Abstract - We have developed and applied novel tools for

functional optical imaging of the brain. By imaging the brain’s

response to stimulus using different modalities, and on different

length scales, we can form a more detailed picture of the

mechanisms underlying healthy and diseased functional brain

activity. One such tool, Laminar Optical Tomography (LOT), is

a new technique for 3D, non-contact, high-resolution functional

imaging of living tissues. LOT has been used to examine the

depth-resolved hemodynamic response to functional activation

in exposed rat cortex. LOT has sufficient spatial and temporal

resolution to resolve the individual vascular compartments

involved in the hemodynamic response (arterial, capillary and

venous). To further validate our observations, we also developed

a video-rate two-photon microscopy system, capable of imaging

at 22 frames per second. We have used this system to create

full-field two-photon movies of both the vascular dynamics and

calcium-dependent neuronal activity at very high resolution. In

addition, we have developed a system for simultaneous

exposed-cortex, multi-spectral 2D optical imaging and fMRI at

4.7T. These experiments have allowed us to relate our optical

findings to the clinically important BOLD signal.

I. INTRODUCTION

Optical imaging provides unrivalled sensitivity to the

functional properties of living tissues. Imaging superficial

tissues using planar-geometry CCD camera-based systems can

provide elegant measures of function e.g. via oxygenation-

dependent hemoglobin changes and via fluorescence contrast

from voltage and calcium sensitive dyes [1-4]. However in

stratified tissues (e.g. the cortex), interpretation of CCD

images is complicated by the lack of depth information and the

distortion of signals from deeper layers due to light scatter.

We have developed a number of optical imaging

techniques to study the exposed living brain, including 2D

hyper-spectral imaging [5], 3D Laminar Optical Tomography

(LOT) [6] and in-vivo video-rate two-photon microscopy [7].

In addition, we have combined 2D optical imaging of the

exposed cortex with simultaneous functional magnetic

resonance imaging (fMRI). These tools allow us to investigate

the mechanisms underlying the hemodynamic response to

stimulus, and its relation to neuronal activity. Understanding

this neuro-vascular interplay is particularly important for

improving interpretation of clinical fMRI results.

*This work was supported in part by the National Institutes of Health

under Grants NS053684-01, NS05118-01 and EB000790-02.

II. LAMINAR OPTICAL TOMOGRAPHY

LOT is a new technique that allows optical imaging

to depths of >2mm with 100-200 micron resolution and

excellent sensitivity to absorption and fluorescence contrast

[6, 8]. The LOT system and non-contact measurement

geometry are shown in Fig 1. LOT uses diffuse optical

tomography-like image reconstruction methods [9]. However,

by restricting the imaging depths to ~ 2mm and using high

measurement densities we can acheive useful resolution for

functional imaging. In addition, since rodent cortex is

generally <2mm thick, penetration is sufficient for cortical

imaging and exceeds depths and fields of view achievable

with two-photon microscopy.

Using LOT we are able to distinguish between

functional signals from the superficial vasculature, and signals

from the capillary beds deeper within the cortex. Fig 2 shows

how LOT’s non-contact geometry, allows simultaneous

electrophysiology recordings. We have also utilized

spatiotemporal analysis techniques to extract the 3D

distribution, and functional time-courses of the response in

major vascular compartments; arteries, capillaries and veins.

Fig 1. Laminar Optical Tomography system for depth-resolved,

high-resolution imaging of absorption and fluorescent contrast. A confocal

microscope-type configuration acquires scanning, non-contact

measurements of multiply scattered light, achieving DOT-type

measurements with high source and detector densities (50x50 source

positions and 50x50x7 detector positions) and very small source-detector

separations (0-2mm).

Functional optical imaging of brain activation: a multi-scale,

multi-modality approach

Elizabeth M. C. Hillman*

Department of Biomedical Engineering

Columbia University

Room 351L ET, 1210 Amsterdam Ave,

New York, NY 10027 eh2245@columbia.edu

Matthew Bouchard, Anna Devor,

Alex de Crespigny, David. A. Boas

Massachusetts General Hospital,

Martinos Center for Biomedical Imaging,

149 13th Street, Charlestown, MA 02129

Fig 2. Simultaneous

LOT &

electrophysiology:

40% isosurfaces show

depth-resolved (HbO)

hemodynamic

response to serial

tactile stimulus of 2

whiskers (D1 and δ).

Timecourses show the

peak HbR (blue),

HbT (green) and HbO

(red) responses.

Multi-unit activity

MUA and local field

potential LFP e-phys

recordings are also

shown:

III. VIDEO-RATE TWO-PHOTON MICROSCOPY

We developed a second imaging system to perform very rapid

full-frame 2-photon microscopy of the vascular compartments

in-vivo [7]. Our 2-photon system design is optimized for

in-vivo imaging and can currently acquire at up to 22 frames

per second. By injecting dextran-conjugated fluorescein

intravenously, the blood’s plasma can be visualized to depths

of ~500 µm in-vivo with ~2µm axial resolution.

Fig 3. Multi-scale

imaging of functional

hemodynamics: 2D

CCD imaging of the

hemodynamic response

to forepaw stimulus

reveals a region of

increased hemoglobin

absorption (top-left).

2-photon microscopy

then closely examines

the active region

(bottom). Repeatedly

imaging a frame

containing an artery,

vein and venule during

stimulus, we can extract

diameter, speed of flow,

and hematocrit changes.

Since red blood cells are not stained by the fluorescein, they

appear as dark shadows in a background of bright

fluorescence. It is therefore possible to image three different

aspects of blood flow using very rapid image-series: 1) The

speed of the red blood cells can be calculated, 2) the number

of red blood cells in the vessel at a given time (HbT(t) and

blood flow) and 3) the diameter of the vessels [10]. We have

observed robust changes in arteriole diameter, and venous

flow during stimulus response, and also employed calcium

sensitive dyes to allow simultaneous imaging of neuronal

firing and local hemodynamic changes on a micron scale.

While the depth penetration of 2-photon microscopy is

limited, we can compare results in the upper layers of the

cortex with the LOT results for the same superficial layers.

IV. SIMULTANEOUS 2D OPTICAL IMAGING AND FMRI

Since a major goal of our studies is to better

understand the neuronal origins of the BOLD effect in fMRI,

in order to properly link our optical imaging findings to fMRI

results, we also designed experiments to simultaneously

acquire optical and fMRI data during functional activation.

2D optical imaging prior to all our functional imaging studies

allows us to bridge results across modalities and verify that we

are indeed observing the same hemodynamic processes.

We designed a third system which consisted of a

CCD camera and a fiber-optic endoscope. A specially

designed MRI-compatible mount was used to hold a dielectric

mirror over the exposed cortex of the rat and the endoscope

both delivered light of either 570nm or 610nm, and also

relayed the image of the cortical surface back to the CCD

camera (Fig 4). Data acquisition was synchronized with a 4.7T

MRI scanner to allow simultaneous acquisition of the BOLD

signal with high resolution 2D hemodynamic imaging of the

same region during electrical whisker stimulation.

Fig 4. Simultaneous fMRI and optical imaging: (top) MRI-compatible

system for optical imaging of exposed cortex via a fiber-optic endoscope.

(middle), raw image through endoscope and peak hemodynamic response

distributions for HbR, HbO and HbT. (bottom) simultaneously acquired

fMRI results (left) oblique plane corresponding to cortical surface, (right)

coronal plane.

By combining multiple high-resolution optical imaging

techniques and exploiting the strengths of each, we have been

able to image the functional brain response to stimulus on

multiple length scales and with different contrast mechanisms.

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