupesh
Experimental eye research
Mitochondrial membrane potential (Ψ) is a critical parameter that can be used to determine cellular well-being. As it is a direct measure of the cell's ATP generating capability, in recent years, this key component in cell biology has been the subject of thousands of biochemical and biophysical investigations. Membrane-permeant fluorescent dyes, like tetramethylrhodamine ethyl ester (TMRE), have been predominantly employed to monitor ΔΨ in cells. These dyes are typically lipophilic cationic compounds that equilibrate across membranes in a Nernstian fashion, thus accumulating into the mitochondrial membrane matrix space in inverse proportion to Ψ. However, the bath loading method practiced for labelling tissue slices with these cationic dyes poses limitations in the form of non-specificity and low signal to noise ratio, which compromises the precision of the results. Therefore, we introduce an alternative way for TMRE loading to image the ΔΨ in tissue slices by utilizing a low resistance glass pipette attached to a pressure injector. This method shows highly precise fluorescent dye labelling of the mitochondria and offers maximum output intensity, in turn enhancing signal to noise ratio.
10.1016/j.exer.2020.108318
Mitochondrial Membrane Potential Assay.
Methods in molecular biology (Clifton, N.J.)
Mitochondrial function, a key indicator of cell health, can be assessed through monitoring changes in mitochondrial membrane potential (MMP). Cationic fluorescent dyes are commonly used tools to assess MMP. We used a water-soluble mitochondrial membrane potential indicator (m-MPI) to detect changes in MMP in various types of cells, such as HepG2, HepaRG, and AC16 cells. A homogenous cell-based MMP assay has been optimized and performed in a 1536-well plate format, which can be used to screen several compound libraries for mitochondrial toxicity by evaluating the effects of chemical compounds on MMP.
10.1007/978-1-0716-2213-1_2
VoltageFluor dyes and fluorescence lifetime imaging for optical measurement of membrane potential.
Gest Anneliese M M,Yaeger-Weiss Susanna K,Lazzari-Dean Julia R,Miller Evan W
Methods in enzymology
Membrane potential is a fundamental biophysical parameter common to all of cellular life. Traditional methods to measure membrane potential rely on electrodes, which are invasive and low-throughput. Optical methods to measure membrane potential are attractive because they have the potential to be less invasive and higher throughput than classic electrode based techniques. However, most optical measurements rely on changes in fluorescence intensity to detect changes in membrane potential. In this chapter, we discuss the use of fluorescence lifetime imaging microscopy (FLIM) and voltage-sensitive fluorophores (VoltageFluors, or VF dyes) to estimate the millivolt value of membrane potentials in living cells. We discuss theory, application, protocols, and shortcomings of this approach.
10.1016/bs.mie.2021.02.009
A guide for membrane potential measurements in Gram-negative bacteria using voltage-sensitive dyes.
Microbiology (Reading, England)
Transmembrane potential is one of the main bioenergetic parameters of bacterial cells, and is directly involved in energizing key cellular processes such as transport, ATP synthesis and motility. The most common approach to measure membrane potential levels is through use of voltage-sensitive fluorescent dyes. Such dyes either accumulate or are excluded from the cell in a voltage-dependent manner, which can be followed by means of fluorescence microscopy, flow cytometry, or fluorometry. Since the cell's ability to maintain transmembrane potential relies upon low and selective membrane ion conductivity, voltage-sensitive dyes are also highly sensitive reporters for the activity of membrane-targeting antibacterials. However, the presence of an additional membrane layer in Gram-negative (diderm) bacteria complicates their use significantly. In this paper, we provide guidance on how membrane potential and its changes can be monitored reliably in Gram-negatives using the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide [DiSC(5)]. We also discuss the confounding effects caused by the presence of the outer membrane, or by measurements performed in buffers rather than growth medium. We hope that the discussed methods and protocols provide an easily accessible basis for the use of voltage-sensitive dyes in Gram-negative organisms, and raise awareness of potential experimental pitfalls associated with their use.
10.1099/mic.0.001227
Membrane-Activated Fluorescent Probe for High-Fidelity Imaging of Mitochondrial Membrane Potential.
Lin Bo,Liu Yunfan,Zhang Xiaoping,Fan Li,Shu Yang,Wang Jianhua
ACS sensors
Mitochondrial membrane potential (ΔΨ) is a key indicator of cell health or injury due to its vital roles in adenosine 5'-triphosphate synthesis. Thus, monitoring ΔΨ is of great significance for the assessment of cell status, diagnosis of diseases, and medicament screening. Cationic fluorescent probes suffer from severe photobleaching or false positive signals due to the luminescence of the probe on non-mitochondria. Herein, we report a lipophilic cationic fluorescent probe [1-methyl-2-(4-(1,2,2-triphenylvinyl)styryl)-β-naphthothiazol-1-ium trifluoromethanesulfonate ()] with the features of aggregation-induced emission and intramolecular charge transfer for imaging ΔΨ in live cells. is enriched on the surface of the mitochondrial inner membrane due to the negative ΔΨ, and its fluorescence is activated in the high-viscosity microenvironment. The false positive signals of emission from on non-mitochondria are therefore effectively eliminated. Moreover, exhibits a Stokes shift of >200 nm, near-infrared (∼675 nm) emission, excellent photostability, and low cytotoxicity, which facilitate real-time imaging in live cells. Cell imaging confirmed that the probe can rapidly and reliably report mitochondrial depolarization (decrement of ΔΨ) during cell damage caused by CCCP and HO as well as mitochondrial polarization (increment of ΔΨ) by oligomycin. Furthermore, the probe successfully detected the reduction of ΔΨ in these cell models of hypoxia, heat damage, acidification, aging, inflammation, mitophagy, and apoptosis caused by hypoxia, heatstroke, lactate/pyruvate, doxorubicin, lipopolysaccharide, rapamycin, monensin, and nystatin, respectively.
10.1021/acssensors.1c01390
Comparative studies on measurement of membrane potential of bacterial cells treated with ZnO nanoparticles by Spectrofluorometry, fluorescence microscopy and flowcytometry.
Khater Maya,Khater Sagar S,Gholap Haribhau,Patil Rajendra,Kulkarni Gauri
Journal of microbiological methods
Many methods are developed to assess antimicrobial action of ZnO nanoparticles (NPs). A large number of methods associated with the use of fluorescent probes are developed, including Spectrofluorometry, fluorescence microscopy, and cytometry. In this study, flowcytometry, Spectrofluorometry and fluorescent microscopy was used to measure membrane potential variation of E. coli and S. aureus cells treated with two different sizes of zinc oxide (ZnO) NPs and were compared with conventional methods. In order to estimate change in membrane potential, E. coli and S. aureus cells were treated with iopnophore agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) and membrane potential was evaluated using fluorescent probe 3,3'-Diethyloxacarbocyanine, iodide (DIOC(3)). All the three methods showed similar results and among these Spectrofluorometry was easy to use and inexpensive to assess the viability of bacterial cells via their membrane potential.
10.1016/j.mimet.2020.105920
Electrophysiology, Unplugged: Imaging Membrane Potential with Fluorescent Indicators.
Accounts of chemical research
Membrane potential is a fundamental biophysical property maintained by every cell on earth. In specialized cells like neurons, rapid changes in membrane potential drive the release of chemical neurotransmitters. Coordinated, rapid changes in neuronal membrane potential across large numbers of interconnected neurons form the basis for all of human cognition, sensory perception, and memory. Despite the importance of this highly orchestrated and distributed activity, the traditional method for recording membrane potential is through the use of highly invasive single-cell electrodes that offer only a small glimpse of the total activity within a system. Fluorescent dyes that change their optical properties in response to changes in biological voltage have the potential to provide a powerful complement to traditional electrode-based methods of inquiry. Voltage-sensitive fluorescent indicators would allow the direct observation of membrane potential changes, significantly expanding our ability to monitor membrane potential dynamics in living systems. Toward this end, we have initiated a program to design, synthesize, and apply voltage-sensitive fluorophores that report on membrane potential dynamics with high sensitivity and speed. The basis for this optical voltage sensing is membrane potential-dependent photoinduced electron transfer (PeT). Voltage-sensitive fluorophores, or VoltageFluors, possess a fluorophore, a conjugated molecular wire, and an aniline donor. At resting potentials, in which the cell has a hyperpolarized or negative potential relative to the outside of the cell, PeT from the aniline donor is enhanced and fluorescence is diminished. At depolarized potentials, the membrane potential decreases the rate of PeT, allowing an increase in fluorescence. We show that a number of different fluorophores, molecular wires, and aniline donors can be employed to generate fast and sensitive VoltageFluor dyes. Multiple lines of evidence point to a PeT-based mechanism for voltage sensing, delivering fast response kinetics (∼25 ns), good sensitivity (>60% Δ/), compatibility with two-photon illumination, excellent signal-to-noise, and the ability to detect neuronal and cardiac action potentials in single trials. In this Account, we provide an overview of the challenges facing the design of fluorescent voltage indicators. We trace the development of molecular wire-based fluorescent voltage indicators within our group, beginning from fluorescein-based VoltageFluor to long-wavelength indicators that use modern fluorophores like silicon rhodamine and carbofluorescein. We examine design principles for PeT-based voltage indicators, showcase the use of our recent indicators for two-photon voltage imaging in intact brains, and explore the development of hybrid indicators that can localize to genetically defined cells. Finally, we highlight outstanding challenges to and opportunities for voltage imaging.
10.1021/acs.accounts.9b00514
Use of a Fluorescence-Based Assay To Measure Escherichia coli Membrane Potential Changes in High Throughput.
Antimicrobial agents and chemotherapy
Bacterial membrane potential is difficult to measure using classical electrophysiology techniques due to the small cell size and the presence of the peptidoglycan cell wall. Instead, chemical probes are often used to study membrane potential changes under conditions of interest. Many of these probes are fluorescent molecules that accumulate in a charge-dependent manner, and the resulting fluorescence change can be analyzed via flow cytometry or using a fluorescence microplate reader. Although this technique works well in many Gram-positive bacteria, it generates fairly low signal-to-noise ratios in Gram-negative bacteria due to dye exclusion by the outer membrane. We detail an optimized workflow that uses the membrane potential probe, 3,3'-diethyloxacarbocyanine iodide [DiOC(3)], to measure membrane potential changes in high throughput and describe the assay conditions that generate significant signal-to-noise ratios to detect membrane potential changes using a fluorescence microplate reader. A valinomycin calibration curve demonstrates this approach can robustly report membrane potentials over at least an ∼144-mV range with an accuracy of ∼12 mV. As a proof of concept, we used this approach to characterize the effects of some commercially available small molecules known to elicit membrane potential changes in other systems, increasing the repertoire of compounds known to perturb membrane energetics. One compound, the eukaryotic Ca channel blocker amlodipine, was found to alter membrane potential and decrease the MIC of kanamycin, further supporting the value of this screening approach. This detailed methodology permits studying membrane potential changes quickly and reliably at the population level.
10.1128/AAC.00910-20
Fluorescence Measurement of Mitochondrial Membrane Potential Changes in Cultured Cells.
Nicholls David G
Methods in molecular biology (Clifton, N.J.)
The mitochondrial membrane potential is the dominant component of the proton-motive force that is the potential term in the proton circuit linking electron transport to ATP synthesis and other energy-dependent mitochondrial processes. Cationic fluorescent probes have been used for many years to detect gross qualitative changes in mitochondrial membrane potentials in intact cell culture. In this chapter I describe how these fluorescence signals may be used to obtain a semiquantitative measure of changes in mitochondrial membrane potential.
10.1007/978-1-4939-7831-1_7
Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels using Live Imaging in HeLa Cells.
Journal of visualized experiments : JoVE
Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (Parkin) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.
10.3791/65304
Fluorescence Imaging of Cell Membrane Potential: From Relative Changes to Absolute Values.
International journal of molecular sciences
Membrane potential is a fundamental property of biological cells. Changes in membrane potential characterize a vast number of vital biological processes, such as the activity of neurons and cardiomyocytes, tumorogenesis, cell-cycle progression, etc. A common strategy to record membrane potential changes that occur in the process of interest is to utilize organic dyes or genetically-encoded voltage indicators with voltage-dependent fluorescence. Sensors are introduced into target cells, and alterations of fluorescence intensity are recorded with optical methods. Techniques that allow recording relative changes of membrane potential and do not take into account fluorescence alterations due to factors other than membrane voltage are already widely used in modern biological and biomedical studies. Such techniques have been reviewed previously in many works. However, in order to investigate a number of processes, especially long-term processes, the measured signal must be corrected to exclude the contribution from voltage-independent factors or even absolute values of cell membrane potential have to be evaluated. Techniques that enable such measurements are the subject of this review.
10.3390/ijms24032435