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RecentProgressinVoltage-SensitiveDyeImagingforNeuroscience

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Nanoscience and NanotechnologyVol. 14, 4733–4744, 2014

www.aspbs.com/jnn

Recent Progress in Voltage-Sensitive Dye

Imaging for Neuroscience

Vassiliy Tsytsarev1�∗� †, Lun-De Liao2� †, Kien Voon Kong3, Yu-Hang Liu2,Reha S. Erzurumlu1, Malini Olivo3�5, and Nitish V. Thakor2�4

1Department of Anatomy and Neurobiology, University of Maryland School of Medicine,

HSF-2, Baltimore, MD 21201, USA2Singapore Institute for Neurotechnology (SINAPSE), National University of Singapore,

28 Medical Drive, #05-COR, Singapore 1174563Singapore Bioimaging Consortium, Agency for Science, Technology and Research, 138667, Singapore

4Department of Biomedical Engineering, Johns Hopkins University,

Traylor 701/720 Rutland Ave, Baltimore, MD 21205, USA5School of Physics, National University of Ireland, Galway, Ireland

Voltage-sensitive dye imaging (VSDi) enables visualization of information processing in differentareas of the brain with reasonable spatial and temporal resolution. VSDi employs different chemicalcompounds to transduce neural activity directly into the changes in intrinsic optical signal. Physi-cally, voltage-sensitive dyes (VSDs) are chemical probes that reside in the neural membrane andchange their fluorescence or absorbance in response to membrane potential changes. Based onthese features, VSDs can be divided into two groups-absorbance and fluorescence. The spatialand temporal resolution of the VSDi is limited mainly by the technical characteristics of the opticalimaging setup (e.g., computer and light-sensitive device-charge-coupled device (CCD) camera orphotodiode array). In this article, we briefly review the development of the VSD, technique of VSDiand applications in functional brain imaging.

Keywords: Brain Imaging, Contrast Agents, Functional Brain Mapping, Functional BrainImaging, Optical Imaging, Voltage-Sensitive Dye Imaging.

CONTENTS1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4733

2. Basic Mechanisms of VSDi . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4735

3. Physico-Chemical Basis of VSD . . . . . . . . . . . . . . . . . . . . . . . . 4736

4. VSDi in Neuroscience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4739

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4741

References and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4741

1. INTRODUCTIONVisualization of the neural activity in vivo is an important

objective in both fundamental and clinical neuroscience.

There are now several optical imaging methods, based

on the changes of the optical properties of the brain tis-

sue that can be used to measure neural activity. These

include intrinsic signal optical imaging, near-infrared

∗Author to whom correspondence should be addressed.†These two authors contributed equally to this work.

optical imaging, functional photoacoustic tomography,

optical coherence tomography and optical imaging with

voltage sensitive dyes (VSDi). Here, we review a wide

variety of VSDi approaches for the study of neural activ-

ities in the brain. Optical techniques enabling the descrip-

tion of brain function at levels ranging from single cells to

neural ensembles are also introduced in this article.

Understanding cortical function after neural activation

allows probing into cerebral neurovascular coupling and

uncoupling functions.1 To date, many neuroimaging tech-

niques are available to provide the information of neural

circuit functions in both laboratory animals and humans.1

VSDi is a powerful technique for studying neural circuit

functions with relatively high spatial (up to 20 �m) and

temporal (up to tens of microseconds) resolution, compa-

rable to electrophysiology techniques.2�3 The first optical

recordings of membrane potentials using VSDi were done

more than three decades ago on the squid giant axon and in

J. Nanosci. Nanotechnol. 2014, Vol. 14, No. 7 1533-4880/2014/14/4733/012 doi:10.1166/jnn.2014.9531 4733

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Recent Progress in Voltage-Sensitive Dye Imaging for Neuroscience Tsytsarev et al.

Vassiliy Tsytsarev has received a Ph.D. in Neuroscience in Saint-Petersburg State Uni-

versity, Russia. He moved to Japan soon after graduation and worked there within seven

years—first in the Brain Science Institute of RIKEN and then in Kyoto University. After

that he moved to the United States and is now working in the University of Maryland

School Of Medicine. Functional brain mapping, neural circuits and different types of the

brain optical imaging are his main scientific interests as well as professional background.

Lun-De Liao received his Ph.D. degree in electrical engineering from National Chiao

Tung University (NCTU), Taiwan in February of 2012. He was a Postdoctoral Researcher

at the Brain Research Center (BRC) in NCTU. He proposed the world first bio-inspired

dry EEG sensors and their corresponding circuit to intelligent image the human brain

under the guidance of Professor Chin-Teng Lin (FIEEE) at BRC in NCTU, Taiwan. He is

the co-founder of MINDO Company for products in wearable and wireless EEG device

(http://mindo.com.tw/). He is currently a Research Scientist and head of the Neurovascular

Imaging Laboratory in Singapore Institute for Neurotechnology (SINAPSE) at National

University of Singapore. He has published over 45 peer-reviewed SCI journal papers,

including Nature: J of Cerebral Blood Flow and Metabolism, Proceedings of the IEEEand Neuroimage journals and 10 issued patents. He was selected/nominated for more

than 30 international awards since 2004. In 2011, he also won the 1st place of Young Investigator’s Awards from

the world association for Chinese biomedical engineers for his contributions on medical imaging and bioelectronics

domain. He was also selected as an Outstanding Research Award of 2012 from National Chiao Tung University,

for his outstanding research performance. He served on organization committee and technical program committee

of many flagship international conferences and workshops. He currently serves as Co-Editors-in-Chief of Journal ofNeuroscience and Neuroengineering, also an Associate Editor of four SCI-index journals. In recent years, his research

interests include neuroimaging, cerebral neuroscience, cognitive neuroscience, in vivo optical microscopy, advanced

sensing techniques and design of optical system.

Kien Voon Kong obtained his B.Sc. degree in Applied Chemistry from University of

Malaya in 2004 and Ph.D. Degree from National University of Singapore in 2009. His

research interests include drug delivery, surface enhanced Raman scattering and nanopar-

ticle based multimodality optical imaging for biomedical applications.

Yu-Hang Liu is currently working toward Ph.D. degree in Electrical and Computer Engi-

neering at National University of Singapore. He received his B.S. degree from National

Central University, Taiwan, in Electrical Engineering (2008). He also received his M.S.

degree from National Chiao Tung University, Taiwan, in Biomedical Engineering (2010).

During his Master studies, he proposed a real-time wireless system of brain computer

interface (BCI) for drowsiness detection to help drivers reduce the risks of fatigue, which

was based on EEG and EOG signals for long term monitoring. He has co-authored 3 jour-

nal and 1 conference papers. He is a Student Member of IEEE and EMBS. Yu-Hang’s

research interests include biomedical optic image, signal processing, embedded system

design of biomedical application and cognitive neuroscience. Now, he is focusing on the

photothrombotic stroke model to propose novel treatment based on electrical stimulation

for protecting the stroke region and recovering the injured area through mechanism of collateral circulation.

4734 J. Nanosci. Nanotechnol. 14, 4733–4744, 2014

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Tsytsarev et al. Recent Progress in Voltage-Sensitive Dye Imaging for Neuroscience

Reha S. Erzurumlu is Professor of Neurobiology and Anatomy at School of Medicine,

University of Maryland Baltimore. He received his Ph.D. in Biological Sciences from

University of California Irvine, and postdoctoral training at Brown University and MIT.

His research focuses on development and plasticity of the sensory systems. He is a world-

recognized researcher in the field of rodent trigeminal system development and plasticity.

Malini Olivo is Head of Bio-Optical Imaging, Singapore Bioimaging Consortium, A ∗STAR, Singapore. She holds a Professorship at the National University of Ireland. She

is a Visiting Professor at Harvard Medical School. She obtained a Ph.D. in Bio-Medical

Physics and is a pioneer in the area of photomedicine in optical diagnostics and ther-

apeutics. She has numerous awards for her contribution to biophotonics in Singapore,

Ireland and US. In 2011, the SPIE conferred an award for her contribution as a woman

in photonics. She publishes extensively and serves in the EU research commission.

Nitish V. Thakor is a Provost Professor, Electrical and Computer Engineering, National

University of Singapore (NUS), and is currently Director of Singapore Institute for

Neurotechnology (SINAPSE), an institute dedicated to neurotechnology research and

development. He is also a Professor of Biomedical Engineering, Electrical and Computer

Engineering, and Neurology at Johns Hopkins and directs the Laboratory for Neuro-

engineering. He received his undergraduate degree from Indian Institute of Technology,

Bombay, India (1974), he worked as an engineer with Philips India for two years, and he

then earned his Ph.D. from the University of Wisconsin, Madison (1981). He first became

a member of the faculty of Northwestern University (1981–1983), and since then he has

been with the Johns Hopkins School of Medicine. He conducts research on neurological

instrumentation, biomedical signal processing, micro and nanotechnologies, neural pros-

thesis, clinical applications of neural and rehabilitation technologies and brain-machine interface. He has authored more

than 250 peer-reviewed publications on these subjects. He was the Editor in Chief of IEEE Transactions on Neural

and Rehabilitation Engineering and presently of the Medical and Biological Engineering and Computing journal. Cur-

rently Dr. Thakor directs the Laboratory for Neuroengineering and is also the Director of the NIH Training Grant on

Neuroengineering. One of his current research projects, in collaboration with a multi-University consortium funded by

DARPA, is to develop next generation neurally controlled upper limb prosthesis. He is actively engaged in developing

international scientific programs, collaborative exchanges, tutorials and conferences in the field of Biomedical Engi-

neering. Dr. Thakor is a recipient of a Research Career Development Award from the National Institutes of Health

and a Presidential Young Investigator Award from the National Science Foundation. He is a Fellow of the American

Institute of Medical and Biological Engineering, International Federation of Medical and Biological Engineering, IEEE

and Founding Fellow of the Biomedical Engineering Society. He is also a recipient of the Technology Award from

IEEE Engineering in Medicine and Biology Socity, Centennial Medal from the University of Wisconsin School of

Engineering, Honorary Membership from Alpha Eta Mu Beta Biomedical Engineering Student Honor Society and

Distinguished Alumnus Award from his alma mater, Indian Institute of Technology, Bombay.

individual leech neurons.4–6 Few years later, VSDi experi-

ments were done in vivo on the mammalian neocortex.7

A typical VSD molecule has a pair of hydrophobic

hydrocarbon chains, which work as anchors on the neu-

ronal membrane. The rest of the molecule is a hydrophilic

group, which align the chromophore perpendicular to the

cell surface.8 The optical signal intensity is linearly corre-

lated with the transmembrane voltage changes, so it allows

measuring neural activity without using electrophysiologi-

cal recordings. In this review article, we review VSDi and

describe some of its applications in neuroscience.

2. BASIC MECHANISMS OF VSDi

The simplest voltage-sensing mechanism is molecular

redistribution (Fig. 1). The change in the membrane

J. Nanosci. Nanotechnol. 14, 4733–4744, 2014 4735

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Recent Progress in Voltage-Sensitive Dye Imaging for Neuroscience Tsytsarev et al.

Figure 1. Three different mechanism of the VSD optical features

changes.9�10 Redistribution (A), reorientation (B) and the direct electrical

modulation of the electronic structure of the dye molecule (C).

potential causes the charged molecule to move in or out

of the cell. This changes the dye concentration in the cell

and subsequently fluorescence.9 The dye molecules do not

have to completely leave the cell, part of a molecule can

be embedded in the membrane.10

Another mechanism is reorientation of the dye

molecule. In this case the dye molecule lies in or on

the membrane with a particular orientation, determined by

the interaction of the intermolecular electric fields. Usu-

ally reorientation is faster than redistribution since it does

not involve a significant movement of the molecule.10 The

most effective for imaging is an electrochemical mecha-

nism which is direct modulation of the electronic structure

of the dye molecule. VSD molecules have large differ-

ences in the dipole moment of their ground state and

their low-lying electronically excited states. The energies

of these states are determined by the voltage and direction

of the transmembrane electric field. Changes in the elec-

tronic structure lead to changes in both the excitation and

emission spectra and cause changes in the light absorption

or photon emission.10 The response time is very fast in

comparison to redistribution or reorientation since it only

involves intramolecular charge changes, without molecular

movement.11

The VSDi was done initially in mollusks: leech

ganglia or the snail.4�12�13 Moving on to the mam-

malian brain in vivo experiments required significant

improvements in VSDi.14 New generation of dyes was

created,15 that is absorbed away from the hemoglobin

absorption peak.1�12�13 Most in vivo studies have been

made in anesthetized16�17 and sometimes freely moving

rodents.18–21 VSDi experiments on visual cortex have been

performed in cats22 and monkeys.23�24

VSDi signals are much difficult to be measured from

in vivo mammalian brain compared to in vitro targets,

because of the noise from the cardiac rhythm and respi-

ratory movements. VSDi reflects fast changes of neural

activity, and requires a frame rate of at least hundreds of

hertz.25�26 The optical signal is thousands of times smaller

than baseline fluorescence; therefore, a large dynamic

range of charge-coupled device (CCD)-camera is much-

needed. Large number of photons must be recorded dur-

ing a short period due to the speed (milliseconds) of the

neuronal pulse to achieve a sufficient signal to noise ratio

(SNR).25�26

Ideal functional brain imaging would be minimal inva-

siveness and a large area of investigation in vivo. One

potential way for realizing this goal is incorporating flu-

orescence imaging system with complementary metal-

oxide-semiconductor (CMOS) implantable image sensors.

This system has been developed and described recently.18

In spite of authors using relatively low signal-to-noise ratio

VSD (RH795), the data recorded in the rat somatosen-

sory cortex area is comparable to those obtained with

electrophysiology.27

3. PHYSICO-CHEMICAL BASIS OF VSDAn alternative imaging technique is based on the fluores-

cence resonance energy transfer (FRET). This technique

employs two molecules: the acceptor and the donor.28

A donor molecule, initially in its electronic excited state,

transfers energy to an acceptor molecule through dipole–

dipole coupling.29 Tsutsui et al. developed a novel sen-

sor, named Mermaid, that shows 40% changes in emission

ratio per 100 mV, allowing for direct visualization of mem-

brane potential29 and improved signal to noise ratio.

In regards to in vivo functional brain mapping stud-

ies, the VSD signal is related to the stained membrane

area. Fluorescence mainly originates from the neuron’s

dendrites and axons but not from the soma. Dendrites

frequently extend across large cortical areas, so the opti-

cal activity pattern is usually larger than the center of

the neural activity, which must be mentioned during data

analysis.30 Usually in VSDi animal experiments the skull

must be opened and the dura mater removed above the

recording site, but in some cases, especially in experiments

with young rodents, the cerebral dura mater can be left

intact.

VSDi needs a special optical system usually called “flu-

orescent microscope” equipped with a large field of view

and relatively long focal length (Fig. 2). Both tempo-

ral and spatial resolution of the system is determined,

mainly, by CCD camera or photodiode array.31 2-photon

microscopy, in combination with VSDi, can increase spa-

tial resolution,32 but due to the limitation of scanning, the

temporal resolution of this combination technique is quite

limited. Several efforts have been made to develop organic

VSDs to improve image contrast (Fig. 3). VSDs can be

used for direct imaging of cellular membrane potentials,

and functional mapping studies in awake mammals.

The key point for visualization of neural activity is

not only the experimental setup, but the dyes themselves.

4736 J. Nanosci. Nanotechnol. 14, 4733–4744, 2014

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Tsytsarev et al. Recent Progress in Voltage-Sensitive Dye Imaging for Neuroscience

Figure 2. Main principle of the VSDi. VSD is able to provide mea-

surements of transmembrane voltage of single neurons or neuronal

ensembles.1�31

Currently, several research groups are actively working

on synthesizing new organic VSDs.7�8�12�13 The relative

response (�F ) to electrical activity in mammalian brains

for these proposed dyes is about 10−3, which is enough

to visualize neural activity after averaging and/or noise

removing. Another team, led by Peter Fromherz devel-

oped the organic VSD family, called “ANNINE.”33 The

dye molecule has a double positively charged chromophore

and two bromide counter ions, as well as styryl-type

VSD.8�33 This probe exhibits high solubility, a strong

membrane binding, and a high voltage-sensitivity in neu-

rons (Fig. 4). However, many of the organic molecules

based chromophore at this time does bear from seri-

ous chemical and photophysical liabilities, such as fluo-

rescence self-quenching and photo-bleaching which can

affect the accuracy of FRET measurements.34 Other lim-

itations include short-term aqueous stability, broad red-

tailed emission spectra via small Stokes shifts, and short

Figure 3. Examples of the different types of organic VSDs.7�8�12�13

excited state fluorescent lifetimes. To tackle these limita-

tions, more robust chromophores are being developed.

Simultaneous recording of the activity of many neurons

over long periods of time is important in studies of neu-

ral networks. The suitability of the two VSDs, RH795 and

Di-4-ANEPPS for recording neural activity of the stom-

atogastric nervous system of crustaceans within 24 h was

achieved recently.35

These authors concluded that both dyes provided suffi-

cient signal-to-noise ratio for recording of the neural activ-

ity in vivo, but Di-4-ANEPPS displayed a higher signal

quality, while RH795 showed weak and slowly developing

phototoxic effects and bleaching. In other words, Di-4-

ANEPPS is better for fast experiments that require higher

signal intensity, in contrast, RH795 is more suitable for

long-term experiments.35

Moreover, the new quantum dots technique provides

a new opportunity for brain neuroscience research. The

use of semiconductor nanocrystals (quantum dots) as flu-

orescent labels for multiphoton microscopy enables mul-

ticolor imaging in complex brain tissue. Quantum dots,

however, are not applicable for the VSDi but works

perfectly for fluorescence angiography probe, cell track-

ing and detection of the antibodies.50 Quantum dots,

semiconductor nanocrystals, are a class of chromophore

that has enabled important advancements in fluorescence

imaging,36�37 because they display many superior optical

qualities desired for imaging applications, and are use-

ful for both single- and multi-color experiments. Size of

quantum dots generally ranges from 2 to 10 nanome-

ters (Fig. 5). Their emission profiles are independent of

excitation wavelength and across the visible spectrum

(Fig. 5).38–43 The high quantum yield, the size-tunable

nature and narrow emission profiles accompanied with

quantum dots are the major advantages that arise from

their unique core/shell architecture.36–39�44�45 The core

semiconductor material of quantum dots has a narrow band

J. Nanosci. Nanotechnol. 14, 4733–4744, 2014 4737

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Recent Progress in Voltage-Sensitive Dye Imaging for Neuroscience Tsytsarev et al.

Figure 4. (A) VSDi images showing single-whisker stimulation fluorescence changes in the mouse barrel cortex. Time after stimulus onset is indicated

at the bottom right corner of each image. Duration of each frame is 5 ms. (B) Change in fluorescence (�F /F (%), ordinate) in response to whisker

stimulation. Fluorescence signal was recorded in the four small (7×7 pixels) square marked on the cortical surface in A, at the last image. (C) areas

activated by whisker stimulation. The number of pixels with the value of fluorescence signal more than 90, 80, 70 and 60% of the maximum has been

counted.8�33

gap. This core material is enveloped within a shell coat-

ing that consists of a different semiconductor material of

higher band gap. This core–shell architecture can curbs

excitation and emission to the core to increase the lumi-

nescence quantum yield of the core emission and reduce

photo-bleaching from the core.36�39�45–47

While toxicity of inorganic semiconductor materials and

nanoparticle continues to be an area of debate,48–52 numer-

ous studies reported modified quantum dots with limited

cytotoxicity. Moreover, substantial effort has gone into

developing different coatings to render quantum dots bio-

compatible (Fig. 6).53–58 Due to robustness of the opti-

cal properties of quantum dots, they are desirable for a

wide range of in vivo studies, immunoassays, cell track-

ing, and FRET.54�55�57�59–73 The most recent development is

the study of quantum dots for neuronal voltage sensing.63

A number of biocompatible surface chemistries have been

developed to deliver quantum dots to cells.42 These sur-

face modifications making quantum dots allow specific

localization inside or adjacent to cellular membranes.

Meanwhile, the membrane simulations also envisage that

localization of quantum dots inside a bilipid layer can

be a free-energetically favorable process with appropriate

surface modification. Quantum dots are also reported to

have voltage-sensitive optical properties (Fig. 7), including

emission spectral broadening, red shifting and decreased

intensity of emission peak.74–78 Quantum dots have greater

cross sections for one- and two-photo absorption than

those of fluorescent proteins or voltage-sensitive dyes in

which far larger cross sections are an important factor to

facilitate voltage detection, suggesting quantum dots can

be a new class for VSD development. Moreover, accord-

ing to the signal detection theory, quantum dots can be

used in reporting voltage dynamics with millisecond pre-

cision in neurophysiological conditions.78 These results

reveal potential avenues for imaging spiking dynamics in

neural networks by using quantum dots as VSD. Undoubt-

edly, quantum dots will also open new avenues of research

and continue the development of nanoparticles based

probes for VSDi that will truly revolutionize neuroscience

research.

Physiological side effects of two very common VSD are

RH-1691 and di-4-ANEPPS. For future in vivo research,

it is noteworthy that di-4-ANEPPS doesn’t change local

field potentials while RH-1691 cause increases in SEP

amplitude for several hours. On the other hand, nei-

ther probe influences the spontaneous neural activity in

the neocortex.79�80 Over the past decades, new geneti-

cally encoded optical voltage sensitive fluorescence pro-

teins (VSFP1) have evolved.81–85 The first generation of

4738 J. Nanosci. Nanotechnol. 14, 4733–4744, 2014

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Tsytsarev et al. Recent Progress in Voltage-Sensitive Dye Imaging for Neuroscience

Figure 5. (A) Absorption and emission of a common organic dye (rho-

damine red) and genetically encoded DsRed2 protein. (B) Absorption and

emission of six different quantum dots (QDs) dispersions. The black line

shows the absorption of the 510-nm-emitting QDs. (C) Photo demon-

strating the size-tunable fluorescence properties and spectral range of the

six QD dispersions plotted in b versus CdSe (Cadmium Selenite) core

size. All samples were excited at 365 nm with a UV source. (D) Compar-

ison of QD size, diameter ∼ 6 nm, to a maltose binding protein (MBP)

molecule (mw ∼ 44,000).38–43 Reprinted with permission from Ref. [42],

I. L. Medintz, et al., Nat. Materials 4, 435 (2005). © 2005, Nature Pub-

lishing Group.

VSFP was shown to optically report changes in the mem-

brane voltage but its application in mammals was limited

by their poor membrane targeting.86�87 The second gener-

ation of voltage-sensitive proteins (VSFP2) was developed

by Tomas Knopfel’s team.85 The same team generated a

third VSFP generation: VSFP3 so-called monochromatic

fluorescent probes.81�82�88

4. VSDi IN NEUROSCIENCEThe application of VSDi in the modern neuroscience is

rather large since a fast neural activity is of great impor-

tance. In clinical practice as well as in animal experiments,

positron emission tomography (PET), functional magnetic

resonance imaging (fMRI),89 near-infrared spectroscopy

(NIRS),90 optical coherence tomography (OCT) and pho-

toacoustic (PA) imaging91–93 have all been used to locate

centers of the neural activity. Nevertheless, all of these

methods have advantages as well as disadvantages. Within

the last few decades, VSDi have been successfully used for

the functional mapping of the visual cortex.12�13�94 Thus,

an implantable metal-oxide-semiconductor (CMOS) sen-

sor with light-emitting diodes (LED) and optic filter was

developed27 and the neural activity in the visual cortex was

visualized in awake mouse in real-time.

Recently, visual information encoding was studied in

the cat visual cortex with VSDi.22 Authors discovered that

the neurons in area 18 code at least two different signals,

while the proportion of these two signals vary dynamically

as a function stimulus property.22 VSDi has been success-

fully employed in investigating the propagation of neural

activities elicited by intracortical microstimulation (ICMS)

in areas 17 and 18 of the cat visual cortex.95 ICMS-evoked

activity and subsequent propagation has been visualized in

the visual cortices contralateral as well as ipsilateral to the

stimulation and patterns were consistent with anatomical

connections.95

During the last decade, the primary visual cortex was

extensively studied by imaging methods with different

types of stimuli, yet not much is known about encod-

ing of visual information. Recently visual image encod-

ing was studied using VSDi in behaving monkeys by

recording the population response evoked in V1 by the

presentation of the natural images during a face/scramble

discrimination task.96 Authors reported that the population

response showed two phases: (1), that was spread over

most of the area of the recording, and (2), which was spa-

tially confined.96 These experiment results present a spatial

encoding of low- and high-level features of visual images

in the V1: the low level is correlated to the image’s basic

local attributes and the high level is related with the per-

ceptual outcome of the visual image processing.96

Neural responses in response to collinear or orthog-

onal arrays of Gabor patches have been visualized by

the VSDi imaging in the awake monkey.24 Using VSDi,

they imaged the neural population responses in V1and V2

areas in fixating monkeys while they were also presented

with collinear or orthogonal arrays of Gabor patches.97

Authors found that collinear effects are mediated by syn-

chronization in a distributed network in V1 and V2.97

To investigate the neuronal mechanisms of perception, the

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Recent Progress in Voltage-Sensitive Dye Imaging for Neuroscience Tsytsarev et al.

Figure 6. In vivo quantum dot excretion is size-dependent. Cysteine-coated ZnS-capped CdSe QDs with a hydrodynamic diameter 5.5 nm were able

to pass through the kidney into the bladder for excretion in the urine53–58 Shown are fluorescence (bottom) images of surgically exposed CD-1 mouse

bladders following injection of different size quantum dots. Reprinted with permission from Ref. [58], H. S. Choi, et al., Nat. Biotechnol. 25 1165

(2007). © 2007, Nature Publishing Group.

neural population responses of the V1 were visualized

by VSDi in the monkey trained on a contour-detection

task.98 Authors observed that the early responses showed

activation patches corresponding to the individual visual

elements while late responses showed contour elements.

It was concluded that these opposing responses in the con-

tour and background might underlie perceptual processing

in V1.98

The activity patterns in V1 reflect the location of visual

elements in the retina. It is still questionable whether

this organization contributes to image recognition.99 VSDi

Figure 7. Quantum dots show a red shift in fluorescence peak and

decrease in fluorescence intensity in response to applied electric

fields.74–78 (A) Schematic represents a quantum dot embedded in a lipid

bilayer; where yellow and green represent the dot core and the exciton

confining shell, respectively. (B) The non-uniformly coated quantum dot

is unscreened by the membrane. (C) the field-induced exciton polarization

and fluorescence red shift in quantum dots in which ‘e’ and ‘h’ represent

the electron and hole that are excited by the incoming light (black arrow).

The pair subsequently recombines and produces fluorescence (colored

arrows). (D) Schematic represents of absorption and emission spectra

of quantum dots by an applied field. Reprinted with permission from

Ref. [63], J. K. Jaiswal, et al., Nat. Biotechnol. 21, 47 (2003). © 2003,

Nature Publishing Group.

was applied in behaving monkeys to investigate whether

the structure of V1 population responses influences shape

judgments.99 Authors used a computational model to

design visual stimuli that had the same shape, but were

predicted to evoke different responses in the V1, and this

prediction was confirmed by VSDi. It was concluded that

the activity patterns of neural responses in the V1 con-

tributes to visual perception.99 The transfer of visual infor-

mation from V1 to the higher level of the visual cortex

is still poorly understood,100 but recently spatiotemporal

dynamics of the neural activity elicited in the mouse V1

by the presentation of simple visual stimuli was studied

by VSDi.100 It is reported that V1 activation is rapidly

followed by the activation of three functional groups

of higher order visual areas organized retinotopically.100

Based on the imaging data, authors hypothesized that the

cortex integrates visual information through across-area

parallel by the serial processing.100

VSDi has been used for functional mapping of the

auditory cortex by many scientists as well.8�94�101 As

well known, one of the main structural principles of the

auditory cortex is tonotopical organization, which means

that isofrequency patterns appear in the form of elongated

areas orthogonal to the rostrocaudal axis. This organization

has been confirmed by VSDi.102 In the guinea pig auditory

cortex a ventrocaudal field that has mirror-symmetric tono-

topy to that of ventrorostral field was identified.103 High

spatial resolution VSDi has been used to examine the cor-

tical representation of interaural time difference (ITD) in

the rat auditory cortex.2

Available data demonstrated that patterns of neural

activity recorded in response to binaural click presenta-

tions can encode ITD in the auditory cortex.2 It was con-

cluded that some neurons in the investigated area receive

information from several neural pools, each of which pro-

cesses separate sound’s feature.2 Topographical representa-

tion is a main feature of the sensory cortices of mammals,

and the somatosensory cortex is no exception.104 VSDi has

4740 J. Nanosci. Nanotechnol. 14, 4733–4744, 2014

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Tsytsarev et al. Recent Progress in Voltage-Sensitive Dye Imaging for Neuroscience

been used to create a functional map of the directional

sensitivity of the barrel field.3 Not only representation of

functions but also the spatial properties of the oscillation

have been successfully studied in the barrel field by VSDi.

Highly distinct results have been obtained by the com-

bination of VSDi with multichannel recordings in the

rat barrel field cortex. Authors demonstrated that single-

whisker stimulation by particular frequency within 10 min

induces long-term potentiation (LTP) of the whisker-to-

barrel pathway.105 This activity-dependent modification is

age-dependent and may play a critical role in the develop-

ment of the functional map in the barrel field.105

The spatiotemporal pattern of spontaneous activity

across the auditory cortex was successfully visualized by

VSDi in the guinea pig.106 Authors found that phase coher-

ence in spontaneous activity was highest between regions

of core and belt areas that had similar frequency tuning.

It was shown that VSDi can reveal spontaneous and stim-

ulus evoked activity across a tonotopically defined cortical

areas.106

VSDI has been employed in studying cortical oscilla-

tions, which arise during different cognitive and behav-

ioral acts. VSDi revealed that gamma-Aminobutyric Acid

(GABAa) disinhibition with bicuculline can progressively

simplify oscillation dynamics in a concentration-dependent

manner. The authors of the study concluded that neural

synchronization can be increased by both GABAA and

GABAB.107

VSDi is used in developmental neurobiology as well.

For example, it was shown that somatosensory cortical

areas responding to single whisker stimulation were 200 to

300 microns in diameter in young (up to 5 days) rats, while

in adult animals the areas activated by the same man-

ner was increased.108 Also, sensory-evoked propagating

waves were observed by VSDi in the sensory cortex.109�110

This method allows investigating the interactions between

evoked waves in rat visual cortex, and the spatiotempo-

ral patterns of depolarization in the neural network due to

wave-to-wave interactions.111 It was hypothesized that the

spiral waves are a sequential activation in space and time,

and is likely to be carried out in the neural network in the

neocortex.110�111

VSDi methods have been successfully employed not

only in sensory neuroscience research but also for the

effects of chronic hypoxia.112 VSDi of the somatosensory

cortex showed no significant decrease in neural activity

in mice which were housed in a hypoxic chamber for a

month. In combination with cerebral blood flow data, this

finding showed that hemodynamic response to neural acti-

vation could be modified using chronic hypoxia.112

Within the last couple of decades, VSDi became a

popular method, especially for epilepsy research.1 Accu-

rately locating centers of the epileptic seizures has great

importance in advancing antiepileptic therapy. For exam-

ple, using 4-aminopyridine (4AP) model of the epilep-

tic seizures, synchronization during focal seizures in the

rat neocortex was investigated.113 The VSDi signal was

analyzed using the nonlinear dynamics-based technique

of stochastic phase synchronization in order to determine

the degree of synchronization during the development and

spread of seizure events.113 Results showed increase in

neural synchronization during seizure activity.113 Undoubt-

edly, future experiments with VSDi in combination with

electrophysiological and different imaging techniques1 will

help us understand the complex relationships between neu-

ral network, cerebral blood flow and glial cells within

epileptic seizure zones.

5. CONCLUSIONSCurrently, VSDi allows visualization of neural activity

in the somatosensory,3�21 auditory2 and visual100 cortices.

In addition, VSDi is widely used for recording epileptic

seizures.101�113 Voltage-sensitive dyes do not exhibit cel-

lular selectivity and bind not only to neurons but also to

astrocyte membranes and show changes in the membrane

potential of glial cells. In vivo, a single pixel of the CCD

camera contains the signals from dendrites and axons of

many neurons. Physically, the signal of VSDi is linearly

related to the stained membrane area. However, most of

the photons are emitted from cortical dendrites and non-

myelinated axons but not from the neural bodies.8 Only

application of the threshold approaches provides relatively

high-resolution functional maps, with a spatial resolution

up to 50 �m.8 In other words, VSDi provides a useful

way for both in vivo and in vitro and functional mapping

patterns of neuronal activity.

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Received: 15 October 2013. Accepted: 15 January 2014.

4744 J. Nanosci. Nanotechnol. 14, 4733–4744, 2014

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