(Chest. 2001;120:233-239.)
© 2001
American College of Chest Physicians
The Role of Laser-Induced Fluorescence in Myocardial Tissue Characterization*
An Experimental In Vitro Study
George E. Kochiadakis, MD;
Stavros I. Chrysostomakis, MD;
Michael D. Kalebubas, MD;
George M. Filippidis, MD;
Ioannis G. Zacharakis, MD;
Theodore G. Papazoglou, MD and
Panos E. Vardas, MD, PhD
*
From the Cardiology Department (Drs. Kochiadakis, Chrysostomakis, Kalebubas, and Vardas), University Hospital of Crete, Heraklion Crete, Greece; and Institute of Electronic Structure and Laser (Drs. Filippidis, Zacharakis, and Papazoglou) Foundation for Research and Technology, Heraklion, Crete, Greece.
Correspondence to: Panos E. Vardas, MD, PhD, Cardiology Department, University Hospital of Crete, PO Box 1352, Heraklion Crete, Greece
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Abstract
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Objective: The fluorescence of tissue when stimulated
by a laser beam is a well-known phenomenon. The resulting emission
spectra depend on the biochemical and structural composition of the
tissue. In this study, we examined the spectra of laser-induced
fluorescence emitted by myocardial tissue.
Methods: We
used an argon-ion laser to stimulate the myocardium of 20 intact sheep
hearts. For each spectral emission, we calculated the intensity in
specific regions in order to characterize the spectra and to reveal
intercavitary and intracavitary morphologic differences.
Results: The statistical analysis showed significant
differences in the emission spectra intensity between atria and
ventricles. The intensity was higher in the atria than in the
ventricles (p < 0.001). The atrial emission spectra were
morphologically different from those of the ventricles. There was no
difference in the intensity or morphology of emission spectra within
each chamber. All measurements showed good reproducibility after a
short period of time.
Conclusions: Laser-induced
fluorescence of myocardial tissue seems to have the characteristics
necessary for tissue recognition. This might prove useful in
identifying cardiomyopathies and transplant rejection, as well as for
myocardial mapping, assisting electrophysiologists in discovering
fibrotic arrhythmogenic foci.
Key Words: ablation • experimental • fluorescence • heart • histology • laser • mapping • transplantation • ventricular arrhythmias
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Introduction
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The
fluorescence of tissue when stimulated by a laser beam is a well-known
phenomenon. The spectrum of the emission depends on the particular
characteristics of the tissue under examination, on its biochemical and
structural composition.1
2
3
4
In recent years, spectroscopy,
the analysis of the emission spectra of tissue, has been used
increasingly for tissue identification (type, composition,
discrimination between healthy and pathologic) with excellent
results.4
5
6
7
8
9
In this project, we studied the emission spectra of laser-induced
fluorescence of myocardial tissue. We examined the basic
characteristics of the spectra (intensity, morphology) recorded from
different anatomic sites. We also assessed the stability of the
measurements, a fundamental criterion for any technique with research
and clinical applications.
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Materials and Methods
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Tissue Samples
For the purposes of this study, we used hearts from 20 young
sheep (age < 6 months). The samples were collected immediately after
the animals were slaughtered, and the hearts were removed together with
the pericardial sac. They were transported in isothermal containers
wrapped in plastic membrane to avoid dehydration. The hearts were
examined in the laboratories of the Foundation for Research and
Technology within, at most, 2 h of the death of the animal. All
the samples appeared healthy to macroscopic examination, and there were
no regions of myocardium with scar tissue (infarcts), as expected from
the young age of the animals.
Experimental Apparatus
The experimental apparatus is shown in Figure 1
. An argon-ion laser emitting at 457.9 nm (Stabilite 2016;
Spectra-Physics; Mountain View, CA) was used for excitation. The
average power irradiating the samples during the experiments was of the
order of 10 milliwatts. The output of the laser was reflected at
right angles by a dichroic mirror (99.3% at 45° at 442 nm)
and was focused on the input of a step index, multimode optical fiber
(SPC500N; Fiberguide Industries; Stirling, NJ). Fluorescence emission
was collected by the same fiber, passed through the dichroic mirror and
was focused at the entrance slit (100 µm) of a 0.10-m spectrograph,
employing a 450 groove mm-1 holographic grating.
Data acquisition and analysis were performed via an optical
multichannel analyzer (Cronin GmbH; Eichenau, Germany), using a
diode array detector (RL1024S; EG&G Reticon; Santa Clara, CA). The
signal from the diode array detector was fed to a computer for
analog-to-digital conversion and further processing. Wavelength
calibration was performed with a mercury lamp. A high-pass filter (490
nm; Schott CG optical filter; CVI Laser Corp, Albuquerque, NM) was
placed in front of the entrance of the spectrograph in order to isolate
the fluorescence signal from reflected laser light. The spectrum
obtained during the experiment represented the time-integrated
fluorescence of the underlying tissue. The optical multichannel
analyzer was operated in the free-scanning mode.
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Figure 1.. Experimental apparatus for measurements of
laser-induced fluorescence from myocardial tissue. All optical
components, except the pumping laser (argon-ion laser emitting at 457.9
nm), are located in a diagnostic module. HPF = high-pass filter;
DM = dichroic mirror.
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Measurements and Data Processing
During examination, the temperature of the samples was that of
the laboratory, ie, 22°C to 24°C. The atria and the
ventricles were opened along their free wall. The preparations were
washed with normal saline solution to remove blood, which can absorb
tissue radiation and might thus alter the emission
spectrum.10
11
Measurements were performed under low
lighting in order to reduce the ambient optical noise. After opening,
the samples were placed in a black container on absorbent gauze soaked
in normal saline solution. The acquisition strategy was to obtain
background corrected fluorescence spectra from each tissue sample. The
optical fiber, within a protective guiding catheter, had its free tip
placed approximately 1 mm from the point of measurement for as long as
was necessary for the recording of the spectrum (3 s). Recordings were
made during laser irradiation of the background, as a measure of the
fluorescence of the fiber, saline solution, and other external sources
of noise. This background spectrum was automatically subtracted from
each sample tissue spectrum. Between measurements, the tip was rinsed
in saline solution in order to avoid accumulation of tissue on the
surface, which might have an effect on the measurements of background
that were made in pure saline solution.
Four measurements were made for each chamber of every heart.
Measurements in the ventricles were made at the apex (left ventricle
[LV1]-right ventricle [RV1]), the midpoint of the interventricular
septum (LV2-RV2), the midpoint of the anterior papillary muscle
(LV3-RV3), and on the free ventricular wall (LV4-RV4). In the atria,
the measurement sites were the free wall (left atrium [LA1]-right
atrium [RA1]), the atrial appendage (LA2-RA2 and LA3-RA3), and the
interatrial septum (LA4-RA4). For each emission spectrum, we calculated
local maximum or minimum spectral intensity within selected regions
(critical points), which were observed as "peaks" and "valleys"
in the spectral plots.
Statistical Analysis
Repeated-measures analysis of variance with one
within-four–level factor was used to assess whether there were
any significant differences among the four sites of each cavity in the
emission spectral intensities of the critical points (intracavity
comparisons). When no significant intracavity differences were found,
the measurements from each critical region for the four different sites
were combined into a single sample.
We then reemployed a repeated-measures analysis of variance design to
test whether the emission spectral intensities in the critical regions
within the four cavities were significantly different (intercavity
comparisons). In case of significant findings, post hoc
tests were used to pinpoint differences. The repeatability of the
measurements, for peak one (P1) only, at three different time points
(initial, 30 min, and 24 h) was assessed with simple linear
regression; correlation coefficients (r) and slopes (b)
close to 1 are indicators of good repeatability. All p values < 5%
were the criterion of significance throughout.
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Results
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Characteristic examples of fluorescence spectra are shown in
Figure 2 . All spectra are composed of a fluorescence band with a wide range of
peaks and valleys: in the regions of 547 to 552 nm (peak one [P1]),
566 to 571 nm (peak two [P2]), 595 to 600 nm (peak three [P3]), 666
to 671 nm (peak four [P4]), 556 to 561 nm (valley one [V1]), 580 to
585 nm (valley two [V2]), and 616 to 621 nm (valley three [V3]), a
high diagnostic potential for tissue characterization is
suggested.
Table 1
Table 1A
gives 95% confidence intervals of average spectral intensities in the
above-mentioned critical points of the emission spectrum, from all
measurement sites. For all these points, the maximum intensity was in
the range of P1 at 545 nm and the spectral intensity values showed a
decreasing sequence from P1 to P2 to V1 to P3 to V2 to P4 to V3.
In each cardiac chamber, there were no significant differences among
the four acquisition sites with regard to spectral intensities in any
of their critical spectral points (four local peaks and three valleys).
Between the chambers, however, there were very significant differences,
both in emission spectral intensities and in morphology of spectral
plot. More specifically, the average fluorescence emission intensities
of all critical points were significantly higher when atria were
compared to ventricles (p < 0.001) and when left cavities were
compared to right (p < 0.001).
Furthermore, the magnitude of differences was not similar for all
critical points. As shown in Figure 3
, the magnitude of the differences in spectra, both between atria and
ventricles and between left and right chambers, tended to decrease as
the spectral wavelength increased. Measurements from all 16 sites (4
sites for each chamber) were used for the comparison among cavities,
since there was no intracavity variation.
Reproducibility of Measurements
There was good reproducibility of the spectral intensity
evaluation in all critical points of the emission spectra in all
cavities when the first and second measurements were compared
(correlation coefficient [r], and slope, [b] very close
to 1), but there was no good correlation between the first and third
measurements (Table 2
).
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Table 2.. Correlation Coefficient (r) and Slope
(b): First vs Second and First vs Third Measurements in All
Critical Regions of the LA and LV*
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Discussion
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Optical spectroscopy is a technique that has been used
increasingly in recent years for tissue
identification.4
5
6
7
8
9
It is based on the fact that tissue
emits photons when excited by a laser beam.1
2
3
4
More
precisely, photon absorption by the tissue causes the excitation of
electrons within its molecules and their elevation to a higher energy
level. The stimulated electrons remain in this higher-energy state for
approximately 10-8 s, after which they decay to
the ground state by the emission of photons (fluorescence).
The emission spectrum of a tissue is the sum of the emission spectra of
the main fluorescent substances: elastin, collagen, and nicotinamide
adenine dinucleotide.1
2
3
4
12
13
There are, however,
many other substances that may contribute to the creation of the
spectrum. In addition, the scattering of the emitted radiation in the
tissue is quite high, leading to alteration of the
signal.5
12
14
All the above factors result in the
creation of artificial peaks and valleys in the fluorescence spectrum
that are characteristic of the particular type of tissue.
In the present study, we investigated the characteristics of the
emission spectra of myocardial tissue under stimulation by an argon-ion
laser, which emits at 457.9 nm. The wavelength used achieves tissue
penetration of the order of hundreds of microns. Even though the
radiation within the tissue falls exponentially
(I = Io x e-kx, where
x = depth), a substantial number of photons pass through the
endothelium and reach the interior sections of the tissue.
Our results show that there are significant differences in the emission
spectra recorded from different anatomic sites within the heart. The
atrial spectra not only have an overall higher intensity, but also a
different morphology from those recorded in the ventricles. This
morphologic difference is due to the fact that the difference between
the mean spectral intensities in the atria and the ventricles is much
greater in the short than in the long wavelength bands of the emission
spectrum. It is worth noting, however, that within each chamber the
emission spectrum is more or less constant and appears to be
independent of the site from which it is recorded. These observations
are made herein for the first time (to our knowledge), and are clearly
crucial if the method is to be used for clinical purposes. Also of
crucial importance is the good reproducibility of our results after 30
min. The worsening reproducibility over a longer period does not
invalidate the method. Tissue and biochemical changes occurring over
that time might be expected to lead to changes in the emission spectra.
At this time, we do not know the reason for the difference in
fluorescence between atrial and ventricular tissue. Myocardial fibers
are known to be closer to the thin layer of ventricular endocardium,
while in the atrial myocardium they lie under a thicker layer of
endothelium.11
Taking into account the low degree of
penetration of the laser beam (a few hundred microns), this difference
could explain the higher spectral intensity of the atrial fluorescence,
but not its different morphology. Further studies are needed to
determine the biochemical and histologic structures that are
responsible for the peaks and valleys in the emission spectrum, in both
the atrial and ventricular myocardium.
Another issue that must be addressed is whether the laser beam
causes damage to the myocardial tissue. Although it is true that the
light energy used is very small (10 to 12 milliwatts) and the time of
illumination (approximately 3 s) is short enough to make any
tissue damage unlikely, this needs further, more detailed
investigation, including in vivo studies.
Comparison With Previous Studies
A small number of earlier studies5
8
11
14
also used
laser spectroscopy for the identification of myocardial tissue. Nilsson
et al14
recorded the emission spectra from the LV of pig
hearts under laser stimulation and compared it with the spectrum of
other tissues. Perk et al5
showed that the
characteristics of the emission spectra from fibrotic
and/or ischemic myocardial tissue under laser stimulation at 308 nm
were different from those of healthy myocardium. The same investigators
in another study11
showed that the emission spectra of the
human nodal conduction system were different from that of surrounding
tissue. In contrast, Aziz and coworkers,8
using a laser of
similar wavelength in dog hearts, found no difference between the nodal
conduction system and surrounding myocardial tissue. In a more recent
study, Morgan et al15
recorded the emission spectra from
the LVs of rats, excited by blue light, and showed that the spectrum
changed consistently as a heart underwent transplant rejection and
could even distinguish the degree of tissue rejection.
The above studies were based on the examination of small histologic
preparations and/or were aimed at answering specific questions. In
contrast, our study is the only one (to our knowledge) to examine whole
and healthy hearts. Thus, we were able to get an overall evaluation of
the emission spectra of myocardial tissue, not only examining the basic
characteristics (intensity, morphology, reproducibility of
measurements), but also relating them to specific anatomic sites.
Limitations of the Study
In this study we used sheep hearts. We came to this decision for
two reasons: first, the ready availability of intact, young sheep
hearts that could be presumed to be healthy; second, because our study
protocol includes a second, in vivo phase in which sheep
will be used. It remains to be investigated to what extent the
constancy of measurements within each cardiac chamber and the
reproducibility of the method apply to human hearts.
The most important limitation of the method arises from the fact that
the depth of penetration of the laser beam is only a few hundred
microns.1
2
3
4
As a result, the information obtained by
spectroscopy reflects the composition of the endocardium and only a
small thickness of myocardial tissue.
At this point, it is worth mentioning that the penetration depth could
have been greater had we used a laser that emits at a higher
wavelength. However, the increase in depth would be at the expense of a
loss in diagnostic capability, since there are not many chromophores in
the tissue that can absorb the higher wavelength and produce
fluorescence. Furthermore, our selection was based on the fact that it
enabled us to use a relatively small device that can be employed within
the laboratory along with standard techniques. The remaining
limitations are due to its being an in vitro study.
Conclusions – Future Applications
Spectroscopy as a means of identifying tissue has only been used
until now for experimental purposes. However, at least with regard to
the myocardial tissue that we examined, it appears to have the
necessary properties to be used for clinical applications as well.
According to our findings and those of other studies, spectroscopy
might prove useful in electrophysiology for the mapping of myocardium
and the detection of fibrotic arrhythmogenic foci. The various
electrophysiologic tests that are used for this purpose have a
sensitivity of 50 to 90%. Spectroscopy, even as a supplementary
examination, could offer significant assistance.
Spectroscopy might also prove useful in the detection of pathologic
myocardial states (cardiomyopathies, myocarditis, right ventricular
dysplasia, intramyocardial fibrosis, etc) or tissue rejection in the
case of a transplanted heart, enhancing and in some cases replacing
biopsy. It should be noted that spectroscopy appears to have two basic
advantages over biopsy: first, the capacity for multiple measurements
from different sites at one session, and second, the instant
availability of results. In vivo studies will be needed in
order to establish whether the above-mentioned clinical applications
are indeed feasible.
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Acknowledgements
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The authors acknowledge the contribution of Gregory
Chlouverakis, PhD, to the statistical analysis.
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Footnotes
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Abbreviations: LA = left atrium; LV = left
ventricle; P1 = peak one; P2 = peak two; P3 = peak three;
P4 = peak four; RA = right atrium; RV = right ventricle;
V1 = valley one; V2 = valley two; V3 = valley three
Received for publication May 22, 2000.
Accepted for publication January 3, 2001.
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References
|
|---|
-
Andersson, PS, Montan, S, Persson, T, et al (1987) Fluorescence endoscopy instrumentation for improved tissue characterization. Med Phys 14,633-636[CrossRef][ISI][Medline]
-
Tsien, RY (1989) Fluorescent probes of cell signaling. Ann Rev Neurosci 12,227-253[CrossRef][ISI][Medline]
-
Papazoglou, TG, Arakawa, K, Grundfest, WS, et al (1990) Laser-induced tissue autofluorescence versus exogenous chemical probe induced fluorescence as an arterial layer detection method: a comparative study. Optic Fiber Med V Spie 1201,16-26
-
Laifer, LI, O’Brien, KM, Stetz, ML, et al (1989) Biochemical basis for the difference between normal and atherosclerotic arterial fluorescence. Circulation 80,1893-1901[Abstract/Free Full Text]
-
Perk, M, Flynn, GJ, Smith, C, et al (1991) Laser-induced fluorescence emission: I. The spectroscopic identification of fibrotic endocardium and myocardium. Lasers Surg Med 11,523-534[ISI][Medline]
-
Lam, S, Palcic, B, McLean, D, et al (1990) Detection of early lung cancer using low-dose Photofrin II. Chest 97,333-337[Abstract/Free Full Text]
-
Laufer, G, Wollenek, G, Hohla, K, et al (1988) Excimer laser-induced simultaneous ablation and spectral identification of normal and atherosclerotic arterial tissue layers. Circulation 78,1031-1039[Abstract/Free Full Text]
-
Aziz, D, Caruso, A, Aguirre, M, et al (1992) Fluorescence response of selected tissues in the canine heart: an attempt to find the conduction system [abstract]. Proc Int Soc Optic Eng 1642,166-175
-
Schomaker, KT, Frisoli, JK, Richter, JM, et al (1990) Discrimination of adenomatous from hyperplastic colonic polyps in vitro by laser-induced fluorescence [abstract]. Lasers Surg Med 2(suppl),5
-
Leon, MB, Lu, DY, Prevosti, LG, et al (1988) Human arterial surface fluorescence: atherosclerotic plaque identification and effects of laser atheroma ablation. J Am Coll Cardiol 12,94-102[Abstract]
-
Perk, M, Flynn, GJ, Gulamhusein, S, et al (1993) Laser induced fluorescence identification of sinoatrial and atrioventricular nodal conduction tissue. Pacing Clin Electrophysiol 16,1701-1712[CrossRef][Medline]
-
Papazoglou, TG (1995) Malignancies and atherosclerotic plaque diagnosis: is laser induced fluorescence spectroscopy the ultimate solution? J Photochem Photobiol B 28,3-11[CrossRef][Medline]
-
Horvath, KA, Torchiana, DF, Daggett, WM, et al (1992) Monitoring myocardial reperfusion injury with NADH fluorometry. Lasers Surg Med 12,2-6[ISI][Medline]
-
Nilsson, AM, Heinrich, D, Olajos, J, et al (1997) Near infrared diffuse reflection and laser-induced fluorescence spectroscopy for myocardial tissue characterization. Spectrochim Acta A Mol Biomol Spectrosc 53A,1901-1912[CrossRef]
-
Morgan, DC, Wilson, JE, MacAulay, CE, et al (1999) New method for detection of heart allograft rejection: validation of sensitivity and reliability in a rat heterotopic allograft model. Circulation 100,1236-1241[Abstract/Free Full Text]
TOP
Abstract
Introduction
P1 = difference in spectral
intensity at P1 between cavities;