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Optical Imaging and Its Role in Clinical Neurology
Susan V. Szapiel, MD
Arch Neurol. 2001;58:1061-1065.
INTRODUCTION
Rapid advances and developments in optical imaging technology during
the past 15 years have resulted in promising innovations in the realm of high-resolution
imaging of the functional architecture of the brain, which has not only contributed
to the elucidation of brain functional architecture and plasticity (see reviews1, 2, 3, 4, 5, 6, 7)
but has also increased our understanding of the temporal and spatial dynamics
of cortical seizure spread,1, 2, 6, 8
led to related technical improvements in neurosurgical procedures for the
surgical resection of seizure foci,8 and may
help to provide more accurate resection of brain tumors9
as well as contribute to our understanding of higher cognitive function.
At present, various methodologies exist for the investigation of brain
structure and function, ranging from single-cell electrode recordings to imaging
the activity of large populations of neurons. However, they vary widely in
their degree of spatial and temporal resolution (Figure 1). Despite being a somewhat invasive technique, optical
imaging of intrinsic signals has the advantage of excellent spatial and temporal
resolution down to approximately 50 to 100 µm and 150 to 200 milliseconds,
respectively, compared with that of functional magnetic resonance imaging
at a few millimeters and 1 to 2 seconds (most commonly). The temporal resolution
of optical imaging can be improved to a few milliseconds with the use of voltage-sensitive
dyes,1, 2 but such dyes are generally
toxic to the brain, precluding their use in human in vivo studies at present.
Such high spatial resolution allows the visualization of some of the basic
building blocks of the brain's anatomical and functional organization, eg,
the ocular dominance (OD) and orientation columns of visual cortex (Table 1 and Figure 2).
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Figure 1. Comparison of temporal and spatial
resolution of various brain mapping techniques. MEG indicates magnetoencephalography;
ERP, evoked response potential; EROS, event-related optical signal; MRI, magnetic
resonance imaging; fMRI, functional MRI; PET, positron emission tomography;
and 2-DG, 2-deoxyglucose. Adapted with permission from Haglund10
and Churchland and Sejnowski.11
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Terminology of Brain Functional Architecture*
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Figure 2. Intrinsic optical image maps.
A, Top panel shows unstimulated cortical surface of monkey visual cortex area
V1 (570-nm light illumination). Bottom panel shows optical image map of right
eye ocular dominance columns (same cortical surface as in top panel; 570 nm).
Dark areas represent activated cortical regions (arrows). Ocular dominance
maps are obtained by dividing right eye by left eye intrinsic signals, after
visual stimulation with a drifting grating in front of each eye separately.
Reprinted with permission from Frostig et al.12
B, Top panel shows unstimulated cortical surface of cat visual cortex area
18. Bottom panel shows optical image map of cat orientation columns. The same
cortical area as seen in the top panel is shown after stimulation with various
oriented lines; the map is obtained by vectorial addition of individual single-condition
(individual stimulations with oriented lines at orientations are shown to
right of Figure) maps. The angle of the resulting vector is color coded according
to scheme at right of figure. Yellow represents sites responding best to moving
horizontal gratings; blue, sites responding best to vertical gratings. Reprinted
with permission from Hubener et al.13 Copyright
1997 by the Society for Neuroscience. C, Optical image map obtained in the
posterior parietal cortex of an awake monkey in response to an expanding optic
flow stimulus. Arrows indicate activated cortical regions in response to the
stimulus. Map was obtained by dividing stimulation in the left visual hemifield
by stimulation in the right hemifield (605 nm) (S.V.S., Jonn McCollum, PhD,
and Ralph Siegel, PhD, unpublished data, 1997). Prestimulation cortex is not
shown.
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Optical imaging can now resolve this functional architecture as well
as other dynamic properties of the cortex in vivo.
By simply shining light on the surface of the brain and by recording
and analyzing the reflected light patterns, one can obtain high-resolution
maps of the functional architecture of the brain on the basis of intrinsic
signals (Figure 2).1, 2, 3, 5, 12, 14
Intrinsic signals are generated as a consequence of the metabolic aftereffects
of neuronal electrical activity, and therefore indirectly reflect neuronal
activity. These signals evolve after a short delay in response to evoked neuronal
activity. The source of intrinsic signals has been attributed to changes in
microvascular blood volume and flow, changes in light absorption or fluorescence
of intrinsic chromophores (eg, hemoglobin), and light-scattering signals from
water and ion movement, etc (see reviews1, 2, 12).
In the visible light range (540-630 nm), the signal seems to come primarily
from an initial increase in deoxyhemoglobin (starting about 200 milliseconds
after the stimulus), followed by an increase in blood volume/flow. A smaller
component of the signal is reflected by a rise in oxyhemoglobin level, starting
about 1.5 seconds after the stimulus onset.12, 13
In the near-infrared range, the predominant signal is more from light scattering.1, 2, 12 Optical imaging may
also be performed using voltage-sensitive dyes that are applied to the brain
or to brain-slice preparations before recording. The dye binds to electrically
excitable membranes and transduces membrane potentials into optical signals
that are captured by a photodiode array directly reflecting neuronal activity,
hence the better temporal resolution.1, 2
HISTORY
The history of optical imaging encompasses the theoretical and practical
contributions and observations of many investigators across several decades.
It had long been known that changes in the optical properties of nervous tissue
occurred with electrical or metabolic activity, but these changes were small
and hard to image. Later developments using extrinsic voltage-sensitive dyes
to stain the tissue and photodiode arrays revealed a fast signal that could
be imaged and that directly reflected neuronal activity (see reviews1, 2).
In 1986, Blasdel and Salama,4 using voltage-sensitive
dyes in the monkey striate cortex, a video camera, and a new analysis method,
showed for the first time spectacular high-resolution images of OD and orientation
columns in vivo in the monkey. They also noted a slow signal during their
recording but apparently did not consider the signal to be contributing significantly
to the images. Later, Grinvald et al5 showed
that this same slow signal, the intrinsic signal, could be used to reveal
functional architecture, specifically, cat orientation columns and rat whisker
barrels, without using dyes.
Frostig et al12 reported high-resolution
(100-150 µm; 200 milliseconds) optical imaging of intrinsic signals
in cat and monkey OD and orientation columns using a CCD camera and suggested
that the major source of the signal (using 570 nm wavelength light) was due
to blood volume changes but was only 5% of the reflection signal. At 600 nm,
the signal was probably related to oxygen delivery and/or saturation state
of hemoglobin and was 30% to 40% of the reflection signal. They also noted
that it temporally preceded the change in blood volume. At the near-infrared
wavelength of 810 nm, they were able to image orientation columns through
dura, and in the infrared range (930 nm), they were able to image through
thinned skull in the cat. Light scattering seemed to play the predominant
role in contributing to the signal in the near-infrared wavelength and contributed
only about 10% of the signal.13
By the use of imaging spectroscopy and laser-Doppler flowmetry in cats,
Malonek et al13 found that after visual sensory
stimulation, there is an initial increase in deoxyhemoglobin (initial dip)
(originating primarily from the capillaries) that later slowly declined and
was associated with an increase in total hemoglobin concentration, followed
by an increase in cerebral blood flow and oxygenated hemoglobin levels as
deoxyhemoglobin levels began to decline. The later increases in blood flow
and oxyhemoglobin concentration were considered to be less localizing to the
areas of neuronal firing compared with the increase in deoxyhemoglobin concentration,
based on earlier observations by Malonek and Grinvald15
that had shown that the increase in oxyhemoglobin was spatially less well
registered with activated cortical columns relative to the early increase
in deoxyhemoglobin levels. It was concluded that imaging methods based solely
on the secondary vascular response, such as positron emission tomography or
flow-sensitive magnetic resonance imaging, may offer lower spatial resolution
than if they are based on the initial deoxyhemoglobin increase.
METHODS
Most in vivo imaging is currently performed in anesthetized animals,
but may also be performed in awake animals and humans. Human optical imaging8 requires some modifications of the animal setup3 (Figure 3).
For imaging of intrinsic signals in cats or monkeys, a craniotomy is performed
over the area of the brain to be imaged, and a small metal chamber is placed
over the site and affixed to the skull with dental cement. The dura is removed
in most cases, and then the chamber is filled with silicone oil and sealed
with a glass cover. This sealed system helps to dampen arterial brain pulsations
that may interfere with imaging. In human studies, a craniotomy is performed,
and a glass plate may be placed over the surface of the cortex to dampen pulsations.8 The brain surface is illuminated using flexible liquid
light guides supplied by an adjustable direct-current output power supply
source and tungsten halogen lamp. Filters are used to produce the desired
light wavelength. A green filter (546 nm) is used to obtain baseline images
of the cortical vascular surface, and an orange filter (603 nm) is often used
for most general imaging. In human studies, this setup has been modified to
contain the light source within the operating microscope. The camera (video
or CCD) is positioned above the cortex and light guides and is mounted on
a vibration-free support. In human studies, the camera is attached to the
operating microscope.8 Lenses are attached
to the viewing end of the camera and focused on the cortical vasculature to
obtain an initial green-light image (Figure
2A-B, top panels). Later, the camera is focused down approximately
a few hundred micrometers until the vessels become blurred, and the filter
is changed to the imaging wavelength desired.
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Figure 3. Optical imaging apparatus (for
animals) is seen in a schematic representation of a typical setup for animal
optical imaging. For human imaging in the operating room, the cylinder seen
between the cat's ears may be replaced by a flat piece of glass over the exposed
cortex to dampen brain pulsations.
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Sensory stimuli (visual, somatosensory, auditory, etc) are delivered
to the animal at a given rate, and the camera collects the reflected light
off the brain surface during stimulations. When a video camera is used, a
frame grabber digitizes the video signal and sends it to a computer, where
the signals are averaged. Various acquisition and analysis procedures may
be used. Image enhancement before digitization may be performed by analog
differential subtraction of a stored "reference image" from the incoming video
image. The signals are later amplified. The differential image may be viewed
in real time on a monitor, which helps with rapid information feedback to
the investigator. Mapping signals or maps of the intrinsic signals are obtained
by various combinations subtracting or dividing the averaged signals from
each other. The reflected light signal from the brain surface is very small
(0.5%-5%) compared with the background activity; therefore, several stimulations
may need to be averaged together to improve the signal-to-noise ratio. To
obtain OD maps, signals obtained from right eye stimulation may be divided
or subtracted from those obtained from left eye stimulation or from the blank
condition. For orientation maps (Figure 2B), vertical grating (vertical lines drifting across the stimulus
monitor)–induced signals may be subtracted from horizontal grating–induced
signals or from a "cocktail" of combined signals collected in response to
several orientations of gratings. Dark areas (light absorption) on the maps
are areas of cortex that have been activated in response to the stimulus (Figure 2).3
CLINICAL APPLICABILITY
At present, optical imaging of brain activity is primarily a research
technique, but there have been several interesting and revealing human studies.
Haglund et al8 obtained optical maps from patients
undergoing seizure surgery and correlated them with surface electrical recordings.
Maps of the cortical surface were obtained during cognitively evoked functional
activity and during electrically evoked epileptiform discharges. In the study
of cognitively evoked activity, an attempt to identify Broca's and Wernicke's
areas was made in patients placed under local anesthesia during testing. Images
were collected at rest, while the patient moved his or her tongue, during
naming exercises, and during surface stimulation to cause speech arrest. The
researchers found that the area involved during naming was different from
that during tongue movement. Such studies may help to better define eloquent
regions of cortex and subsequently may guide more conservative surgical resection.
In the study of epileptiform discharges, Haglund et al8
found that the recorded optical changes showed a graded response relative
to the intensity and duration of the stimulus (surface-stimulating electrodes),
which correlated with the electrical changes. In addition, optical changes
associated with afterdischarge shifted below baseline in several patients,
suggesting a possible inhibitory neuronal population or inhibitory surround
or some other cause.
Optical imaging of epileptiform activity in brain slices has been shown
to be a useful method for studying various aspects of the spatial and temporal
dynamics of seizure spread. In early in vivo studies, it was shown that epileptic
foci were not stationary (see reviews1, 2).
More recently, slice preparations have helped to elucidate the roles of different
cortical laminae involved in seizure onset and propagation.6
Brain-slice preparations from human surgical specimens or animals provide
an opportunity to study activity in all cortical layers compared with in vivo
optical imaging, where imaging is limited to the gyral surface and generally
to the first few hundred micrometers of cortical depth from the surface. Such
preparations as well as in vivo optical imaging also may be useful for testing
the mechanism of action and efficacy of some anticonvulsants and perhaps other
medications used to treat other neurologic disorders.
Another potential use for optical imaging in humans is to define tumor
margins before and during resection. In an interesting in vivo study by Haglund
et al9 in rats, optical imaging of an intravenously
injected dye differentiated tumors and their margins from nearby normal tissue
with a high degree of sensitivity and specificity. The potential benefit of
more accurate surgical resection is undoubtedly desirable.
Although optical imaging may be employed for human psychophysical studies
of higher cognitive function, studies are currently restricted to those individuals
undergoing neurosurgery procedures. Therefore, such studies might be complemented
by the use of event-related optical signal (EROS) imaging using near infrared
(NIR) photons. This technique is now used to image evoked higher cortical
activity through the skull in humans and currently yields a spatial resolution
of a few millimeters (significantly less than that of optical imaging but
similar to that of functional magnetic resonance imaging) and a temporal resolution
of about 20 milliseconds (camera sampling rate).16
NEUROSCIENCE APPLICATIONS
Optical imaging of brain signals has already yielded many exciting new
insights into the functional architecture of the cerebral cortex. In the study
by Blasdel and Salama4 of OD and orientation
columns in monkeys, the authors demonstrated that orientation column functional
architecture was not organized in linear slabs of alternating orientation
columns as had earlier been suggested by Hubel and Wiesel17
(although they speculated that the columns were likely not sharply linear
in organization). Instead, they had more of a rosette appearance. That study
and another one in cats by Bonhoeffer and Grinvald18
led to the demonstration of a more pinwheel type of organization of orientation
columns.
At present, it is well accepted that brain plasticity is no longer solely
restricted to the critical period of development in many organisms, and methods
to measure spatial aspects of receptive field reorganization or expansion
have often been limited to painstakingly slow single-electrode recordings.
Optical imaging has proven to be a useful tool in measuring dynamic changes
in large populations of neurons at one time, and hence is particularly helpful
in investigations not only of temporal but also spatial aspects of plasticity.7 Polley et al7 made
an intriguing observation in adult rat whisker-barrel cortex, in which they
showed plasticity of the cortical representation of whisker barrels as a function
of sensory deprivation and novel environmental stimuli. In that study, they
removed all large whiskers on the rat except one and observed a large-scale
expansion of the remaining whisker's functional cortical representation. However,
when the animal was removed from its cage and put into a new environment to
explore it briefly, there was large-scale contraction of the barrel's cortical
representation. Contraction and expansion of the cortical whisker barrels
reversed upon regrowth of the whiskers.
Unpublished observations by Szapiel et al (Figure 2C), have revealed a stripelike pattern of activity in the
posterior parietal lobe of an awake rhesus monkey in response to an expanding
optic flow stimulus, suggesting a previously unseen functional architecture
for this type of stimulus using optical imaging techniques. Optic flow is
the perception of motion as we move forward or backward through the environment.
When moving forward, objects tend to flow past us in an expanding pattern
from a central focus; moving backward, the pattern appears to be one of contraction.
A moving pattern of expanding or contracting dots emanating from or flowing
toward a central point on a computer screen can simulate optic flow when viewed
by a subject at a specific distance from the screen. The inferior parietal
cortex of the rhesus monkey has been shown to contain neurons that respond
to visual optic flow.
High-resolution optical imaging of intrinsic signals and real-time optical
imaging with voltage-sensitive dyes in vivo and in vitro continue to advance
our understanding of basic brain structure and function, and as a result,
provide another important tool with which to understand and treat neurologic
disorders.
AUTHOR INFORMATION
Accepted for publication January 23, 2001.
I would like to thank Patricia Mitrano, MA, for technical assistance
with the figures.
From the Department of Psychology and the Center for Molecular and
Behavioral Neuroscience, Rutgers–The State University of New Jersey,
Newark.
Corresponding author and reprints: Susan V. Szapiel, MD, Department
of Psychology, Rutgers–The State University of New Jersey, 101 Warren
St, Newark, NJ 07102 (e-mail: szapiel{at}psychology.rutgers.edu).
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