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CHAPTER 22

Probes for Membrane Potential

Molecular Probes™ HandbookA Guide to Fluorescent Probes and Labeling Technologies

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Fluorophores and Their Amine-Reactive Derivatives

The Molecular Probes® HandbookA GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES11th Edition (2010)

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CHAPTER 22

Probes for Membrane Potential

22.1 Introduction to Potentiometric Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 925

Applications for Potentiometric Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 925

Selecting a Potentiometric Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 925

22.2 Fast-Response Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 926

ANEP Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 926

Di-4-ANEPPS and Di-8-ANEPPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 926

Cationic ANEP Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 927

RH Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 927

FRET-Pair Membrane Potential Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 928

Data Table 22.2 Fast-Response Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 928

Product List 22.2 Fast-Response Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 928

22.3 Slow-Response Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929

Carbocyanines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929

DiI, DiS and DiO Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 929

JC-1 and JC-9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930

MitoProbe™ JC-1 Assay Kit for Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 930

MitoProbe™ DiIC(5) and MitoProbe™ DiOC(3) Assay Kits for Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 931

BacLight™ Bacterial Membrane Potential Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 932

Rhodamine 123, TMRM and TMRE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933

Oxonols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933

Oxonol V and Oxonol VI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933

DiBAC (Bis-Oxonol) Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 933

Merocyanine 540 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 934

Data Table 22.3 Slow-Response Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 935

Product List 22.3 Slow-Response Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 936

The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies

IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

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Chapter 22 — Probes for Membrane Potential

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IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes




Section 22.1 Introduction to Potentiometric Probes

hyperpolarization

depolarization

extracellular face

intracellular face

membrane

Fast-response probe

Slow-response probe

hyperpolarization

depolarization

extracellular face

intracellular face

membrane

A.

B.

Figure 22.1.1 Response mechanisms of membrane potential–sensitive probes. Fast-response probes undergo electric �eld–driven changes of intramo-lecular charge distribution that produce corresponding changes in the spectral pro�le or intensity of their �uorescence (represented by color changes in the illustration). Slow-response probes are lipophilic anions (in this illustration) or cations that are translocated across membranes by an electrophoretic mechanism. Fluorescence changes associated with transmembrane redistribution (represented by color changes in the illustration) result from sensitivity of the probe to intracellular and extracellular environments. Thus, potentiometric response speeds directly re�ect the time constants of the underlying pro-cesses—fast intramolecular redistribution of electrons versus relatively slow transmembrane movement of entire molecules.

22.1 Introduction to Potentiometric ProbesPotentiometric optical probes enable researchers to perform mem-

brane potential measurements in organelles and in bacterial cells that are too small for microelectrodes. Moreover, in conjunction with im-aging techniques, these probes can be employed to map variations in membrane potential across excitable cells, in perfused organs 1 and ul-timately in the brain in vivo,2–5 with spatial resolution and sampling frequency that cannot be obtained using microelectrodes.

Applications for Potentiometric Probes�e plasma membrane of a cell typically has a transmembrane po-

tential of approximately –70 mV (negative inside) as a consequence of K+, Na+ and Cl– concentration gradients that are maintained by active transport processes. Potentiometric probes o�er an indirect method of detecting the translocation of these ions, whereas the �uorescent ion indicators discussed in Chapter 21 can be used to directly measure changes in speci�c ion concentrations.

Increases and decreases in membrane potential—referred to as membrane hyperpolarization and depolarization, respectively—play a central role in many physiological processes, including nerve-impulse propagation, muscle contraction, cell signaling and ion-channel gat-ing. Potentiometric probes are important tools for studying these processes, as well as for visualizing mitochondria (which exhibit transmembrane potentials of approximately –150 mV, negative inside matrix) (Section 12.2), for assessing cell viability (Section 15.2) and for high-throughput screening of new drug candidates.

Potentiometric probes include the cationic or zwitterionic styryl dyes, the cationic carbocyanines and rhodamines, the anionic and hybrid oxonols and merocyanine 540. �e class of dye determines factors such as accumulation in cells, response mechanism and toxic-ity. Surveys of techniques and applications using membrane potential probes can be found in several reviews.2,4,6–8

Selecting a Potentiometric ProbeSelecting the best potentiometric probe for a particular applica-

tion can be complicated by the substantial variations in their optical responses, phototoxicity and interactions with other molecules. Probes can be divided into two categories based on their response mechanism:

• Fast-response probes (usually styrylpyridinium dyes, Section 22.2) operate by means of a change in their electronic structure, and con-sequently their �uorescence properties, in response to a change in the surrounding electric �eld (Figure 22.1.1). �eir optical response is su�ciently fast to detect transient (millisecond) potential chang-es in excitable cells, including single neurons, cardiac cells and in-tact brains. However, the magnitude of their potential-dependent �uorescence change is o�en small; fast-response probes typically show a 2–10% �uorescence change per 100 mV.

• Slow-response probes (Section 22.3) exhibit potential-dependent changes in their transmembrane distribution that are accompanied by a �uorescence change (Figure 22.1.1). �e magnitude of their



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Section 22.2 Fast-Response Probes

Figure 22.2.1 di-4-ANEPPS (D1199). Figure 22.2.2 di-8-ANEPPS (D3167).

Our fast-response potential-sensitive probes (see Figure 22.1.1A in Section 22.1) are listed in Table 22.1, along with their charges, optical responses and selected applications.

ANEP DyesDi-4-ANEPPS and Di-8-ANEPPS

�e ANEP (AminoNaphthylEthenylPyridinium) dyes developed by Leslie Loew and col-leagues 1 are among the most sensitive of the fast-response probes. Zwitterionic di-4-ANEPPS (D1199, Figure 22.2.1) and di-8-ANEPPS (D3167, Figure 22.2.2) exhibit fairly uniform 10% per 100 mV changes in �uorescence intensity in a variety of tissue, cell and model membrane sys-tems.2,3 �e millisecond-range temporal characteristics of the ANEP dyes compensate for this modest response amplitude (Figure 22.2.3). Di-4-ANEPPS is internalized in the cell rather rap-idly, precluding its use in all but very short-term experiments, whereas di-8-ANEPPS is better retained in the outer lea�et of the plasma membrane. In addition, although both ANEP dyes exhibit good photostability and low toxicity, di-8-ANEPPS is reported to be slightly more pho-tostable and signi�cantly less phototoxic than di-4-ANEPPS.4–6

Like other styryl dyes, the ANEP dyes are essentially non�uorescent in aqueous solutions and exhibit spectral properties that are strongly dependent on their environment.7 When bound to phospholipid vesicles, di-8-ANEPPS has absorption/emission maxima of ~467/631 nm (Figure 22.2.4), as compared with ~498/713 nm in methanol. �e �uorescence excitation/emission max-ima of di-4-ANEPPS bound to neuronal membranes are ~475/617 nm.8

Both di-4-ANEPPS and di-8-ANEPPS respond to increases in membrane potential (hyper-polarization) with a decrease in �uorescence excited at approximately 440 nm and an increase in �uorescence excited at 530 nm.7,9 �ese spectral shi�s permit the use of ratiometric methods (Loading and Calibration of Intracellular Ion Indicators—Note 19.1) to correlate the change in �uorescence signal with membrane potential.2 Using di-8-ANEPPS, Loew and colleagues were able to follow changes in membrane potential along the surface of a single mouse neuroblas-toma cell in their study of the mechanisms underlying cathode-directed neurite elongation 10 and to de�ne di�erences between transmembrane potentials of neurites and somata.11 Potential-dependent �uorescence emission ratio measurements (ratio of emission intensities at 560 nm and 620 nm following excitation at 475 nm) have also been reported using both di-4-ANEPPS and di-8-ANEPPS 12–15 (Figure 22.2.3). Some other applications are listed in Table 22.1.

22.2 Fast-Response Probes

Figure 22.2.3 Detection of action potentials in intact rabbit hearts using the fast potentiometric probe di-4-ANEPPS (D1199). Excised rabbit hearts were loaded with di-4-ANEPPS by perfusion with dye-containing me-dium. Fluorescence was excited at 488 nm by an argon-ion laser. Emission components at 540 ± 6 nm (green) and >610 nm (red) were detected simultaneously by two pho-tomultipliers (panel A). The ratio of the green to red signals (panel B) displayed a larger fractional change during action potential cycles than either of the component signals; it also followed transmembrane voltage contours recorded simul-taneously by an intracellular microelectrode (panel C). In ad-dition, �uorescence ratio measurements reduce the motion artifacts that typically distort optical signals detected from contracting hearts. Figure reproduced with permission from Am J Physiol Heart Circ Physiol (2000) 279:H1421.

0

0

100

100

200

200

300

300

100

1.06

94

1.00

96

1.02

98

1.04

102

1.08

Time (ms)

Time (ms)

A

B

Green signal

Red signal

Rat

io

-50

-100

0

100 ms

C

Mic

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sign

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Fluo

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REFERENCES1. Nature (1998) 392:78; 2. Nature (2009) 461:930; 3. Nat Rev Neurosci (2008) 9:195; 4. Methods Mol Biol (2009) 489:43; 5. Nat Protoc (2008) 3:249; 6. J Physiol Paris (2010) 104:40; 7. Trends Neurosci (2000) 23:166; 8. Methods (2000) 21:271; 9. Cytometry A (2009) 75:593; 10. Methods Mol Biol (1998) 91:85; 11. Circ Res (2009) 104:670.

optical responses is much larger than that of fast-response probes (typically a 1% �uores-cence change per mV). Slow-response probes, which include cationic carbocyanines and rhodamines and anionic oxonols, are suitable for detecting changes in average membrane potentials of nonexcitable cells caused by respiratory activity, ion-channel permeability, drug binding and other factors.

Calibration of potentiometric probes can be accomplished by imposing a transmembrane potential using valinomycin or gramicidin (V1644, G6888; Section 21.1) in conjunction with externally applied K+ solutions.9,10 �e ultimate test of calibration veracity is quantitative agree-ment with electrophysiological measurements.5,11







Cationic ANEP DyesIn collaboration with Leslie Loew and Joe Wuskell of the University of Connecticut, we

o�er a series of potential-sensitive cationic ANEP dyes.16–18 �e water-soluble di-2-ANEPEQ 19 (JPW 1114, D6923; Figure 22.2.5) can be either microinjected into cells, a mode of delivery that intensi�es the staining of remote neuronal processes, or applied topically to deeply stain brain tissue.20 Microinjection of di-2-ANEPEQ into neurons in ganglia of the snail Helix aspersa pro-duced an approximately 50-fold improvement in voltage-sensitive signals from distal processes over that obtained with conventional absorption– and �uorescence-based staining methods.21 Di-12-ANEPPQ (D6927) is useful for potential-sensitive retrograde labeling of neurons 17,22 us-ing techniques similar to those employed for lipophilic carbocyanine and aminostyryl trac-ers (Section 14.4). Di-3-ANEPPDHQ (D36801, Figure 22.2.6) and di-4-ANEPPDHQ (D36802, Figure 22.2.7) both exhibit very low rates of internalization and good signal-to-noise ratios, and are useful for neural network analysis.23 Di-4-ANEPPDHQ has proven useful for visualizing cholesterol-enriched lipid domains in model membranes.24

RH DyesOriginally synthesized by Rina Hildesheim, the RH dyes include an extensive series of di-

alkylaminophenylpolyenylpyridinium dyes that are principally used for functional imaging of neurons (Table 22.1). �e existence of numerous RH dye analogs re�ects the observation that no single dye provides the optimal response under all experimental conditions.5,25,26 Currently, the most widely used RH dyes are RH 237 (S1109, Figure 22.2.8), RH 414 (T1111, Figure 22.2.9), RH 421 (S1108) and RH 795 (R649, Figure 22.2.10). Physiological e�ects of staining with di�er-ent analogs are not equivalent. For example, staining of the cortex with RH 414 causes arterial constriction, whereas staining with RH 795 does not.27 RH 795 produced negligible side e�ects

Figure 22.2.4 Absorption and �uorescence emission spectra of di-8-ANEPPS bound to phospholipid bilayer membranes.

Figure 22.2.5 Di-2-ANEPEQ (JPW 1114, D6923).

Figure 22.2.6 Di-3-ANEPPDHQ (D36801).

2 Br

CH CHN(CH2CH2CH3)2

HOCH2CH2NCH2CHCH2NOHCH3

CH3

2 Br

CH CHN�(CH2)3CH3�2

HOCH2CH2NCH2CHCH2NOHCH3

CH3

Figure 22.2.7 Di-4-ANEPPDHQ (D36802).

Table 22.1 Characteristics and selected applications of Molecular Probes® fast-response probes.

Dyes (Cat. No.) Structure (Charge) Optical Response Selected Applications

Di-4-ANEPPS (D1199)Di-8-ANEPPS (D3167)Di-2-ANEPEQ (D6923)Di-12-ANEPPQ (D6927)Di-3-ANEPPDHQ (D36801)Di-4-ANEPPDHQ (D36802)

Styryl (cationic or zwitterionic)

FAST; �uorescence excitation ratio 440/505 nm decreases upon membrane hyperpolarization

• Combined optical potentiometric and electrophysiological measurements 1

• Combined potentiometric and Ca2+ measurements 2,3

• Imaging electrical activity from intact heart tissues 4–6

• Mapping of membrane potentials along neurons 7–9 and muscle �bers 10

• Membrane potential changes in response to pharmacological stimuli 11,12

• Two-photon excitation microscopy 13–16

RH 237 (S1109)RH 414 (T1111)RH 421 (S1108)RH 795 (R649)

Styryl (cationic or zwitterionic)

FAST; �uorescence decreases upon membrane depolarization

• Electrical activity of cardiomyocytes and cardiac tissue 17,18

• Functional tracing of neurons 19,20

• Membrane potentials evoked by visual 21 and auditory 22,23 stimuli

1. Biophys J (1998) 74:48; 2. J Biol Chem (2006) 281:40302; 3. Cardiovasc Res (2005) 65:83; 4. Am J Physiol Heart Circ Physiol (2005) 289:H2602; 5. Circ Res (2004) 95:21; 6. Am J Physiol Heart Circ Physiol (2004) 287:H985; 7. Neuron (2008) 58:763; 8. Science (2007) 317:819; 9. Proc Natl Acad Sci U S A (2006) 103:16550; 10. J Membr Biol (2005) 208:141; 11. Am J Physiol (1998) 274:H60; 12. Pharmacol Res (1996) 34:125; 13. Methods Mol Biol (2009) 489:43; 14. J Neurophysiol (2008) 99:1545; 15. J Gen Physiol (2006) 127:623; 16. J Neurosci Methods (2005) 148:94; 17. Am J Physiol Heart Circ Physiol (2008) 294:H1417; 18. Biophys J (2007) 92:448; 19. Nat Protoc (2008) 3:249; 20. J Neurosci Methods (1994) 54:151; 21. Proc Natl Acad Sci U S A (2006) 103:12586; 22. Front Neuroengineering (2009) 2:2.1-; 23. Proc Natl Acad Sci U S A (2006) 103:1918.

(CH CH)3 N�(CH2)3CH3�2O3�(CH2)�N

Figure 22.2.8 RH 237 N-(4-sulfobutyl)-4-(6-(4-(dibutylamino) –phenyl)hexatrienyl)pyridinium, inner salt (S1109).

Figure 22.2.9 N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)pyridinium dibromide (RH 414, T1111).

Figure 22.2.10 RH 795 (R649).








when tested in vitro using hippocampal slices and in vivo using single-unit recordings in cat and monkey visual cortices.28 Electrophysiological measurements indicate a broadening of action potentials that is attributable to the staining of cultured neurons with RH 237.29

Like the ANEP dyes, the RH dyes exhibit varying degrees of �uorescence excitation and emission spectral shi�s in response to membrane potential changes.8 �eir absorption and �uo-rescence spectra are also strongly dependent on the environment.30 Spectra of RH 414 bound to phospholipid vesicles are similar to those obtained on neuronal plasma membranes.8 Using the RH dyes in conjunction with �uorescent Ca2+ indicators allowed the simultaneous optical mapping of membrane potential (with RH 237) and intracellular calcium (with rhod-2 AM; R1245MP, R1244; Section 19.3) in cardiomyocyte monolayers; rhod-FF (R23983, Section 19.3) was used to check for bu�ering of calcium dynamics by the high-a�nity rhod-2 indicator.31

FRET-Pair Membrane Potential SensorsFluorescence resonance energy transfer (FRET) between a mobile lipophilic anion in the

membrane interior and a static donor �uorophore on the membrane surface provides a potential-sensing mechanism that generates a more sensitive �uorescence response than electrochromic dyes and a more rapid temporal response than intracellular–extracellular ion translocation.32,33 A particularly e�ective implementation of this concept uses DiOC18(3) or DiOC16(3) (D275, V22886, D1125; Section 14.4) as the static reference marker in combination with the mobile anion dipic-rylamine. Characterization of this approach by Bradley and co-workers 34 demonstrated depolar-ization-induced �uorescence changes of >50% per 100 mV with submillisecond time constants in whole-cell patch-clamped HEK^s293 cells. In neuronal cultures and brain slices, action potentials generated �uorescence increases relative to the resting baseline signal of >25% per 100 mV.

PRODUCT LIST 22.2 FAST-RESPONSE PROBESCat. No. Product QuantityD6923 di-2-ANEPEQ (JPW 1114) 5 mgD36801 di-3-ANEPPDHQ 1 mgD36802 di-4-ANEPPDHQ 1 mgD6927 di-12-ANEPPQ 5 mgD1199 di-4-ANEPPS 5 mgD3167 di-8-ANEPPS 5 mgR649 RH 795 1 mgS1109 N-(4-sulfobutyl)-4-(6-(4-(dibutylamino)phenyl)hexatrienyl)pyridinium, inner salt (RH 237) 5 mgS1108 N-(4-sulfobutyl)-4-(4-(4-(dipentylamino)phenyl)butadienyl)pyridinium, inner salt (RH 421) 25 mgT1111 N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)pyridinium dibromide (RH 414) 5 mg

DATA TABLE 22.2 FAST-RESPONSE PROBESCat. No. MW Storage Soluble Abs EC Em Solvent NotesD1199 480.66 D,L DMSO, EtOH 497 42,000 705 MeOH 1D3167 592.88 D,L DMSO, EtOH 498 37,000 713 MeOH 1D6923 549.39 D,L DMSO, EtOH 517 36,000 721 EtOHD6927 843.95 D,L DMSO, EtOH 519 36,000 719 EtOH 1D36801 637.50 F,D,L DMSO, EtOH 512 36,000 712 EtOH 1D36802 665.55 F,D,L DMSO, EtOH 512 36,000 712 EtOH 1, 2R649 585.42 D,L DMSO, EtOH 530 47,000 712 MeOH 1S1108 498.72 D,L DMSO, EtOH 515 50,000 704 MeOH 1, 3S1109 496.71 D,L DMSO, EtOH 528 53,000 782 MeOH 1, 3T1111 581.48 D,L DMSO, EtOH 532 55,000 716 MeOH 1For de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. Abs and Em of styryl dyes are at shorter wavelengths in membrane environments than in reference solvents such as methanol. The di�erence is typically 20 nm for absorption and 80 nm for emission, but varies considerably from one dye to another. Styryl dyes are generally non�uorescent in water.

2. The �uorescence excitation/emission maxima of di-4-ANEPPDHQ (D36802) bound to dioleoylphosphatidylcholine (DOPC) bilayer membranes are 468/635 nm. Em is sensitive to membrane cholesterol content. (Biophys J (2006) 90:2563)

3. Abs/Em for these dyes adsorbed on neuronal plasma membranes are 493/638 nm (S1108) and 506/687 nm (S1109). (Biochim Biophys Acta (1993) 1150:111)

REFERENCES1. Biochemistry (1985) 24:5749; 2. Biophys J (1998) 74:48; 3. J Membr Biol (1992) 130:1; 4. Biophys J (1999) 76:2272; 5. Biophys J (1994) 67:1301; 6. P�ugers Arch (1994) 426:548; 7. Biochemistry (1989) 28:4536; 8. Biochim Biophys Acta (1993) 1150:111; 9. Biophys J (1994) 67:208; 10. Neuron (1992) 9:393; 11. Neuron (1994) 13:1187; 12. Am J Physiol Heart Circ Physiol (2000) 279:H1421; 13. Biophys J (2001) 81:1163; 14. Am J Physiol (1996) 270:H2216; 15. Am J Physiol (1998) 274:H60; 16. Neuron (2003) 37:85; 17. J Neurosci Methods (1996) 70:121; 18. Pure Appl Chem (1996) 68:1405; 19. Biol Bull (2000) 198:1; 20. Proc Natl Acad Sci U S A (1997) 94:7621; 21. J Neurosci (1995) 15:1392; 22. J Neurosci Methods (1996) 70:111; 23. J Neurosci Methods (2004) 134:179; 24. Biophys J (2006) 90:2563; 25. Physiol Rev (1988) 68:1285; 26. Annu Rev Neurosci (1985) 8:263; 27. Nature (1986) 324:361; 28. J Neurosci (1994) 14:2545; 29. Biophys J (1995) 69:299; 30. Biophys J (1995) 68:1406; 31. Am J Physiol Heart Circ Physiol (2008) 294:H1417; 32. Chem Biol (1997) 4:269; 33. Biophys J (1995) 69:1272; 34. J Neurosci (2009) 29:9197.






Section 22.3 Slow-Response Probes

22.3 Slow-Response ProbesOur slow-response potential-sensitive probes1 (see Figure 22.1.1B in Section 22.1) are listed in Table 22.2, along with their charges, optical

responses and selected applications.

Table 22.2 Characteristics and selected applications of Molecular Probes® slow-response probes.

Dyes (Cat. No.) Structure (Charge) Optical Response Selected Applications

• DiOC2(3) (D14730)• DiOC5(3) (D272)• DiOC6(3) (D273)• DiSC3(5) (D306)• DiIC1(5) (H14700)

Carbocyanine (cationic) SLOW; �uorescence response to depolarization depends on the staining concentration and detection method

• Bacterial infection 1–3

• Flow cytometry assays of membrane potential 4

• Membrane potential in intact yeast cells 5

• Mitochondrial activity 6–8

• Oxidative stress 9,10

• JC-1 (T3168)• JC-9 (D22421)

Carbocyanine (cationic) SLOW; �uorescence emission ratio 585/520 nm increases upon membrane hyperpolarization

• Apoptotic mitochondrial depolarization 11–13

• Ca2+ regulation by mitochondria 14–16

• Mitochondrial function in brain slices 17 and cultured neurons 18,19

• Mitochondrial response to glutamate excitotoxicity 20,21

• Mitochondrial response to oxidative stress 22

• Tetramethylrhodamine methyl and ethyl esters (T668, T669)

• Rhodamine 123 (R302, R22420)

Rhodamine (cationic) SLOW; used to obtain unbiased images of potential-dependent dye distribution

• Ca2+ regulation by mitochondria 23–25

• Mitochondrial permeability transition 26–28

• Oxidative stress 29,30

• Stem cells 31

• Oxonol V (O266)• Oxonol VI (O267)

Oxonol (anionic) SLOW; �uorescence decreases upon membrane hyperpolarization

• Ion channels and electrogenic pumps 32–36

• Liposomes 37–39

• Plant physiology 40–42

• DiBAC4(3) (B438, B24570)• DiBAC4(5) (B436)• DiSBAC2(3) (B413)

Oxonol (anionic) SLOW; �uorescence decreases upon membrane hyperpolarization

• ATP-sensitive K+ channel activation 43–46

• Combined potentiometric and Ca2+ measurements 47–49

• Confocal imaging of membrane potential 50

• Flow cytometry assays of cell viability 51–54

• Primary vascular smooth muscle cells 47

• Merocyanine 540 (M24571) Merocyanine FAST/SLOW (biphasic response) • Membrane lipid asymmetry 55,56

• Membrane potentials in mitochondria 57,58 and skeletal muscle 59

• Photodynamic therapy• Structure of membrane surfaces 60–62

1. J Biol Chem (2007) 282:7742; 2. Antimicrob Agents Chemother (2005) 49:1127; 3. Appl Environ Microbiol (2002) 68:37; 4. J Biol Chem (2007) 282:18069; 5. Anal Biochem (2001) 293:269; 6. J Biol Chem (2006) 281:13990; 7. J Biol Chem (1998) 273:33942; 8. Cytometry (1998) 33:333; 9. J Biol Chem (2006) 281:6726; 10. Science (2003) 299:1751; 11. Methods Cell Biol (2001) 63:467; 12. J Cell Biol (1997) 138:449; 13. FEBS Lett (1997) 411:77; 14. J Biol Chem (2000) 275:38680; 15. J Physiol (1998) 509:81; 16. Biophys J (1998) 75:2004; 17. Methods (1999) 18:104; 18. J Biol Chem (2009) 284:9540; 19. Mol Biol Cell (2008) 19:150; 20. J Neurosci (1996) 16:5688; 21. Neuron (1995) 15:961; 22. J Biol Chem (2007) 282:24146; 23. Methods Mol Biol (2007) 372:421; 24. J Biol Chem (2001) 276:23329; 25. J Neurosci (2000) 20:7290; 26. J Biol Chem (2009) 284:15117; 27. Biochem J (1999) 343; 28. Biophys J (1998) 74:2129; 29. J Biol Chem (2009) 284:14476; 30. J Biol Chem (2009) 284:18754; 31. Nat Methods (2010) 7:61; 32. J Biol Chem (1993) 268:23122; 33. Biochemistry (1990) 29:3859; 34. Biochim Biophys Acta (1990) 1023:81; 35. Biochim Biophys Acta (1990) 1017:221; 36. Biochim Biophys Acta (1989) 980:139; 37. Biochim Biophys Acta (1993) 1146:87; 38. Biochem Biophys Res Commun (1990) 173:1008; 39. Biophys Chem (1989) 34:225; 40. J Biol Chem (1992) 267:21850; 41. Plant J (1992) 2:97; 42. Biophys J (1999) 76:360; 43. Br J Pharmacol (2000) 129:1323; 44. J Physiol (1999) 517:781; 45. J Physiol (1997) 502:397; 46. P�ugers Arch (1997) 434:712; 47. Circ Res (2009) 104:670; 48. J Neurosci (2007) 27:8238; 49. Methods (2000) 21:335; 50. Exp Cell Res (1997) 231:260; 51. Methods Cell Biol (2001) 64:553; 52. J Appl Microbiol (1998) 84:988; 53. Yeast (1998) 14:147; 54. J Microbiol Methods (1998) 32:45; 55. Biochim Biophys Acta (1991) 1062:24; 56. Proc Natl Acad Sci U S A (1986) 83:3311; 57. J Biol Chem (1991) 266:803; 58. J Membr Biol (1991) 123:23; 59. J Gen Physiol (1990) 95:147; 60. Biochim Biophys Acta (1993) 1146:169; 61. Biochim Biophys Acta (1992) 1107:245; 62. J Cell Physiol (1989) 138:61.

CarbocyaninesDiI, DiS and DiO Derivatives

Indo- (DiI), thia- (DiS) and oxa- (DiO) carbocyanines with short alkyl tails (<7 carbon at-oms) were among the �rst potentiometric �uorescent probes developed.2 �ese cationic dyes ac-cumulate on hyperpolarized membranes and are translocated into the lipid bilayer.3 Aggregation within the con�ned membrane interior usually results in decreased �uorescence, although the magnitude and even the direction of the �uorescence response is strongly dependent on the concentration of the dye and its structural characteristics.1,4 While the distribution of extracell-ularly applied dye is dependent on both the plasma and mitochondrial membrane potentials, the primary determinant is the latter. Very low applied concentrations (<100 nM) are required to obtain mitochondrial signal speci�city and avoid potential-independent background derived from staining of the endoplasmic reticulum and other intracellular membranes.

DiOC6(3) (D273, Figure 22.3.1) has been the most widely used carbocyanine dye for mem-brane potential measurements,5 followed closely by DiOC5(3) (D272, Figure 22.3.2); see Table 22.2 for selected references. In �ow cytometry measurements, the detected intensity of carbocyanine

Figure 22.3.1 3,3´-dihexyloxacarbocyanine iodide (DiOC6(3), D273).

Figure 22.3.2 3,3´-dipentyloxacarbocyanine iodide (DiOC5(3), D272).

N

O O

N(CH2)�(CH2)�

CH CH CH

CH3CH3

�








�uorescence is dependent not only on the membrane potential, but also on cell size. In some cas-es, measurements of forward light scatter have been used to normalize the optical changes for cell size variability. A �uorescence ratio method (Figure 22.3.3) that exploits a potential-dependent red shi� in the emission spectrum of DiOC2(3) (D14730, Figure 22.3.4) has been developed for membrane potential measurements in bacteria 6,7 (B34950). �e 633 nm light–excitable indodi-carbocyanine DiIC1(5) 8 (H14700, Figure 22.3.5) enables analysis of mitochondrial potential in apoptotic cells in combination with �uoresceinated annexin V 9 (A13199, Section 15.5), a method we have utilized in two of our MitoProbe™ Assay Kits for �ow cytometry (M34150, M34151; see below). Carbocyanine dyes, particularly thiacyanines such as DiSC3(5) (D306, Figure 22.3.6), can inhibit respiration 4,10 and may therefore be relatively cytotoxic.11

JC-1 and JC-9JC-1 (5,5 ,ʹ6,6 -́tetrachloro-1,1́ ,3,3 -́tetraethylbenzimidazolylcarbocyanine iodide, T3168;

Figure 22.3.7) exists as a green-�uorescent monomer at low concentrations or at low membrane potential. However, at higher concentrations (aqueous solutions above 0.1 µM) or higher po-tentials, JC-1 forms red-�uorescent “J-aggregates,” which exhibit a broad excitation spectrum and a very narrow emission spectrum (Figure 22.3.8). Because J-aggregate formation increases linearly with applied membrane potential over the range of 30–180 mV, this phenomenon can be exploited for potentiometric measurements 12,13 (Table 22.2). JC-1 is more speci�c for mitochon-drial versus plasma membrane potential and more consistent in its response to depolarization than some other cationic dyes such as DiOC6(3) and rhodamine 123.14

Various types of ratio measurements are possible by combining signals from the green-�uorescent JC-1 monomer (absorption/emission maxima ~514/529 nm) and the red-�uorescent J-aggregate (Figure 22.3.9) (absorption/emission maxima ~585/590 nm), which can be e�ectively excited anywhere between 485 nm and its absorption maximum. Optical �lters designed for �uorescein and tetramethylrhodamine can be used to separately visualize the monomer and J-aggregate forms, respectively. Alternatively, both forms can be observed simultaneously using a �uorescein longpass optical �lter set. For �ow cytometry, JC-1 can be excited at 488 nm and detected in bivariate mode using the green channel for the monomer and the red channel for the J-aggregate form 15,16 (Figure 22.3.10).

JC-1 is widely used for detecting mitochondrial depolarization in apoptotic cells 14,15,17–19 (MitoProbe™ JC-1 Assay Kit, M34152, described below) and for assaying multidrug-resistant cells 20 (Section 15.6). It is also frequently employed for mitochondrial function assessment in cell-based high-throughput assays.21,22 We have discovered another carbocyanine dye, JC-9 (3,3 -́dimethyl-α-naphthoxacarbocyanine iodide, D22421; Figure 22.3.11), with potential-de-pendent spectroscopic properties (Figure 22.3.12) similar to those of JC-1 for detecting mito-chondrial depolarization in apoptotic cells.23,24

MitoProbe™ JC-1 Assay Kit for Flow Cytometry�e MitoProbe™ JC-1 Assay Kit (M34152) provides the cationic dye JC-1 and a mitochondrial

membrane potential disrupter, CCCP (carbonyl cyanide 3-chlorophenylhydrazone), for the study of mitochondrial membrane potential. Use of JC-1 �uorescence ratio detection allows researchers to make comparative measurements of membrane potential and to determine the percentage of mitochondria within a population that respond to an applied stimulus (Figure 22.3.13). Subtle heterogeneity in cellular responses can be discerned in this way.13,15,18,25 For example, four dis-tinct patterns of mitochondrial membrane potential change in response to glutamate receptor activation in neurons have been identi�ed using confocal ratio imaging of JC-1 �uorescence.26

Figure 22.3.3 A) Two-color �ow cytometric analysis of Staphylococcus aureus populations stained with 30 µM DiOC2(3) (D14730) in the presence (red) or absence (blue) of the metabolic uncoupler carbonyl cyanide m-chlorophenyl-hydrazone (CCCP). Note the variability (~100-fold range) of the green and red �uorescence intensities. B) The same data expressed as red/green �uorescence intensity ratios. Ratio values are calculated by subtracting the logarithmic green �uorescence channel value from the corresponding loga-rithmic red �uorescence channel value. Figure supplied by Howard M. Shapiro, Harvard Medical School, Boston, MA.

100 101 102 103 104

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en �

uore

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ce (5

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Figure 22.3.7 5,5´,6,6´-tetrachloro-1,1´,3,3´-tetra ethyl-benzimidazolylcarbocyanine iodide (JC-1; CBIC2(3), T3168).

N

O O

NCH2CH3CH2CH3

�

CH CH CH

Figure 22.3.4 3,3´-diethyloxacarbocyanine iodide (DiOC2(3), D14730).

Figure 22.3.5 1,1´,3,3,3´,3´-hexamethylindodicarbocyanine iodide (DiIC1(5), H14700).

(CH CH)2 CHN

� �

N

(CH2)2(CH2)2

C�CH3 CH3

Figure 22.3.6 3,3´-dipropylthiadicarbocyanine iodide (DiSC3(5), D306).







Figure 22.3.8 Cultured human pre-adipocytes loaded with the ratiometric mitochondrial potential indicator JC-1 (T3168) at 5 µM for 30 minutes at 37°C. In live cells, JC-1 exists either as a green-�uorescent monomer at depolarized membrane po-tentials or as an orange-�uorescent J-aggregate at hyperpolar-ized membrane potentials. Cells were then treated with 50 nM FCCP, a protonophore, to depolarize the mitochondrial mem-brane. Approximately 10 minutes after the addition of the uncou-pler, the cells were illuminated at 488 nm and the emission was collected between 515–545 nm and 575–625 nm. Image contrib-uted by Bob Terry, BioImage A/S, Denmark.

Each MitoProbe™ JC-1 Assay Kit provides:

• JC-1• Dimethylsulfoxide (DMSO)• CCCP• Concentrated phosphate-bu�ered saline (PBS)• Detailed protocols

Su�cient reagents are provided for 100 assays, based on a labeling volume of 1 mL.

MitoProbe™ DiIC(5) and MitoProbe™ DiOC(3) Assay Kits for Flow Cytometry�e MitoProbe™ DiIC1(5) and MitoProbe™ DiOC2(3) Assay Kits (M34151, M34150) pro-

vide solutions of the far-red–�uorescent DiIC1(5) (1,1́ ,3,3,3 ,́3 -́hexamethylindodicarbocyanine

Figure 22.3.10 Bivariate JC-1 (T3168) analysis of mitochondrial membrane potential in HL60 cells by �ow cytometry. The sen-sitivity of this technique is demonstrated by the response to depolarization using K+/valinomycin (V1644) (bottom two pan-els). Distinct populations of cells with di�erent extents of mitochondrial depolarization are detectable following apoptosis-inducing treatment with 5 µM staurosporine for 2 hours (top right panel). Figure courtesy of Andrea Cossarizza, University of Modena and Reggio Emilia, Italy.

100 101 102 103 104

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Control Staurosporine

Valinomycin Valinomycin +staurosporine

Figure 22.3.11 3,3´-dimethyl-α-naphthoxacarbocyanine iodide (JC-9; DiNOC1(3), D22421).

Figure 22.3.12 A viable bovine pulmonary artery endothe-lial cell incubated with the ratiometric mitochondrial poten-tial indicator, JC-9 (D22421). In live cells, JC-9 exists either as a green-�uorescent monomer at depolarized membrane potentials, or as a red-�uorescent J-aggregate at hyperpo-larized membrane potentials.

Figure 22.3.9 Absorption and �uorescence emission (excited at 488 nm) spectra of JC-1 in pH 8.2 bu�er containing 1% (v/v) DMSO.

Figure 22.3.13 Flow cytometric analysis of Jurkat cells us-ing the MitoProbe™ JC-1 Assay Kit (M34152). Jurkat cells were stained with 2 µM JC-1 for 15 minutes at 37°C, 5% CO2, and then washed with phosphate-bu�ered saline (PBS) and analyzed on a �ow cytometer using 488 nm excitation with 530 nm and 585 nm bandpass emission �lters. Untreated cul-tured cells are shown in panel A. Panel B shows cells induced to apoptosis with 10 µM camptothecin for 4 hours at 37°C.

A

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iodide) and green-�uorescent DiOC2(3) (3,3 -́diethyloxacarbocyanine iodide) carbocyanine dyes, respectively, along with a mitochondrial membrane potential uncoupler, CCCP, for the study of mitochondrial membrane potential. �ese DiIC1(5) and DiOC2(3) carbocyanine dyes penetrate the cytosol of eukaryotic cells and, at concentrations below 100 nM, accumulate primarily in mitochondria with active membrane potentials. In the case of DiOC2(3), this accumulation is accompanied by a shi� from green to red emission due to dye stacking (Figure 22.3.3), allowing the use of a ratiometric parameter (red/green �uorescence ratio) that corrects for size di�erences when measuring membrane potential in bacteria.6,7 DiOC2(3) can be paired with other reagents, such as the far-red–�uorescent allophycocyanin annexin V (A35110, Section 15.5) or TO-PRO®-3 nucleic acid stain (T3605, Section 8.1) for multiparameter study of vitality and apoptosis 27 (Figure 22.3.14).

�e MitoProbe™ DiIC1(5) and MitoProbe™ DiOC2(3) Assay Kits provide:

• DiIC1(5) (in Kit M34151) or DiOC2(3) (in Kit M34150)• CCCP• Detailed protocols for labeling cells

Each kit provides su�cient reagents for 100 assays, based on a labeling volume of 1 mL.

BacLight™ Bacterial Membrane Potential Kit�e BacLight™ Bacterial Membrane Potential Kit (B34950) pro-

vides a �uorescent membrane-potential indicator dye, DiOC2(3), along with a proton ionophore (CCCP) and premixed bu�er. At low concen-trations, DiOC2(3) exhibits green �uorescence in all bacterial cells, but it becomes more concentrated in healthy cells that are maintain-ing a membrane potential, causing the dye to self-associate and the �uorescence emission to shi� to red. �e red- and green-�uorescent bacterial populations are easily distinguished using a �ow cytome-ter (Figure 22.3.15). CCCP is included in the kit for use as a control because it eradicates the proton gradient, eliminating bacterial mem-brane potential.7,27

�e BacLight™ Bacterial Membrane Potential Kit contains:

• DiOC2(3) in dimethylsulfoxide (DMSO)• CCCP in DMSO• Phosphate-bu�ered saline (PBS)• Detailed protocols

Using the recommended reagent dilutions and volumes, this kit provides su�cient DiOC2(3) to perform approximately 100 individual assays by �ow cytometry; su�cient CCCP is provided for 30 depolar-ized control samples. �e BacLight™ Bacterial Membrane Potential Kit is designed to assay bacterial concentrations between 105 and 107 organisms per mL. Note that DiOC2(3) and CCCP are inhibitors of respiration, rendering the cells nonculturable beyond the brief time window required for staining and analysis.

Using the BacLight™ Bacterial Membrane Potential Kit, we have detected membrane potentials in all bacteria tested (including loga-rithmically growing cultures of Micrococcus luteus, Staphylococcus aureus, Bacillus cereus, Staphylococcus warnerii, Escherichia coli and Salmonella choleraesuis), although the magnitude varies with species (Figure 22.3.16).

Figure 22.3.14 Flow cytometric analysis of camptothecin-treated Jurkat cells stained with DiOC2(3) (D14730, M34150) and allophycocyanin annexin V (A35110). Jurkat cells were in-cubated for 4 hours with camptothecin at 37°C, 5% CO2, then stained with 50 nM DiOC2(3) and allophycocyanin annexin V. Cells were analyzed on a �ow cytometer using 488 nm and 633 nm excitations with 530 nm and 660 nm bandpass emission �lters.

Allophycocyanin �uorescence

DiO

C2(

3) g

reen

�uo

resc

ence

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Figure 22.3.16 Detection of membrane potential in various bacteria with the BacLight™ Bacterial Membrane Potential Kit (B34950). Red/green �uorescence ratios were calcu-lated using population mean �uorescence intensities for gram-positive (Micrococcus lu-teus, Staphylococcus aureus, Bacillus cereus and Staphylococcus warnerii) and gram-negative (Escherichia coli and Salmonella choleraesuis) bacteria incubated with 30 µM DiOC2(3) for 30 minutes in either the presence or absence of 5 µM CCCP, according to the kit protocol.

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+ CCCP

Figure 22.3.15 Response of Staphylococcus aureus to valinomycin and external potassium ions, as measured by �ow cytometry using the BacLight™ Bacterial Membrane Potential Kit (B34950). Samples containing S. aureus were treated with 5 µM valinomycin in di�erent con-centrations of potassium bu�er, and then stained using 30 µM DiOC2(3) for 30 minutes, ac-cording to the kit protocol. Data are expressed either using a ratiometric parameter based on the formula provided in the kit protocol (n, right axis) or as the ratio of population mean red-�uorescence intensity/mean green-�uorescence intensity (d, left axis).

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Rhodamine 123, TMRM and TMRERhodamine 123 (R302, R22420; Figure 22.3.17) is widely used as a structural marker

for mitochondria (Section 12.2) and as an indicator of mitochondrial activity (Section 15.2, Figure 22.3.18). Highly selective, potential-dependent staining of mitochondria is obtained by setting the extracellular K+ concentration close to intracellular values (~137 mM), thereby depo-larizing the plasma membrane.28

TMRM (T668, Figure 22.3.19) and TMRE (T669, Figure 22.3.20), the methyl and ethyl esters of tetramethylrhodamine, are closely related to rhodamine 123. �ey are primarily mi-tochondrial membrane potential sensors.29,30 As with rhodamine 123, accumulation of these cationic dyes in mitochondria results in diminished �uorescence due to self-quenching (Figure 22.3.18).30 TMRM and TMRE cross the plasma membrane more rapidly than rhodamine 123, and their strong �uorescence allows the use of low probe concentrations, thus avoiding aggrega-tion. Because their �uorescence is relatively insensitive to the environment, spatially resolved �uorescence of TMRM and TMRE presents an unbiased pro�le of their transmembrane dis-tribution that can be directly related to the plasma membrane potential via the Nernst equa-tion.30–34 TMRE has been successfully used in a high-throughput screening assay for drugs that a�ect mitochondrial membrane potential in live cells.35 TMRM has been used in conjunction with X-rhod-1 AM, (X14210, Section 19.3) for simultaneous confocal imaging of mitochondrial membrane potential and calcium in rat cardiomyocytes.36

OxonolsOxonol V and Oxonol VI

�e anionic bis-isoxazolone oxonols (O266, Figure 22.3.21; O267, Figure 22.3.22) accumu-late in the cytoplasm of depolarized cells by a Nernst equilibrium–dependent uptake from the extracellular solution.37 �eir voltage-dependent partitioning between water and membranes is o�en measured by absorption rather than �uorescence. Of the oxonols studied by Smith and Chance,38 oxonol VI (O267) gave the largest spectral shi�s, with an isosbestic point at 603 nm. In addition, oxonol VI responds to changes in potential more rapidly than oxonol V and is therefore considered to be the better probe for measuring fast potential changes.39

DiBAC (Bis-Oxonol) Dyes�e three bis-barbituric acid oxonols, o�en referred to as DiBAC dyes, form a family of

spectrally distinct potentiometric probes with excitation maxima at approximately 490 nm (DiBAC4(3); B438, B24570; Figure 22.3.23), 530 nm (DiSBAC2(3), B413; Figure 22.3.24) and

Figure 22.3.17 Rhodamine 123 (R302).

Figure 22.3.19 Tetramethylrhodamine, methyl ester, per-chlorate (TMRM, T668).

Figure 22.3.20 Tetramethylrhodamine, ethyl ester, per-chlorate (TMRE, T669).

Figure 22.3.18 Staining of rat cortical astrocytes by rhodamine 123 (R302, R22420). Potential-dependent accumulation of the cationic dye in mitochondria results in a relatively weak �uorescence signal due to self-quenching (left panel). Dissipation of the mitochondrial membrane potential by the uncoupler FCCP is marked by increasing �uorescence (middle panel) and subsequent redistribution of the dye throughout the cell (right panel). Images courtesy of Michael Duchen, University College, London.

Figure 22.3.21 Oxonol V (bis-(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol, O266).

(CH CH)2 CHNO O

N

OH O

Figure 22.3.22 Oxonol VI (bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol, O267).

(CH CH)2 CHNO O

N

OH O

(CH2)2CH3

(CH2)2CH3








590 nm (DiBAC4(5), B436; Figure 22.3.25). Several papers have referred to these dyes simply as “bis-oxonol” and it is not always possible to determine which of the dyes was employed; however, DiBAC4(3) (Figure 22.3.23) has been used in the majority of publications that cite a “bis-oxonol.”

�ese dyes enter depolarized cells where they bind to intracellular proteins or membranes and exhibit enhanced �uorescence and red spectral shi�s.40 Increased depolarization results in more in�ux of the anionic dye and thus an increase in �uorescence (Figure 22.3.26). Conversely, hyperpolarization is indicated by a decrease in �uorescence (Figure 22.3.27). In contrast to cationic carbocyanines, anionic bis-oxonols are largely excluded from mitochondria and are primarily sensitive to plasma membrane potential. Potential-dependent �uorescence changes generated by DiBAC4(3) are typically ~1% per mV.41,42 Interactions between anionic oxonols and the cationic K+-valinomycin complex complicate the use of this ionophore when calibrating po-tentiometric responses.43 Oxonol dyes have known pharmacological activity against various ion channels and receptors.44,45 It is therefore important to establish, as is the case in any experiment using �uorescent probes, that experimental observations with implied physiological signi�cance are independent of the externally applied probe concentration.

Merocyanine 540Although merocyanine 540 (M24571) was among the �rst �uorescent dyes to be used as

a potentiometric probe,46 its use for this application has declined with the advent of superior probes. A signi�cant disadvantage of merocyanine 540 is its extreme phototoxicity; consequent-ly, it is now more commonly used as a photosensitizer.47–56

Merocyanine 540 exhibits a biphasic kinetic response to membrane polarization changes. It binds to the surface of polarized membranes in a perpendicular orientation, reorienting as the membrane depolarizes to form non�uorescent dimers with altered absorption spectra.57,58 �is fast (microseconds) reorientation is followed by a slower response caused by an increased dye uptake.

Merocyanine 540 is also a useful probe of lipid packing because it binds preferentially to membranes with highly disordered lipids.59,60 Fluorescence of merocyanine 540 is sensitive to heat-induced changes in the organization of membrane lipids.61

Figure 22.3.27 Detection of ATP-sensitive potassium (KATP) channel activation in isolated capillaries from guinea pig hearts using DiBAC4(3) (B438, B24570), a slow potentio-metric probe. Application of a K+ channel opener (HOE 234) induced membrane hyperpolarization, resulting in a net e�ux of intracellular DiBAC4(3), which is registered as a de-crease of �uorescence intensity. These e�ects were reversed by subsequent treatment with the channel blocker gliben-clamide. Figure reproduced with permission from J Physiol (1997) 502:397.

0 10 20 30

0.8

0.4

0.5

0.6

1.1

Time (minutes)

Rel

ativ

e D

iBA

C4(

3) �

uore

scen

ce

25155

1.0

0.9

0.7

0.3

100 nM HOE 2341 M glibenclamide

Figure 22.3.26 NIH 3T3 �broblast undergoing pro-gressive depolarization induced by the stepwise in-crease of KCl concentration from 5 mM (bottom pan-el) to 120 mM (top panel). The cell culture was loaded with DiSBAC2(3) (B413) and examined under a confo-cal laser-scanning microscope after each appropriate medium change, keeping a constant plane of section (Exp Cell Res 231, 260 (1997)). The image was contrib-uted by Rita Gatti, Institute of Histology and General Embryology, University of Parma, Parma, Italy.

[K+] = 120 mM

[K+] = 90 mM

[K+] = 60 mM

[K+] = 30 mM

[K+] = 5 mM

Figure 22.3.23 bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3), B438).

Figure 22.3.24 bis-(1,3-diethylthiobarbituric acid)trime-thine oxonol (DiSBAC2(3), B413).

CH CH CHN

NN

N��

O

O

OH

O

CH2CH3CH3CH2

CH3CH2 CH2CH3

Figure 22.3.25 bis-(1,3-dibutylbarbituric acid)pentam-ethine oxonol (DiBAC4(5), B436).







1. J Photochem Photobiol B (1996) 33:101; 2. Biochemistry (1974) 13:3315; 3. J Membr Biol (1986) 92:171; 4. Biophys J (1989) 56:979; 5. Biochim Biophys Acta (1998) 1404:393; 6. Methods (2000) 21:271; 7. Cytometry (1999) 35:55; 8. Cytometry (2000) 41:245; 9. J Biol Chem (2007) 282:18069; 10. J Biol Chem (1981) 256:1108; 11. Biochem Pharmacol (1993) 45:691; 12. Biochemistry (1991) 30:4480; 13. Proc Natl Acad Sci U S A (1991) 88:3671; 14. FEBS Lett (1997) 411:77; 15. Cytometry A (2005) 68:28; 16. Biochem Biophys Res Commun (1993) 197:40; 17. J Biol Chem (2008) 283:5188; 18. Nat Protoc (2007) 2:2719; 19. Methods Cell Biol (2001) 63:467; 20. J Biomol Screen (2008) 13:185; 21. Proc Natl Acad Sci U S A (2008) 105:7387; 22. Nat Biotechnol (2008) 26:343; 23. Toxicol Lett (2009) 191:246; 24. J Am Chem Soc (2005) 127:8686; 25. Exp Cell Res (1996) 222:84; 26. J Neurosci (1996) 16:5688; 27. Antimicrob Agents Chemother (2000) 44:827; 28. Methods Cell Biol (1989) 29:103; 29. Trends Neurosci (2000) 23:166; 30. Biophys J (1999) 76:469; 31. Methods Enzymol (1999) 302:341; 32. Neuron (1995)

15:961; 33. Biophys J (1998) 74:2129; 34. Am J Physiol (1997) 272:C1286; 35. J Biomol Screen (2002) 7:383; 36. Methods Mol Biol (2007) 372:421; 37. Biochim Biophys Acta (1987) 903:480; 38. J Membr Biol (1979) 46:255; 39. Biophys Chem (1989) 34:225; 40. Chem Phys Lipids (1994) 69:137; 41. J Physiol (1997) 502:397; 42. Biochim Biophys Acta (1984) 771:208; 43. Cytometry A (2009) 75:593; 44. J Neurosci (2010) 30:2871; 45. Mol Pharmacol (2007) 71:1075; 46. Annu Rev Biophys Bioeng (1979) 8:47; 47. J Immunol Methods (1994) 168:245; 48. Pharmacol �er (1994) 63:1; 49. Arch Biochem Biophys (1993) 300:714; 50. Bone Marrow Transplant (1993) 12:191; 51. Cancer Res (1993) 53:806; 52. Free Radic Biol Med (1992) 12:389; 53. Biochim Biophys Acta (1991) 1075:28; 54. J Infect Dis (1991) 163:1312; 55. Photochem Photobiol (1991) 53:1; 56. Cancer Res (1989) 49:3637; 57. Biochemistry (1985) 24:7117; 58. Biochemistry (1978) 17:5228; 59. Biochim Biophys Acta (1993) 1146:136; 60. Biochim Biophys Acta (1983) 732:387; 61. Biochim Biophys Acta (1990) 1030:269.

DATA TABLE 22.3 SLOW-RESPONSE PROBESCat. No. MW Storage Soluble Abs EC Em Solvent NotesB413 436.54 L DMSO, EtOH 535 170,000 560 MeOH 1B436 542.67 L DMSO, EtOH 590 160,000 616 MeOH 1B438 516.64 L DMSO, EtOH 493 140,000 516 MeOH 1, 2B24570 516.64 L DMSO, EtOH 493 140,000 516 MeOH 1, 2, 3D272 544.47 D,L DMSO 484 155,000 500 MeOHD273 572.53 D,L DMSO 484 154,000 501 MeOHD306 546.53 D,L DMSO 651 258,000 675 MeOHD14730 460.31 D,L DMSO 482 165,000 497 MeOH 4D22421 532.38 D,L DMSO, DMF 522 143,000 535 CHCl3 5H14700 510.46 L DMSO 638 230,000 658 MeOH 6M24571 569.67 D,L DMSO, EtOH 555 143,000 578 MeOHO266 384.39 L DMSO, EtOH 610 135,000 639 MeOH 1O267 316.36 L DMSO, EtOH 599 136,000 634 MeOH 1R302 380.83 F,D,L MeOH, DMF 507 101,000 529 MeOHR22420 380.83 F,D,L MeOH, DMF 507 101,000 529 MeOH 3T668 500.93 F,D,L DMSO, MeOH 549 115,000 573 MeOHT669 514.96 F,D,L DMSO, EtOH 549 109,000 574 MeOHT3168 652.23 D,L DMSO, DMF 514 195,000 529 MeOH 7For de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.

Notes1. Oxonols may require addition of a base to be soluble.2. Fluorescence of DiBAC4(3) increases about 3-fold relative to H2O on binding to proteins (Abs = 499 nm, Em = 519 nm). (Chem Phys Lipids (1994) 69:137)3. This product is speci�ed to equal or exceed 98% analytical purity by HPLC.4. QY for DiOC2(3) in MeOH = 0.04. Abs = 478 nm, Em = 496 nm in H2O. (Biochemistry (1974) 13:3315)5. JC-9 exhibits long-wavelength J-aggregate emission at ~635 nm in aqueous solutions and polarized mitochondria.6. DiIC1(5) in H2O; Abs = 636 nm, Em = 658 nm.7. JC-1 forms J-aggregates with Abs/Em = 585/590 nm at concentrations above 0.1 µM in aqueous solutions (pH 8.0). (Biochemistry (1991) 30:4480)

REFERENCES








PRODUCT LIST 22.3 SLOW-RESPONSE PROBESCat. No. Product QuantityB34950 BacLight™ Bacterial Membrane Potential Kit *for �ow cytometry* *100 assays* 1 kitB436 bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC4(5)) 25 mgB438 bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) 25 mgB24570 bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) *FluoroPure™ grade* 5 mgB413 bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3)) 100 mgD273 3,3´-dihexyloxacarbocyanine iodide (DiOC6(3)) 100 mgD22421 3,3´-dimethyl-α-naphthoxacarbocyanine iodide (JC-9; DiNOC1(3)) 5 mgD272 3,3´-dipentyloxacarbocyanine iodide (DiOC5(3)) 100 mgD14730 DiOC2(3) (3,3´-diethyloxacarbocyanine iodide) 100 mgD306 DiSC3(5) (3,3´-dipropylthiadicarbocyanine iodide) 100 mgH14700 1,1´,3,3,3´,3´-hexamethylindodicarbocyanine iodide (DiIC1(5)) 100 mgM24571 merocyanine 540 25 mgM34151 MitoProbe™ DiIC1(5) Assay Kit *for �ow cytometry* *100 assays* 1 kitM34150 MitoProbe™ DiOC2(3) Assay Kit *for �ow cytometry* *100 assays* 1 kitM34152 MitoProbe™ JC-1 Assay Kit *for �ow cytometry* *100 assays* 1 kitO266 oxonol V (bis-(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol) 100 mgO267 oxonol VI (bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol) 100 mgR302 rhodamine 123 25 mgR22420 rhodamine 123 *FluoroPure™ grade* 25 mgT3168 5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; CBIC2(3)) 5 mgT669 tetramethylrhodamine, ethyl ester, perchlorate (TMRE) 25 mgT668 tetramethylrhodamine, methyl ester, perchlorate (TMRM) 25 mg



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