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EMBO reports 4, 9, 838–843 (2003)
doi:10.1038/sj.embor.embor924
AOP Published online: 22 August 2003
Shedding light on bioscience
Symposium on Optical Imaging: Applications to Biology and
Medicine
Mary J. Cole1, Melinda Pirity2, 3 & Anna-Katerina Hadjantonakis4
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1 Department of Physiology, Genentech Inc.,
1 DNA Way, San Francisco, California
94080, USA
2 Department of Molecular Genetics, Albert Einstein
College of Medicine, 1300 Morris Park Avenue,
Bronx, New York 10461, USA
3 Institute of Genetics, Biological Research Center
of the Hungarian Academy of Sciences, PO Box 521, Szeged
6701, Hungary
4 Department of Genetics and Development, College
of Physicians and Surgeons of Columbia University, 701 West 168th
Street, New York, New York 10032,
USA
To whom correspondence should be addressed
Anna-Katerina Hadjantonakis Tel: +1 212 305 4791; Fax: +1 212 923 2090;
akh39@columbia.edu
Received 31 March 2003; Accepted 22 July 2003; Published online 22 August 2003.
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This dynamic symposium, held on 11–16 February 2003 in Taos,
New Mexico, was the first Keystone meeting to focus on optical techniques and
their use in biology and medicine. It was organized by D. Becker, D. Farkas and
S. Fraser and attracted almost 100 participants from both academia and
industry. Fluorescence imaging and its applications, ranging from
nano-bioscience to small-animal imaging and imaging of disease progression in
humans, were the main topics, with opportunities for further discussion in the
cantinas of the town and on the ski slopes of Taos mountain.
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Introduction
Advances in molecular and cellular biology techniques, combined with
concomitant developments in a kaleidoscope of biologically specific fluorescent
markers, improvements in detector sensitivity and the design of dedicated
small-animal imaging systems, has resulted in a powerful toolbox to visualize
and quantify biological processes at the cellular and subcellular level, in
both in vitro and intact living organisms. More specifically, imaging of
cellular and molecular processes, such as gene expression and
protein–protein interactions, monitoring of cell trafficking and
targeting, optimization of drug and gene therapy and assessment of development
and disease progression, are all now possible using optical techniques.
Presentations at the 'Keystone Symposium on Optical Imaging' covered all of
these aspects.
Probes and techniques for dynamic cellular imaging
R. Tsien (San Diego, CA, USA) gave the keynote address and set the
stage for the meeting by illustrating the broad applicability and superior
sensitivity of many newly developed fluorescent probes (Zhang et al., 2002). He introduced the Aequoria
victoria-derived green fluorescent protein (GFP) and spoke of recent
efforts to expand its repertoire through the generation of biologically
compatible spectral variants. Several of these wavelength-shifted fluorescent
proteins have been developed in Tsien's laboratory, including 'Citrine', a
yellow fluorescent protein (YFP) mutant with reduced pH sensitivity and
superior photostability compared with other YFPs. Tsien also discussed how
recent work from his lab has succeeded in generating a monomeric red
fluorescent protein, mRFP1.
Real-time functional imaging of live specimens demands reporter
assays that function as a direct read-out of a physiological process. Tsien
described how fluorescence resonance energy transfer (FRET)-based assays might
be exploited for these purposes. Briefly, FRET measures the energy transfer
between two chromophores (donor and acceptor) whose emission spectrum and
absorption spectrum, respectively, overlap. The degree of energy transfer is
highly distance dependent and is therefore useful for monitoring changes in the
interaction and conformation of the molecules. Tsien described the use of FRET
in several assays that use the visible fluorescent proteins cyan (CFP) and YFP
as the donor and acceptor, respectively, one of which was used to measure
kinase/phosphatase activity.
He went on to describe the biarsenical–tetracysteine system,
an alternative to fluorescent-protein labelling. Here, a non-fluorescent
membrane-permeable biarsenical dye, FIAsH (fluorescent arsenical helix binder;
508/528 nm) or ReAsH (resorufin-based red arsenical helix binder; 593/608 nm),
is engineered so that it forms a fluorescent covalent complex with any
intracellular protein that contains a short engineered tetracysteine motif.
This small peptide tagged onto the end of, or possibly even integrated into, a
protein sequence enables biarsenical dyes to bind. This is especially useful in
cases in which the comparatively large size of fluorescent proteins might
hinder wild-type protein function. He illustrated the potential of this system
through a 'pulse-chase' experiment, in which sequential labelling with
different biarsenical dyes was used to follow the dynamics of connexin-43
protein turnover at the gap junctions of cells grown in culture. ReAsH is
particularly promising as it can also be used for electron microscopy, and
therefore represents a genetically encoded targetable tag that, under intense
illumination, can oxidize diamenobenzine (DAB) into a polymer that can be
stained with osmium tetroxide.
T. Jovin (Göttingen, Germany) also disussed many of the probes
from Tsien's talk, particularly fluorescent proteins. He warned that the
accuracy of FRET is often compromised by the lack of a priori knowledge
about the amount of chromophore present, and, when used in an imaging
configuration, by the difficulty of quantifying FRET efficiencies for every
pixel in the field of view. He presented two approaches for circumventing these
limitations; energy migration FRET (emFRET) and photochromic FRET (pcFRET;
Fig. 1). In emFRET (Clayton et
al., 2002), a single label with a small 'Stokes shift' (the
difference in wavelength between excitation maximum and fluorescence emission
maximum) is used. The interchange of excitation energy between identical
molecules is probed by measuring the change in emission anisotropy (the change
in polarization between the excitation light and emitted fluorescence) due to
homotransfer (concentration depolarization). This incarnation of FRET improves
the ease and reliability of fluorophore concentration measurements without the
need for cumbersome calibration of fluorescence signals, cell numbers and cell
size. Jovin's group is applying this technique to assess dimerization of erbB
(erythroblastic leukaemia viral oncogene homologue) receptor tyrosine kinases
on cell surfaces. pcFRET involves the use of specially designed acceptor
molecules whose absorption state can be reversibly switched in response to
illumination at a particular wavelength (Fig. 1A,B). By
switching the molecule between the two states, FRET can be switched on and off
in a localized and reversible manner. Thus, a reference measurement (donor
fluorescence only, FRET off) can be made in addition to the FRET-induced
acceptor-fluorescence signal. As this process is reversible, re-examination of
the same region at different time points is possible (Fig.
1C,D). Repeated quantitative FRET measurements are therefore possible,
even under the unknown donor–acceptor stoichiometries that are typical in
microscopy experiments. It is expected that the pcFRET approach will be
extended to include the development of nucleic acid and intramolecular
protein-based FRET probes, thus complementing the abilities of the FlAsH and
ReAsH labels described by Tsien.
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Figure 1
Photochromic FRET. (A) The photochromic compound (a
diheteroarylethene) is converted from a colourless open form to a fluorescence
resonance energy transfer (FRET)-competent acceptor closed form on irradiation
with ultraviolet (UV) light. The open form lacks absorbance in the visible
range. Thus, the overlap with the emission of the donor (in this example,
Lucifer Yellow covalently coupled to the acceptor) is negligible. (B)
The acceptor (closed form) absorbance overlaps well with the donor emission and
FRET is possible. (C) Donor emission spectra for the two states of the
acceptor. (D) Multiple FRET cycles generated by alternating exposure to
UV and visible light.
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S. Simon (New York, NY, USA) discussed recent work using quantum
dots (QDs), which have recently emerged as useful markers for biological
applications (Chan et al., 2002). QDs are
semiconductor nanocrystals  10–15 nm in diameter and are perhaps one
of the best recent examples of technology migration from the physics laboratory
into the domain of biology. Initial QD research focused on the photophysics of
these nanostructures and their potential application in micro- and
optoelectronics. However, more recent developments in coating technology have
resulted in biocompatible QDs that are non-toxic, have more specific binding
properties and are stable for long periods in the watery cellular milieu.
Variations in the size of the QD result in a change in its
fluorescence-emission wavelength, with larger dots having longer emission
wavelengths. In addition, dots of all sizes can be excited with a single source
in the ultraviolet/blue range of the spectrum. This spectral versatility,
combined with improved biocompatibility, is opening the door to the use of QDs
for a variety of multicolour imaging experiments in the cell and developmental
biology arenas. Other advantages are that QDs are resistant to photobleaching,
have quantum efficiencies that exceed those of most organic dyes, and can be
coupled to biomolecules of interest. Simon discussed the use of QDs for the
long-term imaging of multiple fluorophores in living cells (Jaiswal et al., 2003), in the context of his work on
the dynamics of molecules involved in signal transduction and exo- and
endocytosis. In addition to discussing the use of QDs as labels, he highlighted
the virtues of total internal reflection fluorescence microscopy (TIR-FM) for
live-cell imaging. Simon showed that this technique allows the acquisition of
high-resolution images with high signal-to-noise ratios in real-time, which are
all necessary to resolve the detailed kinetics of individual cell-surface
events (Rappoport & Simon, 2003).
Intense interest in QDs provoked an impromptu open-microphone
session during which potential future applications of QDs were discussed, along
with a few caveats. Because of their bright, spectrally narrow fluorescence
emission and the increasing availability of QDs at longer wavelengths, it is
anticipated that QDs will allow direct translation of cell-based assays into
in vivo animal imaging investigations. Caveats include the possibility
that the blinking (such that fluorescence emission is not constant) of quantum
dots will preclude their use for some applications, for example, fluorescence
correlation spectroscopy. Concerns were also raised about their potential to
form aggregates, their significant size, and whether they could be targeted to
confined subcellular compartments. Despite these limitations, QDs will probably
come to the fore as widely used markers for several biological
applications.
D. Walt (Boston, MA, USA) described his most recent work on the
development of high-density optical fibre arrays for cell-based sensing. By
etching the face of a fibre bundle, high-density arrays of microwells can be
formed in which single cells can be trapped. Trapping cells that have been
genetically modified to respond to a specific analyte results in a biosensor
that can be used to monitor, for example, the expression of different reporter
genes. Walt also discussed the possibility that this work could be extended to
include cell selection using optical tweezers.
A final talk on the cellular applications of optical imaging was
given by R. Singer (Bronx, NY, USA). He described the concurrent visualization
of gene expression and dynamic nuclear structures to image the transcriptional
states of individual cells. This approach links cell biology and genetics by
correlating sub-nuclear physical topology with gene expression (Levsky & Singer, 2003). The methodology combines
advances in computational fluorescence microscopy with multiplexed fluorescent
probe design, and facilitates the visualization of several genes in situ
at high resolution (Fig. 2A). Interestingly, Singer
showed that population variability is often observed, as variable subsets of
genes are actively transcribed in individual cell nuclei within a single
population.
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Figure 2
Multimodal images. (A) Simultaneous detection of many genes
in a single human colon adenocarcinoma cell G2 nucleus (DAPI stained) with a
pseudo-coloured representation of 17 transcription sites detected in
situ. The image is 'flattened' so that all 12 of the 0.5- m Z sections
are displayed on the background, which is the DAPI counterstain from the middle
image of the stack. Gene identity is denoted by colour and the Z location is
recorded by the adjoining number. Lower numbers represent closer proximity to
the cover slip. (B) Two colour-coded depth projections generated from a
stack of multiphoton laser scanning microscopy Z sections taken through a
6.5-day-old mouse embryo constitutively expressing a histone–green
fluorescent protein fusion. Colours denote fluorescence at different depths of
the Z stack. (C) Two images of 9.5-day-old mouse embryos. Surface view
(top) and an embryo with a sagittal cut exposing internal structures (bottom).
The technique used to produce these images is surface-imaging microscopy and
the images were rendered in ResView 3.1. (D) Imaging tumour burden and
annexin-V binding after a single dose of chemotherapy. In vivo
bioluminescence imaging was used to assess tumour burden in an orthotopic
lymphoma model as a means of standardizing treatment groups before chemotherapy
(whole-body image in background). After therapy, 9mTc-annexin-V
binding was imaged using bioluminescence imaging to assess chemotherapy-induced
tumour-cell death in the same animal. (E) Three-dimensional model
highlighting portions of the limbic system of the adult human brain. Similar
models have been generated for the mouse brain. These models can be measured
morphometrically and can also be used to represent population statistics. K,
kidney; SP, spleen.
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In vivo embryo and animal imaging
The need to translate the sensitivity and specificity of
fluorescence-based cellular-imaging techniques into those used for transgenic
animal models, and potentially into a clinical setting, was acknowledged by
many of the meeting participants. In particular, the availability and relative
ease of generating mutants, combined with the establishment of protocols for
in vitro culture and the optical transparency of developing vertebrate
embryos, make genetically encoded fluorescent proteins ideal markers for
developmental biology applications (Hadjantonakis et
al., 2003).
Confocal laser scanning microscopy and multiphoton laser scanning
microscopy (MPLSM) applied to embryogenesis were central to S. Fraser's
(Pasadena, CA, USA) talk. Fraser showed that the fluorescent vital dye BODIPY
ceramide could be used to follow the movement of cells in the region of the
rhombic lip (the structure that ultimately gives rise to the cerebellum) in
zebrafish embryos. He then went on to describe a yeast Gal4–VP16-based
method for generating transgenic fish that robustly express fluorescent
proteins, which permits in vivo imaging of individual cells. He also
discussed the methodology of surface-imaging microscopy (Fig.
2C) for the high-resolution three-dimensional (3D) reconstructions of
embryos (Ewald et al., 2002). Finally,
experiments that pave the way for the unequivocal separation of spectrally
overlapping fluorochromes using linear unmixing and emission and excitation
fingerprinting were described.
M. Chalfie (New York, NY, USA), whose group was the first to use GFP
in a heterologous system, spoke of recent work that integrates imaging and
genetic approaches to explore the distribution and function of touch receptors
in the nematode Caenorhabditis elegans; the transparency of this
organism makes it well suited for imaging the expression of genetically encoded
fluorescent proteins.
However, the mouse is now the premier mammalian model organism. K.
Hadjantonakis (New York, NY, USA) described new strains of mice that express
subcellularly localized fluorescent proteins, which facilitate high-resolution
imaging in living embryos (Fig. 2B). E. Jones (Pasadena,
CA, USA) spoke about the use of high-speed line-scanning techniques to
determine blood flow velocities in living mouse embryos and illustrated this
using a line of transgenic mice in which GFP is exclusively expressed in
embryonic blood cells. Both Hadjantonakis and Jones discussed the development
of whole- embryo in vitro culture protocols that enable time-lapse
imaging of fluorescent proteins.
Fluorescent-protein-expressing mice and optical imaging techniques
have greatly advanced the field of neurobiology in recent years (Lichtman & Fraser, 2001). Two talks discussed the use
of optical imaging for monitoring the development of the nervous system in
mice. L. Katz (Durham, NC, USA) showed the use of MPLSM for imaging the
architecture of the mouse olfactory bulb. His work combined optical-imaging
techniques with the use of genetically modified (fluorescent protein-expressing
transgenic and knockout) mice, electrophysiology (targeted intracellular
recordings) and neuroanatomy. Intrinsic signal imaging was performed through
the thinned skull of a mouse and was used to investigate the spatial coding
information within the olfactory epithelium, in which approximately 1,200
G-protein-coupled receptors are each expressed in distinct populations of
sensory neurons. Different odorants elicit different responses by affecting
only a subset of sensory neurons, and these axons converge on the main
olfactory bulb where the information is processed. Katz described the success
of his laboratory in mapping the responses to different molecules and mixtures,
the latter being represented by the additive patterns of the constituent
components, in wild-type and mutant mice.
J. Lichtman's (St Louis, MO, USA) talk focused on the use of a
series of transgenic mice in which the Thy1 promoter drives expression of a
variety of fluorescent proteins, which have a wide range of expression patterns
presumably due to transgene position effects. Repeated imaging of a
neuromuscular junction allowed the investigation of the dynamics of
neuromuscular junction synapse formation. Lichtman was able to draw several
important conclusions about the guidance of neurons during injury and about the
basic mechanisms of neuronal branching and synapse maturation on the basis of
these studies. For example, a large amount of branch retraction takes place
during the first week after birth, during maturation of the nervous system, and
axons branch into muscle fibres in newborn mice more than do those of
1-week-old pups.
M. Cahalan (Irvine, CA, USA) continued the intravital imaging theme.
He described the use of MPLSM for the imaging of cells in the lymph nodes of
living mice. Naive T cells were collected from donor animals, labelled and
injected through the tail vein into recipient animals. Subsequent imaging and
analysis of the velocities at which both T and B cells travel through the
lymphatic system revealed that T-cell behaviour changed after antigen
challenge. Extending this work to the imaging of dendritic cells allowed the
dynamic interactions between T cells and the dendrites of dendritic cells to be
visualized.
Imaging disease states: from animal models to
humans
The discussion of imaging in mice was not limited to fluorescence
techniques, and several talks were devoted to bioluminescence imaging (BLI;
Contag & Ross, 2002). C. Contag (Stanford, CA,
USA) gave an overview of recent advances in the field of small-animal
luciferase imaging. Luciferase genes have been cloned from several organisms,
including bacteria, firefly (Photinus pyralis) and coral
(Renilla). In the firefly, the conversion of luciferin to oxyluciferin
in the presence of a luciferase catalyst, ATP and oxygen results in emission of
photons (bioluminescence). The resulting broad spectral emission peaks at 560
nm and includes a significant component greater than 600 nm, which makes it
suitable for in vivo imaging. In the case of in vivo BLI, animal
models (usually murine) are used, in which the biological entities of interest
(for example, tumour cells and immune cells) express the luciferase gene
(luc). The subsequent administration of luciferin, most commonly by
intraperitoneal (i.p.) injection, results in the emission of photons from sites
of luc expression. The photons are detected using sensitive cameras,
such as intensified charged coupled devices (CCDs) or cooled, back-thinned
CCDs. The potential for combining BLI with other imaging techniques was shown
by S. Mandl (Stanford, CA, USA) who gave a short talk describing the use of BLI
in combination with single-photon emission computed tomography for assessment
of apoptosis and tumour-cell loss in mice (Fig. 2D).
In addition to describing the application of in vivo BLI to
several disease models, including examples drawn from immunology, oncology and
neuroscience, Contag discussed some of the challenges that still need to be
overcome. The current two-dimensional (2D) planar-imaging configuration used in
most laboratories is limited in its ability to provide true quantitative
measurements of disease progression, for example tumour volume. Tomographic 3D
reconstruction would provide a means of addressing this shortcoming and could
potentially be achieved through a back-projection-based method as was presented
at the meeting. Efforts to push the emission wavelength of firefly luciferase
further into the red spectral range, thus improving its penetration of
biological tissue, were also described. Modification of both the substrate and
the substrate-binding cleft can push the emission wavelength to between 620 and
675 nm; however, at present, the resulting reduction in emission intensity
outweighs any potential benefit of the wavelength increase.
T. Wyss-Coray (Stanford, CA, USA) discussed the use of a
luciferase-based in vivo biosensor to monitor the transforming growth
factor- (TGF-) signalling pathway. TGF-1 has been proposed to
be a primary organizer of the central nervous system (CNS) injury response. In
Alzheimer's disease, the variation in activation of TGF-1 prompted by
cellular damage is thought to be the cause of variability in disease outcome.
To gain insight into how TGF- and its signalling pathway are regulated in
the CNS, transgenic mice expressing luciferase under the control of the
TGF- responsive Smad-binding cis-acting element (SBE) were
generated. In this study, a kainate-induced excitotoxic injury increased the
bioluminescent signal in the hippocampus and cortex of SBE-luciferase mice,
which, in turn, indicates that the TGF-1 signalling pathway targets gene
expression in both these locations.
The neurodegenerative disease theme was continued by W. Pralong
(Zurich, Switzerland), who discussed progress in the use of gene therapy for
the treatment of Parkinson's disease. Pralong explained that the delivery of
neurotrophic factors into the brain parenchyma is expected to result in
neuroprotection and brain repair. However, because neurotrophic factors do not
cross the blood–brain barrier, they must be administered by stereotactic
injection of an encapsulated lentiviral vector into the brain. Pralong
described his use of luciferase to monitor such gene therapy approaches.
Glial-cell-derived neurotrophic factor (GDNF) was administered to mice, induced
through tetracycline regulation and the resulting expression was monitored
through the bioluminescence signal.
The above examples illustrate the huge volume of optical imaging
data that is being produced. Indeed, the need for a multi-modal,
multi-dimensional and multi-spectral atlas of mouse development was mentioned
by S. Fraser. A. Toga (Los Angeles, CA, USA) presented a mouse atlas project
that is already in progress
(http://www.loni.ucla.edu/SVG/index.html), which has successfully
combined micro-magnetic resonance imaging, histology, in situ
hybridization and immunohistochemistry data into a digital atlas of the C57Bl/6
mouse brain. Through the use of incorporated image-processing tools, the atlas
aims to facilitate the correlation of gene-expression patterns with anatomical
and molecular information drawn from a variety of imaging technologies. Such a
project is also being undertaken for the human brain (Fig.
2E).
Skin imaging was addressed in several presentations and posters. R.
Anderson (Boston, MA, USA) gave an overview of some of the challenges that are
presented by this methodology, using skin cancer and microcirculation imaging
as examples, and discussed the palette of techniques available. The most
popular of these are infrared reflectance confocal microscopy (RCM) and optical
coherence tomography (OCT), which are complementary in terms of achievable
depth penetration (RCM, 0.3 mm; OCT, 2mm) and resolution (RCM, 1 m;
OCT, 10 m), respectively. The modification of these techniques to
include additional read-outs such as fluorescence was discussed, along with the
potential benefits of using two-photon excitation to enhance penetration of the
excitiation source. Fluorescence imaging of skin and angiogenesis was the
central theme of D. Becker's (Pittsburgh, PA, USA) presentation. She described
the use of fluorescent dyes conjugated to antibodies against markers of
interest to determine the effects of inhibiting the expression of basic
fibroblast growth factor (FGF) and FGF receptor 1 in a murine model. A similar
approach was also used for imaging gene expression in melanoma precursor
lesions obtained from patients undergoing chemotherapy.
J. Fujimoto (Boston, MA, USA) showed that OCT has successfully been
implemented in the clinic for several diseases (Fujimoto
et al., 2000). This technique is essentially an optical
analogue of ultrasound imaging, and it probes variations in tissue-refractive
index so that high-resolution imaging of tissue microstructure is possible. He
described the recent developments in ultra-broadband laser sources that have
led to improvements in the resolution that is attainable using OCT, now down to
1–15 m. OCT has been successfully used for the diagnosis of acute
macular degeneration in human subjects and for skin imaging. Imaging of deeper
structures necessitates the integration of fibre-optic catheters and endoscopes
into the OCT imaging system. Fujimoto presented examples of intravascular OCT
imaging, including clot and plaque formation in a rabbit aorta. He suggested
that the current 'biopsy plus histology' gold standard for the imaging of
neoplastic changes could potentially be replaced by an in situ optical
biopsy using OCT.
Intravital microscopy allows high-resolution imaging of tumours and
the surrounding vasculature. In turn, this enables imaging of the effects of
cancer therapeutics at the microscopic level (Brown et
al., 2001). E. Brown (Boston, MA, USA) introduced the use of
second-harmonic generation (SHG) microscopy for intravital imaging. SHG relies
on the conversion of incident light at a frequency of value  , to photons
at a frequency of 2 (the second harmonic) by a nonlinear material. The
local nonlinear properties of a material are dependent on its molecular
structure and hyperpolarizability, thus the SHG signal is dependent on the
intrinsic molecular properties of the specimen, and requires no additional dyes
or reagents. Also, the non-resonant nature of this technique in combination
with near-infrared excitation wavelengths render it potentially less
biologically phototoxic. As the SHG signal is generated only at the focal point
of the incident excitation light, this technique also provides inherent optical
sectioning abilities. Brown has applied intravital microscopy to the imaging of
collagen I, which is an extracellular-matrix component that is intrinsic to
certain tumours and has a dominant role in drug transport. Its presence
therefore often determines the efficacy of treatments for certain types of
cancer. Brown was able to detect collagen-derived SHG signals by imaging a
dorsal chamber murine-tumour model. The SHG signal was found to vary among
tumour types and correlated with collagen content. This technique could be
extended to the measurement of collagen density in tumours and to assess its
impact on effective drug delivery.
Conclusions
Due to space constraints, we could not include all of the
presentations given and we apologize to the participants whose work could not
be mentioned. Hopefully, the concepts and presentations highlighted in this
report provide a flavour of the meeting and a glimpse into the future of this
field. Also, we hope that we have succeeded in conveying some of the excitement
(and anticipation) that has been generated by the broad range of optical
techniques that are now at the disposal of biologists and clinicians. However,
the best illustration of this excitement is probably the great enthusiasm with
which the presentations at this symposium were received. Hopefully, there will
be an opportunity for Keystone Symposia to revisit this illuminating and
rapidly expanding field in the not too distant future.
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Acknowledgements
We are grateful to D. Becker, D. Farkas and S. Fraser for putting
together a superb meeting. We thank A. North and T. Swayne for comments on the
manuscript, and C. Contag, E. Jares-Erijman, A. Ewald, S. Fraser, T. Jovin, S.
Mandl, R. Singer, and A. Toga for providing images used in the figures. A.-K.H.
is a fellow of the America Heart Association.
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