Photobleaching of Arterial Fluorescent Compounds: Characterization of Elastin, Collagen and...

Photochemistry and Photobiology, 1999, 69(6): 71 3-721

Photobleaching of Arterial Fluorescent Compounds: Characterization of Elastin, Collagen and Cholesterol Time-resolved Spectra during Prolonged Ultraviolet Irradiation

Laura Marcu1,2, Warren S. GrundfesV and Jean-Michel 1. Maarek*’ ‘Department of Biomedical Engineering, University of Southern California, Los Angeles, CA and ‘Laser Research and Technology Development Laboratory, Cedars Sinai Medical Center, Los Angeles, CA

Received 1 December 1998; accepted 19 March 1999

ABSTRACT

To study the photobleaching of the main fluorescent compounds of the arterial wall, we repeatedly measured the time-resolved fluorescence of elastin, collagen and cholesterol during 560 s of excitation with nitrogen laser pulses. Three fluence rate levels were used: 0.72,7.25 and 21.75 pW/mm2. The irradiation-related changes of the fluorescence intensity and of the time-resolved fluorescence decay constants were characterized for the emission at 390, 430 and 470 nm. The fluorescence intensity at 390 nm decreased by 25-35% when the fluence delivered was 4 mJ/mm2, a common value in fluorescence studies of the arterial wall. Cholesterol fluorescence pho- tobleached the most, and elastin fluorescence photo- bleached the least. Photobleaching was most intense at 390 nm and least intense at 470 nm such that the emission spectra of the three compounds were markedly dis- torted by photobleaching. The time-resolved decay constants and the fluorescence lifetime were not altered by irradiation when the fluence was below 4 mJ/mm2. The spectral distortions associated with photobleaching complicate the interpretation of arterial wall fluorescence in terms of tissue content in elastin, collagen and cholesterol. Use of the time-dependent features of the emission that are not altered by photobleaching should increase the accuracy of arterial wall analysis by fluorescence spectroscopy.

INTRODUCTION

Laser-induced fluorescence spectroscopy is a widely ex- plored technique for medical diagnosis that could be used in situ for nondestructive optical biopsy. An application of con- siderable interest is the monitoring of atherosclerosis pro- gression (1,2). In some studies, the fluorescence spectra of artery tissue reflected the biochemical composition of the arterial wall (3-5): elastin fluorescence was dominant in

*To whom correspondence should be addressed at: Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90089-145 1, USA. Fax: 213-740-0343; e-mail: jmaarek@ bmsrs.usc.edu

0 1999 American Society for Photobiology 003 1-8655/99 $S.OO+O.OO

healthy arterial wall whereas plaque collagen was responsible for the emission of advanced (collagenous) atherosclerotic lesions (3). While most studies focused on the spectral content of the arterial wall emission, a few investigators (6- 8) observed that the time-dependent decay of the fluorescence measured with time-resolved techniques provided additional information that could enhance the characterization of atherosclerotic lesions.

Prolonged irradiation of a tissue reduces the intensity of the fluorescence emission due to irreversible photobleaching of the tissue fluorophores (9-1 1). Photobleaching of arterial tissue was described by Chaudhry et al. (9). who reported a decrease in human aorta fluorescence during prolonged excitation with 476 nm laser light. The intensity of collagen fluorescence decreased more than that of elastin as a function of irradiation time (12), suggesting that photobleaching affected each fluorescent compound of the arterial wall in a unique way. Photobleaching has also been reported for other tissues such as skin in studies that compared the fluorescence fading of tissue to that of photodynamic therapy agents (1 1). Fluorescence fading unevenly affected the emission spectrum of skin collagen to reflect the preferential destruction of some collagen fluorophores over others (10). In general, however, there is only limited information available on the photobleaching characteristics of fluorescent compounds in tissue.

In this paper, we report that photobleaching describes an irreversible decrease of the fluorescence intensity that results from exposure to the excitation laser. Photobleaching is caused by the chemical change of an excited fluorophore into a species that does not fluoresce because of its inability to absorb excitation photons (1 1,13). For simple molecules with one fluorophore, photobleaching does not change the fluorophore lifetime ( 12). Complex fluorescent molecules such as elastin and collagen have more than one fluorophore (14-16). The different fluorophores are likely to photobleach at different rates with irradiation. Because the time-dependent fluorescence decay of these molecules is a composite of the decay characteristics of the different fluorophores, the lifetime could change with photobleaching to reflect a decreased contribution of the fluorophores most altered by irradiation. Photobleaching of the fluorescent compounds in artery tissue could therefore be accompanied by both spectral and lifetime changes that would complicate the interpre-

713

714 Laura Marcu et a/.

FL

FO (fluorescence)

MONO- CHROMATOR pm

SAMPLE ?,+, PREAMP* ENERATOR

Tngger Signal

Figure 1. Time-resolved fluorescence spectroscopy setup wed to measure the photobleaching characteristics of arterial fluorescent compounds. BS 1 and BS 2, quartz beam splitters: PD I and PD 2. photodetectors: PMT. multichannel plate photomultiplier tube: Pre- amp., I GHz preamplifier.

tation of fluorescence measurements in terms of tissue composition. To our knowledge. the effect of photobleaching on the spectral and temporal emission characteristics of elastin and collagen has been investigated only in a limited way (17).

In addition to elastin and collagen, certain lipids that ac- cumulate in the arterial wall during the development of atherosclerosis have heen shown to emit fluorescence (12.18,19). These fluorescent lipids include the lipopigment ceroid, free cholesterol. and cholesterol esters. Little is known of their photobleaching characteristics at the wavelengths and excitation intensities commonly used to probe arterial tissue samples.

This study had two goals: first, to investigate the photobleaching characteristics of the main fluorescent compounds of the arterial wall during prolonged irradiation with nitrogen laser pulses, and second. to determine levels of irradiation that minimize the distortion of the time-resolved spectra.

MATERIALS AND METHODS LiLq/tt &/ii.ur? nrtrl,Pir~,rc,sc,r,ic.e durectiot,. In the experimental setup (Fig. I ) . the output pulsc of a nitrogen laser (EG&G. model 2100: A = 337 nm: pulse width = 3 ns FWHM: pulse repetition rate = 1 0 Hz) was focused on thc illumination channel (diameter = 1.4 mm; 200 bni \ilicn fiherz: NA = 0.22) of a fluorescence probe (Ori- el. model 77558) and directed to the sample (spot size = 8 mm’) from aboLe. A ring of 200 p i silica fibers around the illumination channel i n thc fluorescence probe (center-to-center distance = 0.9 mni) captured the tranhient fluorc\cence pulse emitted by the sample. The emission was guided toward the input slit of a scanning mono- chroniator (Oriel. model 77200: F/4.4: UV-vis grating = 1200 groobeshni: bandpa,\ = 5 nm FWHM) equipped with a stepper-

motor wavelength drive (Oriel, model 77228). A gated multichannel plate photomultiplier tube (Hamamatsu, model R2024U; rise time = 0.3 11s: high voltage = 2000-2200 V) at the exit slit of the mono- chroniator detected the dispersed fluorescent transient. The photomultiplier output was amplified with a preamplifier (EG&G Ortec, model 9306; rise time = 0.35 ns; bandwidth = 100 kHz-1 GHz) and digitized with a digital oscilloscope (Tektronix, model TDS 620A: bandwidth = %SO0 MHz; sampling rate = 2 X lo9 samples/ s ) . A fraction of the laser output pulse was directed with two quartz beam splitters toward two fast silicon detectors. One detector (New- port. 8 18-BB-2 1 ) was connected to a gate-and-delay generator (EG&G Ortec. model 416A) and used to gate the photomultiplier ( 5 ks gate duration). The other detector (EG&G Parc, model 2100199) triggered the oscilloscope and monitored the pulse-to-pulse variations of the laser output energy. Data collection was automated with a personal computer that controlled scanning of the output wavelength on the monochromator and data sampling on the oscilloscope.

Soruples. Fluorescence measurements were recorded from purified samples of the principal fluorescent compounds in normal arterial wall and atherosclerotic lesions: elastin, collagen and choler- terol. Samples of type I insoluble bovine Achilles tendon collagen. bovine neck ligament elastin and cholesterol (Sigma Chemical, St. Louis. MO) were placed in a nonfluorescing cell open to atmosphere and studied at room temperature. Measurements were performed on dry specimens and specimens hydrated with 0.9% saline solution. For the latter, saline was thoroughly mixed with the sample to wet i t and permeate it for 5 min before the fluorescence data were col- lected.

E.\;Derirwri/tr( procedures. Fluorescence transients were recorded during 560 s (150 transients) of uninterrupted pulsed laser illumination for three wavelengths of emission: 390, 430 and 470 nin. These wavelengths were selected based on the shape of the emission spectra of elastin. collagen and cholesterol (3,8,20) to investigate photobleaching near the peak of emission (-390 nm) and at two wavelengths along the tail of the spectra in the visible range. Each fluorescence transient represented the average of 16 consecutive fluorescence pulses computed by the oscilloscope circuitry. A fluorescence transient required 3.7 s to be captured by the oscilloscope, transferred to the computer and stored on the computer disk. The data acquisition program was started before firing the laser, and sani- pling of the fluorescence transients began at thc first trigger signal to the oscilloscope (tirst laser pulse). In this way, the first fluorescence transient of a measurement sequence represented the initial fluorescence response of an area of the sample that had never been exposed to laser irradiation. A new site was used for each measurement sequence.

Three levels of laser pulse energy were used: 0.58, 5.80 and 17.30 pJ/pulse. The corresponding fluence rate levels determined consid- ering the pulse repetition rate of the laser and the geometry of the excitation amounted to 0.72, 7.25 and 21.75 pW/mm2. For these fluence rate levels, the total fluence on the illuminated site at the end of the measurement sequence, i.e. the total energy delivered per unit area. ranged from 0.4 I to 12.18 mJ/nim’. These levels of irradiation were selected to encompass the range of fluence used in many fluorescence studies of artery tissue (4,5,17,2 I ) . The pulse-to- pulse variability of the laser energy output was less than 5%.

After each measurement sequence, the monochromator was tuned to a wavelength slightly below the laser line. Thc average of 16 laser pulses reflected by the sample was used to represent the temporal profile of the laser pulse.

To assess the variability of the fluorescence decay associated with photobleaching, five measurement sequences were performed with the monochromator tuned to pass the wavelength 390 mi. This test wa5 performed on a new site for each dry substance and for each of the three fluence rate levels used in the study. The emission intensity at the end of the measurement period varied by less than 3% from sequence to sequence.

Data ana!\.sis. The analysis comprised two steps. First, we estimated the fluorescence intensity corresponding to each fluorescence transient and computed the reduction in intensity with prolonged irradiation (total fluence). Second. we computed the fluorescence impulse response function associated with each fluorescence transient and examined how its time decay characteristics were affected by fluence. The fluorescence intensity associated with a fluorescence

Photochemistry and Photobiology, 1999, 69(6) 715

Table 1. Normalized fluorescence emission intensity at the end of the irradiation period (average over the last 20 s)

Fluence rate level (p.W/mm2)

Hydrated Wavelength Dry Compound (nm) 0.72 7.25 21.75 0.72 7.25 2 1.75

Elastin 390 430 470

Collagen 390 430 470

Cholesterol 390 430 470

0.9 1 0.91 0.93

0.83 0.84 0.92

0.79 0.84 0.89

0.71 0.77 0.81

0.67 0.75 0.81

0.63 0.77 0.85

0.50 0.54 0.61 0.47 0.58 0.71

0.43 0.56 0.79

0.88 0.88 0.95

0.75 0.75 0.87

0.79 0.85 0.92

0.75 0.81 0.87 0.42 0.54 0.70

0.7 1 0.83 0.87

0.61 0.64 0.71

0.3 1 0.38 0.55

0.56 0.70 0.87

transient was computed by numeric integration of the area under the corresponding curve. The intensity value was then normalized with respect to the intensity of the first fluorescence transient in the measurement sequence (time 0).

The fluorescence impulse response function was retrieved by numeric deconvolution using the laser pulse profile as input and the fluorescence transient as output. The deconvolution technique has been described in previous work (8,19,22). Briefly, the impulse response function was expanded on the basis of discrete-time Laguerre functions (8,22). The numeric parameters of the expansion were it- eratively adjusted to minimize the quadratic distance between the measured fluorescence transient and its computed counterpart, i.e. the convolution of the impulse response function with the laser pulse. The decay of the fluorescence impulse response function (h(t)) with time was characterized using parameters derived by approxi- mating function h(t) with a double exponential model:

h ( t ) = aie-'"l + a2e-'h2.

The number of exponential functions was selected on a representative set of fluorescence data by verifying the absence of trends in the residuals (23.24) for a double exponential fit. In addition, the decrease in the weighted residual sum of squares was not significant for a triple exponential fit when compared with a double exponential fit (25). In the double exponential model, parameters T, and T~ represented the fast-term and the slow-term decay constants. Ratio A = a, l (n , + a2) denoted the fractional contribution of the fast-decay component to the fluorescence impulse response function. The average lifetime was estimated as the instant at which the double exponential fit crossed the lle mark.

With the present experimental setup and the deconvolution meth-

040 100 200 300 1M) 5W 6W Irradiation lime (seconds)

Figure 2. Decay of the fluorescence emission measured at 390 nm as a function of irradiation time for elastin, collagen and cholesterol. Photobleaching intensified when the fluence rate level was increased from 0.72 to 21.75 pW/rnm2. For each curve, the fluorescence intensities are normalized relative to the intensity of the first measurement point in the irradiation sequence. Note that one graphic symbol is shown for every other measurement point in Figs. 2-5.

od, decay constants down to 1 ns could be retrieved with accuracy (8,22). Subnanosecond decays could also be retrieved, albeit with an overestimation of the decay constants. The five measurement sequences analyzed to estimate the variability of the emission intensity were also processed to assess the variability of the time-dependent decay constants (<lo% for T,, <5% for T~).

To study the variations of the time-dependent decay parameters 7,. T~ and A with photobleaching, their average values over 10 data points were computed for four time periods evenly spread within the 560 s duration of the measurement sequence. The averages were used as estimates of T , , T* and A at the beginning, first third, second third and end of the photobleaching period. Variations of the decay parameters T ~ , T, and A as a function of irradiation time and fluence rate level were studied by two-way analysis of variance. Data are reported as mean 2 SE. The level of significance used was P < 0.05.

RESULTS The fluorescence emission intensity at the end of the irradiation period is presented in Table 1 for the different experimental conditions of the study. The following observations are made.

Effect of irradiation on fluorescence intensity decay for dry substances

Fluorescence emission decreased as a function of irradiation time for the three fluence rate levels of the study (Fig. 2, Table 1). At 390 nm, elastin exhibited the smallest change in emission intensity with irradiation time. Cholesterol emission decreased the most. The same fluorescence intensity data are represented as a function of fluence for elastin and cholesterol (Fig. 3) to show that the decrease in the emission intensity varied as a function of the fluence rate level. For elastin and cholesterol, high-energy pulses produced a larger decrease of the fluorescence intensity than did low-energy pulses for the same fluence delivered to the sample. In contrast, collagen fluorescence emission decreased with fluence without being influenced by the fluence rate level.

Effect of hydration

The effect of irradiation on fluorescence intensity was different for the hydrated compounds from that for the dry compounds. For elastin and cholesterol, photobleaching was less intense when the compounds were hydrated (Fig. 4). In contrast, the photobleaching of hydrated collagen was more intense than that of dry collagen. The effect of hydration


Fluencw (mllrnm2)

0 2 4 6 E 10 12 14 Flwnce (ml/rnm2)

".l

Figure 3. Decay of the Huorescence intensity measured at 390 nm as a function of Huence for elastin (top panel) and cholesterol (bot- tom panel). Fading of the fluorescence was more intense when the fluence rate level was higher from the beginning of the irradiation period (inset: 0-1 mJ/mm'). Note that in the raw data, the first point of each sequence corresponded to a different fluence that depended on the fluence rate level. To obtain a common level of normalization. a biexponcntial fit of the raw data wa\ computed for each fluence rate level. The biexponential approximation was extrapolated to a fluence of zero and the extrapolated value was used to normalize the raw fluorescence intensities.

was observed for all fluence rate levels and all wavelengths tested in the study (Table 1).

Effect of emission wavelength

For all fluence rate levels, photobleaching was less intense for the emission at 430 and 470 nm compared with the fading observed at 390 nm (Fig. 5 , Table 1). The effect of wavelength on fluorescence fading was observed for both the dry and the hydrated compounds. Cholesterol was the compound for which the extent of photobleaching varied the most with emission wavelength. For elastin, photobleaching was least dependent on the emission wavelength.

Fluwncw (mJlmm') 05

hydrated

hydraled 030 100 200 XK) 4W 5M) 600

Irradiation time (swconds)

Figure 4. Fluorescence intensity changes at 390 nm ar a function of irradiation time for dry and for hydrated compounds when the fluence rate level was 21.75 pW/mm' The top horizontal scale In- dicates the accumulated fluence during the irradiation sequence

mm2, decay parameters T ~ , T? and A remained invariant during the measurement period ( T ~ = 1.3 ? 0.1 ns, T~ = 5.4 -C 0.1 ns, A = 0.59 +: 0.01 at 390 nm). Increasing the fluence rate level did not change the time-dependent decay parameters or their invariance with fluence.

The time-dependent decay parameters of collagen change minimally with fluence for the lower fluence rate levels of the study (Fig. 6c,d). In contrast, collagen fluorescence decay changed with irradiation when the fluence rate level was 21.75 p,W/mm? parameters T,, T~ and A measured at 390 nm decreased as a function of the irradiation time. The impulse response function decayed faster at the end of the irradiation period than it did at the beginning, with the average lifetime decreasing from 5.5 -t 0.3 to 4.8 ? 0.3 ns. The fluence rate level had a significant effect on the time-dependent decay constants, which were largest at 0.72 p,W/mm2 and decreased when the fluence rate level was increased. The effect was observed from the start of the irradiation period: decreased from 5.0 2 0.1 to 4.1 ? 0.1 ns and T? decreased from 12.6 2 0.6 to 9.8 ? 0.3 ns when the fluence rate level was raised from 0.72 to 21.75 pW/mm'. The average lifetime decreased from 5.9 ? 0.3 to 5.5 ? 0.3 ns.

Fl~enm (rm/mm2) 2.2 4.37 6 54 8.71 1088 13.05

I

Effect of fluence and fluence rate level on time-dependent decay

The time-dependent decay parameters of elastin did not vary with irradiation time at any of the fluence rate levels of the study (Fig. 6a,b). When the fluence rate level was 0.72 kW/

" -0 100 200 300 400 500 6W Inadialion lime (seconds)

Figure 5. Decay of the fluorescence intensity measured at 390, 430 and 470 nm as a function of irradiation time when the fluence rate level was 21.75 pW/mm*. Photobleaching of the fluorescence was less extensive for the higher wavelengths. Data shown were col- lected on dry compounds.


ELASTIN ELAS” (HYDRATED)

Figure 6. Time-resolved fluorescence parameters T,, T~ (left panels) and A (right panels) as a function of irradiation time for elastin (a, b), collagen ( c , d) and cholesterol (e, 0 in dry form. For the three compounds, the time-resolved parameters (mean f SE) were inde- pendent of the irradiation time when the fluence rate level was 0.72 bW/mm2 (0 ) and when it was 7.25 p,W/mm2 (*). For collagen and cholesterol, parameters T? and A varied with irradiation time when the fluence rate level was 21.75 pW/mmZ (0) leading to a progres- sive decrease in the fluorescence lifetime.

The time-dependent decay parameters of cholesterol had similar values for all three fluence rate levels at the beginning of the irradiation sequence (Fig. 6e,f). Irradiation had little effect on cholesterol fluorescence decay for the lower values of the fluence rate level. When the fluence rate level was 21.75 ~~.w/mm~, the time-resolved fluorescence of cholesterol changed with prolonged irradiation primarily through an increase of parameters and A with fluence. These variations were associated with a decrease in the fluorescence lifetime from 1.6 -t 0.1 to 0.5 I 0.1 ns.

The time-dependent decay parameters measured on hydrated elastin and hydrated cholesterol were nearly identical to the values obtained on the dry compounds (Fig. 7). Decay constants T , and T~ were lower for hydrated collagen than for dry collagen (Fig. 7c,d), in agreement with previous observations (8,19). The variations of the time-dependent decay parameters observed on hydrated compounds were less extensive than those described for dry compounds. The only significant change was that of the decay constants of collagen, which decreased when the fluence rate level was raised from 0.72 to 21.75 p W / m 2 as they had for dry collagen.

For all compounds, the time-dependent decay parameters varied slightly when the emission wavelength was increased to 430 and 470 nm, in agreement with previous observations (8,19). At these wavelengths, irradiation time and fluence rate level minimally changed the fluorescence decay parameters. The only significant effect was observed for collagen: parameters T , , T~ and A were largest when the fluence rate level was 0.72 pW/mm2, and they decreased when the fluence rate level was raised to 21.75 pW/mm’. The trend re- sembled that observed at 390 nm and was associated with a decrease in the fluorescence lifetime.

DISCUSSION This study examined how the fluorescence emission of the main fluorescent compounds of arterial tissue is altered by

Figure 7. Time-resolved fluorescence parameters 7,. T~ (Left panels) and A (right panels) as a function of irradiation time for elastin (a,b), collagen (c,d) and cholesterol (e,D in hydrated form. Symbols are the same as in Fig. 6. Collagen decay parameters decreased slightly when the fluence rate level was raised to 21.75 FWlmm?.

prolonged UV irradiation with the pulsed laser source also used to excite the fluorescence. The principal findings were as follows. First, fluorescence emission intensity decreased markedly with fluence even when the light dose was in the range used for spectroscopic studies of arterial tissue. Sec- ond, the fading of the emission was compound-dependent, hydration-dependent and wavelength-dependent such that the fluorescence spectra of elastin, collagen and cholesterol were each altered in a unique way with prolonged pulsed UV irradiation. Third, the time-dependent characteristics of the fluorescence emission were not changed by UV irradiation at low fluence rate levels. The fluorescence impulse response function of collagen and cholesterol became shorter with irradiation at the highest fluence rate level used in the study.

Arterial photobleaching and irradiation

The intensity of the fluorescence emission decreased with irradiation for the three fluence rate levels of the study. For elastin and collagen, the compounds primarily responsible for the fluorescence of the arterial wall (3,4), the emission intensity at 390 nm decreased by 10-20% when the total fluence reached 0.4 mJ/mm2. Fading of the 390 nm fluorescence exceeded 50% when the total fluence was 12 mJ/mm2. Fluorescence spectroscopy has been widely investigated for quantification of artery tissue composition using pulsed and continuous-wave laser sources with excitation at 306-476 nm (4,5,21,26). Table 2 summarizes the conditions of irradiation for representative studies of artery tissue and fluorescent compounds. Most investigators limited the fluence to values between 0.1 mJ/mm’ (18) and 12 mJ/mm2 (5). Our results concur with these estimates and suggest that 0.1 mJ/ mm2 would minimally photobleach artery samples but that fluences of 2 1 2 mJlmm2 would substantially fade the response of arterial fluorescent compounds directly exposed to the excitation beam. Chaudhry et al. (9) determined that the fluorescence of artery samples irradiated with 476 nm light (Ar laser) decreased irreversibly when the fluence exceeded


Table 2. excitation resulted in much larger fluence rate levels than pulsed excitation for similar levels of fluence

Characteristics of irradiation in the present experiments and representative studies of arterial wall fluorescence; note that CW

Excitation Fluence rate Fluence Study CW/pulsed A (nm) level (FW/mm') (mJ/mm') Samples

~~ ~~~

This study Pulsed 337 0.72-2 1.75 0.4-12 Fluorescent compounds Baraga er a/. (4) Pulsed 306-3 I0 11.8 1.3 Artery-fluorescent compounds Oraevsky et a/. ( 18) Pulsed 308 NA 0.1 Artery-fluorescent compounds Anderson-Engels ef t i / . ( 7 ) Pulsed 337 255 46 Artery-fluorescent compounds Garrand et 01. ( 5 ) c w 325 16 X 10"8 X lo3 4-1 2 Artery-fluorescent compounds Cutruzzola et t i / . (21 ) cw 3'5 1.5 X 1W5.1 X 10' 0.5-4 Artery Chaudhry ef (I/. (9) cw 476 0.3 X 10'-12.8 X 10' 70 Artery

70 mT/mm?. Their threshold is far above the limit suggested by our study of fluorescent compounds and by the fluence levels selected by other groups (4.5.21 ). Optical absorption and scattering in tissue disperse the excitation photons and result in an average local fluence that is less than that found at the entry point of the laser, thereby limiting the fading of the emission. Thus, fluorescent compounds and artery tissue could have different photobleaching thresholds. Further- more, visible light at 476 nm penetrates biologic tissues far more than UV laser light (306-337 nm) because tissue absorption decreases with increasing wavelengths. In conditions of decreased absorption, the photobleaching thresholds of fluorescent compounds and arterial tissue would be higher. This would explain why the photobleaching threshold at 476 nm (9) was much higher than the fluence limits in studies that used UV excitation (5.18,21).

Photobleaching is caused by a chemical transformation of the fluorophore in the excited state (or long-living triplet state) into a nonfluorescent species ( 1 3,27,28). The chance that a fluorophore photobleaches increases with the amount of time it spends in the excited state, which is related to the excitation energy, the absorption cross section of the fluorophore and the excited state lifetime (13). For all fluorophores, fluorescence fading increases when the excitation intensity or the duration of irradiation is increased. For a given fluence. fluorophores photobleach more or less depending on their absorption cross sections at the wavelength of excitation and on their excited state lifetimes. We found that collagen fluorescence faded more intensely than did elastin fluorescence for all fluence rate levels. A similar finding was reported for collagen and elastin in artery tissue studied by microfluorimetry ( 12) at 350 nm with a pulse energy density (4 pJ/mm') comparable to the highest value (2.2 kJ/mm2) in our experiments. These results are supported by the longer excited state lifetime of collagen (5.9 ns at 390 nm) compared with that of elastin (2.3 ns) and could also reflect larger absorption cross sections for the fluorophores in collagen compared with those for elastin. The short lifetime of cholesterol (1.6 ns) combined with the intense photobleaching of its fluorescence at 390 nni suggests that the absorption cross section of cholesterol fluorophores exceeds those of fluorophores in elastin and collagen for excitation at 337 nm.

Effect of fluence rate level

For elastin and cholesterol, photobleaching was more intense when the fluence rate level increased, whereas it was inde- pendent of the fluence rate level for collagen. While the lat-

ter observation is consistent with a model of photobleaching for which the number of bleached molecules is related to the number of excitation photons (13), the former is not. It suggests a role for the rate of arrival of the excitation photons not considered in the model. Deviations from the theoretical model have been reported for other fluorescent compounds in complex environments (11,29). Data on the photobleaching of skin autofluorescence (29) suggest an influ- ence of the fluence rate level in the same direction as that observed in the present study. An opposite behavior was reported for the photosensitizer protoporphyrin IX in mouse skin: photobleaching decreased when the fluence rate level was increased for the same light dose (1 1). Additional studies are needed to examine whether the presence of multiple fluorophores with overlapping spectra and the propagation of light in the medium (1 1) contribute to these deviations.

Photobleaching and hydration Photobleaching of the fluorescence emission was reduced for elastin and cholesterol and accentuated for collagen when the compounds were studied in hydrated form. Environmen- tal factors such as the polarity and the dielectric constant of the medium surrounding the fluorophore affect electron-vi- brational transitions, which changes the quantum yield and the fluorescence lifetime (30). Photobleaching, which de- pends on the lifetime and the absorption cross section of the fluorophore, changes to the extent that these characteristics are changed by hydration. In previous studies (8,19), we observed that collagen fluorescence had a shorter lifetime when the compound was hydrated as opposed to when it was dry, a result that was confirmed in the present experiments. No hydration-related differences of the time-dependent decay were observed for elastin or cholesterol. It is not possible to precisely identify the mechanisms responsible for the photobleaching and lifetime changes associated with hydration from the present data. However, our results point to a significant effect of hydration on the fluorescence characteristics of arterial matter. It is likely that elastin, collagen and cholesterol photobleach differently in purified form and in the environment of the arterial wall. Important characteristics of the fluorescence fading (such as its dependence on the emission wavelength) identified in our experiments are likely to also be observed when the fluorescent compounds are found in the arterial wall.

Photobleaching and emission wavelength Photobleaching of the fluorescence was most intense at 390 nm and it decreased at 430 and 470 nm. This trend was

Photochemistry and Photobiology, 1999, 69(6) 71 9

observed for the three arterial compounds and for all fluence rate levels. Previous studies of arterial fluorescence (3,4,19,24) have shown that elastin, collagen and cholesterol have emission spectra that extend from about 350 to 550 nm when excited with ultraviolet radiation (306-337 nm). The emission peak (elastin: 410 nm; collagen: 385 nm; cholesterol: 375 nm) is near the wavelength (390 nm) at which we observed the strongest fluorescence fading. Thus, photobleaching causes a distortion of the emission spectra, which become flattened and broader than the spectra of the unbleached compounds. Furthermore, we found that the photobleaching of collagen and cholesterol fluorescence was more dependent on the emission wavelength than was that of elastin. In the unbleached state, the emission spectra of collagen and cholesterol are narrower than is that of elastin. The fluorescence intensity at 470 nm relative to the peak is about 0.15 for collagen and cholesterol and 0.50 for elastin (4,8,20). The results of this study (Table 1 , Fig. 5) indicate that after irradiation with 12 mJ/mm2, the relative intensities at 470 nm would change to 0.25 and 0.60, respectively. Ir- radiation to a higher fluence would further diminish the bandshape differences between elastin, collagen and cholesterol. Because it affects the spectrum of each compound in a unique manner, accidental photobleaching would have a deleterious effect in studies that aim at determining arterial wall composition by relating the emission of a tissue sample to that of constitutive fluorescent compounds ( 3 3 . How- ever, it might be possible to take advantage of the wavelength dependence of the fluorescence fading to investigate tissue composition. In such a scheme, elastin, collagen and other fluorescent compounds would be differentiated by their rates of photobleaching at different wavelengths.

Photobleaching in tissue compounds with multiple fluorophores

The origin of the fluorescence of elastin, collagen and cholesterol has been investigated by different groups (14- 16,31,32). At least two fluorophores with emission peaks in the blue and the yellow ranges have been identified in elastin (14,3 1). The fluorophore responsible for the blue emission (excitation max. = 340 nm; emission max. = 410 nm) is thought to be pyridinoline, one of the compounds responsible for the fluorescence of collagen (14,15). In insoluble bovine Achilles tendon collagen (the type of our study), pyridinoline fluorescence peaked at 395 nm and it overlapped with the emission of other fluorophores at around 370 nm (16). For cholesterol and other steroids, structural features of the molecule have been identified as specific chromophores (32) that could be responsible for the fluorescence emission (20). In insoluble collagen, ultraviolet radiation (300-320 nm) attenuated the emission of the 395 nm fluorophore more intensely than that of the 370 nm fluorophores (16). In a related compound, acid-soluble collagen from skin (lo), four fluorophores were identified that each presented a specific pattern of photodestruction that depended on the wavelength of the ultraviolet excitation. These studies suggest that the fluorescence spectra of elastin, collagen and cholesterol represent the sum of the emissions of different fluorophores that contribute in different proportions to the detected signals at 390, 430 and 470 nm. We speculate that

these fluorophores photobleach at different rates when exposed to 337 nm radiation. For each compound, the main contributor to the 470 nm emission would be less destroyed than the fluorophore most responsible for the 390 nm emission. In this way, different amounts of photobleaching would be observed for different wavelengths of emission.

Time-dependent fluorescence decay and photobleaching

The presence of several fluorophores in collagen and cholesterol would also explain the variations of the time-dependent fluorescence decay during photobleaching. Preferential photodestruction of the fluorophores with longer-lasting emissions would progressively reduce the experimental fluorescence lifetime, as we observed when the fluence rate level was 21.75 kW/mm2. However, it is possible that an experimental artifact also contributed to the change in the time- dependent fluorescence parameters. Given that marked photodestruction occurred during each excitation pulse and that the pulse had a finite duration, the fluorescence emission generated during the second half of each pulse was reduced compared with the emission observed in the absence of photobleaching. This effect would change the shape of the fluorescence transient and depress its trailing edge to give the appearance of a shorter impulse response function. This artifact would explain why the experimental lifetime of collagen emission appeared shorter from the very first points of the measurement sequence when the fluence rate level was 21.75 pW/mm’. The emission continued to shorten due to preferential destruction of the fluorophores with longer-lasting emissions. With the limited temporal resolution of our instrumentation, the artifact was most noticeable for collagen, the substance with the longest fluorescence lifetime.

Two exponential components were identified in the time- resolved fluorescence decay of elastin (7, = 1.3 ns, T~ = 5.4 ns at 390 nm) and collagen ( T ~ = 5.0 ns, = 12.6 ns). In an earlier study (7), three exponential components were identified in the decay of these compounds (elastin: T, = 2.11 ns, T~ = 6.60 ns, T~ = 0.38 ns at 380 nm; collagen: rl = 4.99 ns, T~ = 9.94 ns, T~ = 0.78 ns). It is possible that with the sampling rate of our experimental system, the subnanosecond component could not be resolved properly, such that the fitting procedure collapsed the triple exponential decay into two components. Our method of analysis computes the multiexponential fit from the fluorescence impulse response function expanded on the Laguerre basis, whereas the fit is determined by deconvolution of the laser input and fluorescence output (33) in other studies. A certain amount of smoothing originating in the Laguerre expansion (22) could explain in part the differences between the two studies. As Table 2 shows, the level of irradiation was much higher in the earlier study (7), which could also have contributed to different estimates of the fluorescence decays.

Consequences for fluorescence-based optical biopsy

To understand how the fluorescence emission of the arterial wall is affected by prolonged UV irradiation, the wall struc- ture and the propagation of light at the wavelengths of excitation and emission must be considered (9,27,34). While this analysis is beyond the scope of the present work, certain predictions can be made based on our experimental findings.

720 Laura Marcu eta/.

Atherosclerosis is accompanied by compositional changes and thickening of the arterial intima, the innermost layer in contact with the blood stream, which one would want to characterize using fluorescence spectroscopy. Therefore, we assume that the optical biopsy is conducted with a catheter system threaded in the arterial circulation. The excitation beam is directed toward the intima and it propagates toward the tunica media, the central layer in the artery cross section. In such a scheme, the fluorescence emission from superficial layers in the intima will bleach more intensely than that from layers in the media such that the contribution of the latter will be relatively enhanced by photobleaching. This may be a disadvantage for near-normal arterial wall and early atherosclerotic lesions because photobleaching will exacerbate the contribution of the elastin-rich media at the expense of the thin intima. Conversely, for intermgdiate lipid-rich lesions, bleaching of the superficial fluorescence will facilitate the detection of fluorescent lipids in the core of the thickened intima. For advanced collagenous lesions, fading of the collagen emission will be accompanied by a shortening of the fluorescence lifetime when a high fluence rate level is used. This effect and the change for a less peaked spectrum (due to the different amounts of photobleaching at different wavelengths) will reduce the spectrotemporal differences between advanced collagenous lesions and near-normal elastin-rich arterial wall.

These considerations could have important implications for the development of a nondestructive optical biopsy method based on the analysis of arterial wall fluorescence. The geometry of excitation delivery and fluorescence collection influences the shape and intensity of the fluorescence spectrum due to selective fluorescence reabsorption in the arterial wall (34). Our study shows that depth-related variation of the local fluence rate and its effect on fluorescence photobleaching introduces additional complexity to the interpretation of arterial fluorescence spectra. When the fluence rate level is sufficiently low, the time-dependent features of the fluorescence emission remain constant even when the spectral shape is altered by photobleaching. Thus, incorporating a capability for time-resolved fluorescence spectroscopy should increase the accuracy of an optical biopsy device. The fluence that minimizes photobleaching will have to be determined experimentally on artery samples, with 0.4 mll mmr being a reasonable estimate for excitation at 337 nm.

CONCLUSION

This study demonstrated that photobleaching decreases the emission intensity of the compounds responsible for artery tissue fluorescence even when the total fluence is in the range commonly used in fluorescence studies of the arterial wall. Photobleaching reduces the spectral differences between elastin and collagen, which could complicate the dif- ferentiation by fluorescence spectroscopy between elastin in healthy arterial wall and collagen in advanced atherosclerotic plaque. The time-dependent characteristics of the fluorescence emission are relatively unaffected by photobleaching, which suggests that measurement of the time-resolved spectra would improve the sensitivity of an optical biopsy system for diagnosis of atherosclerosis.

AcknoMledgements-This work was supported in part by the Amer- ican Heart Association, Greater Los Angeles Affiliate (#1082-GI), the Biomedical Simulation Resource, University of Southern Cali- fornia. and the Medallions Group, Cedars Sinai Medical Center. The authors are grateful to Mr. Thanassis Papaioannou for his technical assistance during the experimental measurements.

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