Direct Near-infrared Luminescence Detection of Singlet Oxygen by sdfgsg234


									Photochemistry and Photobiology, 2002, 75(4):              382–391

Direct Near-infrared Luminescence Detection of Singlet Oxygen
Generated by Photodynamic Therapy in Cells In Vitro and Tissues In
Mark Niedre1, Michael S. Patterson2 and Brian C. Wilson*1
Department of Medical Biophysics, Ontario Cancer Institute/University of Toronto, Toronto, Canada and

Hamilton Regional Cancer Center/McMaster University, Hamilton, Canada

Received 26 June 2001; accepted 9 January 2002

ABSTRACT                                                                     therapy utilizes a photosensitizing drug, usually administered
                                                                             systemically or topically, which may be preferentially local-
Singlet oxygen (1O2) is believed to be the major cytotoxic
                                                                             ized in, for example, solid tumors. The photosensitizer is
agent involved in photodynamic therapy (PDT). Mea-
                                                                             then irradiated with a light source tuned to a wavelength to
surement of 1O2 near-infrared (NIR) luminescence at
                                                                             match the absorption spectrum of the drug. The subsequent
1270 nm in biological environments is confounded by the
                                                                             photochemical reaction results in oxygen-mediated destruc-
strongly reduced 1O2 lifetime and probably has never
                                                                             tion or modification of the target tissue.
been achieved. We present evidence that this is now pos-
                                                                                The main cytotoxic agent in PDT is widely believed (3)
sible, using a new NIR-sensitive photomultiplier tube.
Time-resolved 1O2 luminescence measurements were                             to be singlet oxygen [1O2 (1 g)], a highly reactive oxygen
made in various solutions of aluminum tetrasulphonated                       species that oxidizes biological substrates. Critical sites of
phthalocyanine (AlS4Pc) and Photofrin. Measurements                          action for 1O2 in PDT include mitochondria, DNA and lipid
were also performed on suspensions of leukemia cells in-                     membranes (4,5). Sustained exposure of the treated tissue to
                                                                             1O results in breakdown of cellular microstructures and cell
cubated with AlS4Pc, and a true intracellular component                         2

of the 1O2 signal was clearly identified. Time-resolved                       death.
                                                                                1O is produced by the following Type-II photochemical
analysis showed a strongly reduced 1O2 lifetime and an                            2

increased photosensitizer triplet-state lifetime in the in-                  pathway (6):
tracellular component. In vivo measurements were per-                                            S0     h       → S1                     (1a)
formed on normal skin and liver of Wistar rats sensitized
with 50 mg/kg AlS4Pc. In each case, a small but statisti-                                                S1 → T1                         (1b)
cally significant spectral peak was observed at 1270 nm.
The 1O2 lifetime based on photon count rate measure-                                             T1    3O
                                                                                                         2      → S0   1O
                                                                                                                         2               (1c)
ments at 1270 nm was 0.03–0.18 s, consistent with pub-                       where S0, S1 and T1 are the photosensitizer ground state, first
lished upper limits. We believe that these are the first                      excited singlet state and first excited triplet state, respective-
direct observations of PDT-generated intracellular and in                    ly and 3O2 and 1O2 are the ground-state triplet and excited
vivo 1O2. The detector technology provides a new tool for                    singlet states of oxygen, respectively. Once produced, a mol-
PDT research and possibly clinical use.                                      ecule of 1O2 can undergo nonradiative decay, oxidize sur-
                                                                             rounding biomolecules or undergo radiative decay at around
INTRODUCTION                                                                 1270 nm. The time-resolved measurement of this near-infra-
                                                                             red (NIR) emission is a commonly used method for deter-
Photodynamic Therapy (PDT) is a minimally invasive treat-                    mining 1O2 lifetimes and quantum yields in solution (7).
ment modality for cancer and other conditions (1,2). The                        Measurement of the NIR luminescence during PDT treat-
                                                                             ment is potentially of value as a direct dosimetry metric (8),
¶Posted on the web site on January 28, 2002.                                 so that the ability to detect 1O2 luminescence in true biolog-
*To whom correspondence should be addressed at: Department of                ical environments has been attempted previously. Several in-
  Medical Biophysics, Ontario Cancer Institute, 610 University Av-           vestigators have reported positive results from cells in sus-
  enue, Toronto, ON M5G 2M9, Canada. e-mail: wilson@oci.                     pension (9,10) or red cell ghosts (11). However, these have
Abbreviations: AlS4Pc, tetrasulfonated aluminum phthalocyanine;              either required the use of deuterium oxide (D2O) to increase
  BSA, bovine serum albumin; D2O, deuterium oxide; FWHM, full                the lifetime of 1O2 and eliminate absorption of the 1270 nm
  width at half maximum; HSA, human serum albumin; MCS, mul-                 luminescence by H2O or did not adequately distinguish be-
  tichannel scaler; NaN3, sodium azide; NIR, near infrared; 1O2,             tween intracellular and extracellular 1O2. Hence, the results
  singlet oxygen; OD, optical density; OPO, optical parametric os-
  cillator; PDT, photodynamic therapy; PMT, photomultiplier tube;            do not reflect optical and photophysical conditions present
  PS, photosensitizer.                                                       in vitro or in vivo and so should not be interpreted as suc-
    2002 American Society for Photobiology 0031-8655/02   $5.00 0.00         cessful 1O2 measurements in biological environments.

                                                                                                       Photochemistry and Photobiology, 2002, 75(4) 383

   Attempts to measure 1O2 luminescence have also been
made in vivo by several investigators. These have either pro-
duced outright negative results (6) or in one case (12) pro-
duced apparently positive results in a single animal that were
not subsequently reproduced. Hence, the direct measurement
of 1O2 luminescence in any biological environment has prob-
ably never been reliably achieved.
   These failures have been attributed to the strongly de-
creased lifetime of 1O2 in cells and tissues caused by rapid
quenching by biomolecules, combined with a lack of ade-
quately sensitive detectors at NIR wavelengths, because, as
will be discussed subsequently (see Eq. 5), the total 1O2 lu-
minescence emission is proportional to the lifetime. The life-
time of 1O2 in vivo has been estimated by various methods.
We previously placed an upper limit of 500 ns on the life-
time, based on the known sensitivity of a germanium pho-
todetector in an NIR luminescence instrument (6). Moan and
Berg estimated the lifetime as approximately 10–40 ns, on                                     Figure 1. Schematic of the experimental system used for 1O2 lu-
the basis of the diffusion distance of 1O2 in cell membranes                                  minescence detection. Inset: purpose built animal holder used for in
as determined by the photobleaching rate of one photosen-                                     vivo experiments. Excitation light was delivered through the circular
sitizer because of 1O2 photogenerated in a second (13),                                       port (a), and measurements made through a second port at 90 (not
                                                                                              seen). The animal was held in the hemispheric cylinder (b).
whereas Baker and Kanofsky estimated the lifetime in cells
to be approximately 200 ns using the values determined in
detergent-dispersed cells of increasing concentration and ex-                                                                         [1O2 ](t)
trapolating to in vivo cell density (14).                                                                                L 1270 (t)                                (4)
   In this paper, we report on the use of a novel photomul-
                                                                                              where R is the 1O2 radiative lifetime in a given solvent. The total
tiplier tube (PMT) in detecting 1O2 luminescence in cells in                                  number of photons emitted after a single excitation pulse is the in-
vitro and in tissues in vivo. This PMT is uniquely sensitive                                  tegral of Eq. (4) over all t:
in the NIR region and, we believe, for the first time has                                                                              N [S0 ]     D D
allowed detection of 1O2 luminescence in true biological me-                                                          L 1270 (t) dt                                (5)
dia during PDT.
                                                                                                 Note that the luminescence signal decreases with the 1O2 lifetime,
                                                                                              creating the fundamental challenge in detecting the emission in bi-
MATERIALS AND METHODS                                                                         ological media. As will be shown experimentally, there are other
                                                                                              potential sources of light emission in the 1270 nm region besides
Theory. As described by Patterson et al. (6), three coupled differ-                           1O luminescence, including photosensitizer fluorescence and phos-
ential equations can be written describing the kinetics of the afore-                         phorescence, autofluorescence from the biological medium and fluo-
mentioned PDT photochemical reactions:                                                        rescence from optical components in the system. Here, time-resolved
                      d[S1 ]                           1                                      or time-gated detection was used to reduce this fluorescence back-
                                       [S0 ]               [S1 ]                       (2a)   ground signal, exploiting the fact that this is generally prompt com-
                       dt                              S                                      pared with the 1O2 emission. Any residual fluorescence, although
                      d[T1 ]                           1                                      minimal in most cases, was subtracted as described subsequently.
                                       [S1 ]               [T1 ]                       (2b)      Apparatus. The optical excitation and detection system is shown
                       dt          S                   T                                      schematically in Fig. 1. A tunable pulsed laser system (OPO Rain-
                                                                                              bow 355, OPOTEK Inc., Carlsbad, CA) comprising a nonlinear op-
                     d[1O2 ]           D                   1
                                           [T1 ]                [1O2 ]                 (2c)   tical parametric oscillator (OPO) pumped by the second (532 nm)
                       dt          T T                      D                                 and third (355 nm) harmonics of a Q-switched Nd:YAG laser was
where      is the local fluence rate (photons per second per square                            used as the excitation source, tuned to the appropriate wavelength
centimeter),     is the photosensitizer ground state absorption cross                         to excite the photosensitizer. The laser light was passed through a
section (cm2), T is the photosensitizer triplet state quantum yield,                          bandpass filter centered at the excitation wavelength (630 nm, 10
  D is the O2 quantum yield, S is the photosensitizer singlet-state
                                                                                              nm bandpass or 670 nm, 10 nm bandpass, OD4 blocking filters, CVI
lifetime, T is the photosensitizer triplet-state lifetime and D is the                        Laser Corp., Albuquerque, NM) and focused onto the sample using
1O lifetime. The concentrations of each of the photosensitizer states
                                                                                              an f/1 lens (PLCX-25.4-13.1-UV, CVI). The pulse duration was 20
([S0], [S1], [T1]) and of 1O2 ([1O2]) are expressed in molecules per                          ns, the pulse repetition frequency 10 Hz and the pulse energy at the
cubic centimeter.                                                                             sample 1 mJ. This resulted in an average power at the sample of
   For an excitation pulse,      N (t), where N is number of photons                          10 2 mW over a 3 mm diameter spot and an instantaneous power
per square centimeter incident on the sample at time t         0, and                         of 50 kW during the laser pulse. Light from the sample was col-
assuming that the triplet-state molecules are created instantaneously                         lected using an f/1 lens (BICX-25.4-23.9-UV, CVI) set at 90 to the
after excitation (valid for s K T), it can be shown that the 1O2                              excitation beam. A 1000 nm longpass filter (model 58867, OD3
concentration at time t is:                                                                   blocking, Oriel, Stratford, CT) and an 800 nm longpass filter (57361,
                                                                                              OD2 blocking, Oriel) were used to remove unwanted scattered ex-
         [1O2 ](t)   N [S0 ]   D


  Hence, for a sufficiently short excitation pulse width, such that
                                                                                   ]    (3)   citation light and fluorescence from the sample.
                                                                                                 Four bandpass filters at 1201 nm (9 nm bandpass, OD6 blocking,
                                                                                              Andover Corp., Lawrence, MA), 1272 nm (18 nm bandpass, OD6
                                                                                              blocking, Andover), 1301 nm (10 nm bandpass, OD4 blocking, CVI)
     K T and pulse K D, this equation approximates the 1O2 lu-
                                                                                              and 1329 nm (10 nm bandpass, OD4 blocking, CVI) were mounted
minescence time decay curve. The 1O2 luminescence emission (pho-
                                                                                              side by side on a sliding stage in front of the detector. For simplicity,
tons per cubic centimeter per second) at time t is then:
                                                                                              these will be referred to as the 1200, 1270, 1300 and 1330 nm filters,
384 Mark Niedre et al.

respectively. The 1270 nm filter corresponds to the peak of the 1O2       that the background corrected signal was caused only by 1O2 lumi-
luminescence spectrum, whereas the 1300 nm filter is off peak but         nescence, a solution of PS and BSA ([BSA]/[AlS4Pc]           200) was
still within the 1O2 emission band ( 15% of peak). The 1200 nm           prepared. NaN3 was then added to the solution so that the final
and 1330 nm filters, which lie outside the 1O2 band, were used to         concentration of NaN3 in the cuvette was 0.5 M, and the full time
determine the background fluorescence. The latter also served to          signal was measured. Because the NaN3 completely quenches the
check for a potential water-absorption artifact (see subsequent dis-     1O luminescence but has little effect on the photosensitizer triplet
cussion). For some later experiments, two additional filters at 1250      state, the intent was to rule out the (unlikely) possibility that the
nm (10 nm bandpass, OD4 blocking, CVI) and 1285 nm (10 nm                triplet-state phosphorescence has a spectral peak in the 1270 nm
bandpass, OD4 blocking, CVI) were added to provide further spec-         region and so could be mistaken for the 1O2 signal, for which the
tral resolution. The overall numerical aperture of the detection sys-    kinetics would be similar. The rationale was that the NaN3 would
tem was approximately 1.                                                 completely quench the 1O2 luminescence but not affect any photo-
   The detector was a liquid nitrogen–cooled PMT (model R5509–           sensitizer (PS) triplet-state phosphorescence.
42, Hamamatsu Corp., Bridgewater, NJ). This has a uniquely broad            Experiments in cell suspensions. AML5 or P388 leukemia cells
spectral response from 300 to 1400 nm and therefore enabled ex-          were grown to confluence in suspension (in -mem or RPMI media,
tremely sensitive detection in the 1200–1330 nm range. Its rapid         respectively, with 10% fetal bovine serum (GIBCO, Life Technol-
temporal response (3 ns) allowed photon counting of the 1O2 lumi-        ogies Inc., Rockville, MD) and incubated with 3 or 6 M AlS4Pc
nescence. The operating voltage was set at 1500 V, at which the          for 24 h. Immediately before measurements, the cells were spun at
dark current was 1 nA, resulting in negligible dark counts. The          5000 rpm for 5 min, resuspended in fresh medium, agitated for 10
PMT output was amplified and converted to a voltage pulse using           min, spun a second time and resuspended in fresh media, in order
a high-speed current preamplifier (model SR445, Stanford Research         to minimize the residual photosensitizer in the media. It should be
Systems, Sunnyvale, CA). A multichannel scaler (MCS; model               noted that a large amount of the original PS used for incubation was
SR430, Stanford) connected to a personal computer was used for           washed out during this step. The suspension was then rapidly trans-
time-resolved single photon counting, with a typical temporal reso-      ferred to the measurement cuvette at a concentration of 50          106
lution of 80 ns. For some experiments, a dual-channel photon coun-       cells per milliliter. This will be referred to as the ‘washed’ cell
ter (SR-400, Stanford) was used instead of the MCS to give the           suspension.
time-integrated luminescence signal.                                        Two checks were made to discriminate between AlS4Pc in the
   The PMT has a quantum efficiency of 0.9% at 1270 nm and, when          cells and photosensitizer that may have leaked from the washed cells
operated in photon-counting mode, approximately 2            104 dark    into the media before or during the luminescence measurements
counts per second. For a continuous source, the minimum detectable       (typically 30 min). Firstly, the washed suspension was spun down
signal (SNR 1) with a 1 s integration time is therefore 2.5 10 15        immediately after the luminescence measurement, and cells that had
W. This is approximately an order of magnitude lower than the            not been incubated with AlS4Pc were added to the supernatant at
signal detectable with a liquid nitrogen–cooled germanium detector       the same concentration and a second scan performed. This procedure
(6) under comparable conditions. It is primarily this improvement in     re-created the same light scattering conditions in the sample. The
sensitivity that has enabled the successful detection of 1O2 lumines-    difference between these two sets of scans then represented the frac-
cence from photodynamic sensitizers in cells and tissues, as reported    tion of the 1O2 signal originating from the cells in the washed sus-
subsequently.                                                            pension. Secondly, NaN3 was added to the washed cell suspension
   Data collection. At each of the NIR detection wavelengths the         after the initial scan so that the final concentration of NaN3 in the
signal was summed over many laser pulses (at 10 Hz), typically           cuvette was 0.5 M. The suspension was then rescanned immediately
600–2400, giving data collection times of 60–240 s per wavelength.       at 1270 nm only (completed within 2 min). Assuming that this time
Measurements were made in either ‘‘time-integrated’’ or ‘‘time-re-       delay was not sufficient to allow significant NaN3 diffusion into the
solved’’ mode. In the former, the delayed luminescence in the time       cells but was long enough to completely quench the 1O2 in the me-
interval 5 s in solution and 10 s in vivo after the laser pulse          dia, this determined the fraction of 1O2 signal originating from the
was summed, thereby removing the fast fluorescence component.             cells only. Subsequent scans were performed 7 and 15 min later to
This yielded the time-integrated spectrum of the light emitted from      look for loss of signal as the NaN3 diffused into the cells.
the sample. Because the absolute luminescence intensities varied            For each set of cell suspension experiments, time-resolved scans
from sample to sample (particularly in vivo), each spectrum was          were also performed on control, unincubated cells to determine the
corrected for the system response at each wavelength and normal-         background signal at each wavelength. In all cases, experiments
ized to the signal at 1200 nm. The mean spectrum for each control        were repeated in triplicate.
solution, cell suspension or tissue without photosensitizer was then        Experiments in vivo. The feasibility of 1O2 luminescence detec-
subtracted from that of the corresponding photosensitized sample.        tion from photosensitizer in tissue in vivo was investigated in a small
   For time-resolved measurements, the complete time curve was           number of Wistar rats (Charles River Laboratories Inc., Wilmington,
measured at 1270 nm, and the background (i.e. the time curve from        MA). Four rats were injected i.p. with 50 mg/kg AlS4Pc, and four
a control sample at 1270 nm) was subtracted from it. Equation (6),       were uninjected controls. One of each was used in each experimental
a simplified version of Eq. (3), was then chi squared–fitted to the        run to minimize the effect of any changes in instrument response.
data using commercial software (mmnlfit.m, Matlab 5, The Math-            At 24 h after injection, the animals were anesthetized by i.p. injec-
works Inc., Natick, MA), with T, D and A ( N [S0] D) as free             tion of 4.8 mg Xylazine (Bayer Inc., Toronto, ON, Canada) and 30
parameters.                                                              mg Ketalean (MTC Pharmaceuticals, Cambridge, ON, Canada). In

             [1O2 ]   A
                          (   T

                                      D)   [
                                                           D   ]
  Experiments in solution. Solutions of photosensitizer were mea-
                                                                         six animals (three drugged, three control) the skin was irradiated,
                                                                         for which the abdomen was shaved and depilated (Nair, Carter-Hor-
                                                                         ner Inc., Mississauga, ON, Canada). For the final two of these, mea-
                                                                         surements were taken also with the two additional filter wavelengths
sured in quartz cuvettes (1    1    4 cm), firstly for 2.5 M tetra-       (1250 and 1285 nm). In the remaining two animals the liver was
sulfonated aluminum phthalocyanine (AlS4Pc; Porphyrin Products,          irradiated, for which an upper abdominal incision was made to ex-
UT) in methanol and water, and Photofrin (QLT Phototherapeutics,         pose the liver surface, as described by Patterson et al. (16).
Vancouver, BC, Canada) in methanol to confirm that the lifetimes             For the measurements the animals were placed in a purpose-built
were in agreement with literature values. Subsequently, all solution     holder (see Fig. 1, insert). The laser beam (3 mm diameter) was
studies were done with 6 M AlS4Pc. The biophysical complexity            directed at 45 to the tissue surface and the signal measured at 90
was then increased by adding bovine serum albumin (BSA; Sigma            to the incident beam. Measurements were repeated at two to four
Chemical Co., St. Louis, MO) in water up to a molar ratio of [BSA]/      different points on each tissue. Immediately after completing the
[AlS4Pc]     400, to provide a protein-rich environment to bind the      measurements, the animals were euthanized by intracardiac injection
photosensitizer and possibly quench 1O2.                                 of T-61 (Hoechst Roussel Vet, Whitby, SK, Canada). Liver and skin
   In order to confirm that the 1270 nm signal was due to 1O2, a          samples outside the treated regions were removed for spectrofluor-
known 1O2 quencher (15), sodium azide (NaN3; Sigma), was added           imetric determination of the photosensitizer concentration using a
to AlS4Pc in water up to a concentration of 2 M. As a final check         previously established protocol (17).
                                                                                Photochemistry and Photobiology, 2002, 75(4) 385

                                                                         curves, together with the best fits of Eq. (6) to the data. The
                                                                         derived photosensitizer triplet and 1O2 lifetimes are listed in
                                                                         Table 1, together with the values reported by Patterson et
                                                                         al. (6), obtained by frequency-domain 1O2 luminescence
                                                                         spectroscopy, and Krasnovsky (18), obtained by time-re-
                                                                         solved 1O2 luminescence measurements. Our values gener-
                                                                         ally agree well with the published data, indicating that the
                                                                         time-resolved system performed correctly and supporting the
                                                                         interpretation of the 1270 nm signal as caused by 1O2 lu-
                                                                            The 1O2 detection limit was estimated using Eq. (5) for
                                                                         the case of AlS4Pc in water. The irradiation volume was
                                                                         taken as a cylinder of length 1 cm and diameter 3 mm. For
                                                                         2.5 M AlS4Pc solutions the optical density was 0.11, so
                                                                         that the volume was irradiated approximately uniformly
                                                                         throughout. The total number of photosensitizer molecules
                                                                         in this volume was      1014, which is much less than N a
                                                                         1.1 1016, where N is the number of photons per pulse and
                                                                           a (  0.25 cm 1) is the photosensitizer absorption coefficient
                                                                         at the irradiation wavelength, measured on a spectrometer
                                                                         (model UV160V, Shimadzu, Kyoto, Japan). Hence, all pho-
                                                                         tosensitizer molecules are excited during one 20 ns pulse,
                                                                         whereas the probability of more than one excitation cycle
                                                                         per molecule is low because the triplet-state yield is sub-
                                                                         stantial and T k 20 ns. The 1O2 quantum yield, D, was
                                                                         taken as 0.38 (19), and its radiative lifetime in water, R, was
                                                                         taken as 5.55 s (20). This yields 2           107 molecules of
                                                                         1O that underwent radiative decay. The total number of pho-
                                                                         tons counted at 1270 nm (background subtracted) over 1200
                                                                         laser pulses was 16.0      0.1 103, so that each count corre-
                                                                         sponded, on average, to 1.3      0.1     103 1O2 molecules un-
                                                                         dergoing luminescence decay. Hence, for a minimum signal
                                                                         of 25 counts (SNR 5:1), the limiting detection sensitivity was
                                                                            3 104 molecules. This is of the same order of magnitude
                                                                         as an estimate based on the geometrical efficiency of the
                                                                         detector system ( 1%), the PMT quantum efficiency at 1270
                                                                         nm ( 1%), the transmission of 1270 nm photons through
                                                                         about 4 mm of water ( 30%) and the optical components
                                                                         ( 20%) and the projection of the 1 cm long irradiation vol-
                                                                         ume onto the 3 mm wide photocathode ( 30% overlap). For
                                                                         these values, 2 107 luminescence photons generated would
                                                                         yield 40 counts.
                                                                            Figure 3 summarizes the time-resolved measurements
                                                                         when NaN3 was added in increasing concentration to AlS4Pc
Figure 2. 1O2 luminescence from simple solutions of AlS4Pc and           in water. For [NaN3]       50 mM, the signal was detectable
Photofrin in solution. A: Time-integrated spectra ( 5 s) for 2.5         but too noisy to analyze. It was observed that D decreased
  M AlS4Pc in methanol, 2.5 M AlS4Pc in H2O and 2.5 M Pho-
tofrin in methanol; errors are smaller than the symbol sizes. B: Time-
                                                                         rapidly at first and then more slowly, whereas T appeared
integrated spectra ( 5 s) for 2.5 M AlS4Pc in H2O with additional        to decrease slightly. The strong reduction in D observed is
measurements made at 1250 and 1285 nm; errors are smaller than           consistent with quenching of 1O2, and D follows a Stern–
the symbol sizes. C: Time-resolved measurements at 1270 nm for           Volmer quenching relationship. Figure 3b shows a linear re-
2.5 M AlS4Pc in methanol, 2.5 M AlS4Pc in H2O and 2.5 M                  lationship between the inverse lifetime and the concentration
Photofrin in methanol, showing the best fits of Eq. (6).
                                                                         of NaN3 as expected. The dashed line shows a quenching
                                                                         constant of kq     (4.4   2.0)     108 s 1 M 1 for 1O2. This is
RESULTS                                                                  consistent with the quenching constant of 5.76          108 s 1
                                                                         M 1 for eosin Y and 5.83        108 s 1 M 1 for Rose Bengal
Experiments in solution
                                                                         measured by Hall and Chignell (15). In addition, the PS
Figure 2a shows the time-integrated spectra for AlS4Pc and               triplet state appeared to be quenched slowly with a quench-
Photofrin in solution. Figure 2b shows the spectra measured              ing constant, kq, of 1.3     107 s 1 M 1. Slow quenching of
for AlS4Pc in water with the two additional filters added. In             the PS triplet state by NaN3 as a secondary effect has been
each case, a peak was found at 1270 nm, consistent with 1O2              observed previously (15), although a literature search failed
luminescence. Figure 2c shows the 1270 nm time-resolved                  to reveal rate constants for comparison.
386 Mark Niedre et al.

Table 1.   Photosensitizer triplet state ( T) and 1O2 ( D) lifetimes (microseconds) for photosensitizers in solution, compared with published

                                                    Fits to Eq. 6                                              Published values
    and solvent                           T                                D                            T                                D

AlS4Pc in water                     2.4       0.3                    3.0       0.3                2.4       0.5*                  3.0†
AlS4Pc in methanol                  0.7       0.2                   10.2       0.5                0.3       0.2*                  8.5        0.5*
Photofrin in methanol               0.4       0.2                   10.6       0.5                0.3       0.2*                  9.0        0.5*

*Patterson et al. (6).
†Krasnovsky et al. (18).

  Figure 4 shows the results of adding BSA. For [BSA]/                          crease in T in aqueous phosphate buffered saline solutions
[AlS4Pc]    400, T increased from 2.4        0.3 to 20   2 s.                   of sulphonated aluminum phthalocyanine bound to human
Similar increases in triplet-state lifetime have been reported                  serum albumin (HSA) and Aveline et al. (22) reported that
previously and attributed to binding to albumin and conse-                       T increased when HSA was added to aqueous solutions of
quent shielding of the triplet state from diffusion of oxygen                   benzoporphyrin derivative (BPD-MA). Over the same BSA
molecules: for example, Foley et al. (21) reported an in-                       concentration range, our measured value of D decreased
                                                                                from 3.0     0.3 to 0.8    0.2 s, presumably because of 1O2
                                                                                quenching. A literature search failed to reveal another report
                                                                                of this effect, but the decrease in D seems plausible on the
                                                                                basis of the known effects of quenching by proteins in bio-

Figure 3. 1O2 luminescence for solutions of 6 M AlS4Pc in water
with increasing NaN3 concentration. A: Time-resolved measure-
ments at 1270 nm, showing the best fits ( 5 s) to 0, 2.5, 12.5 and               Figure 4. 1O2 luminescence for solutions of 6 M AlS4Pc in water
100 mM NaN3 concentrations. B: 1/ T and 1/ D vs NaN3 concentra-                 with increasing BSA:AlS4Pc molar ratio. A: Time-resolved mea-
tion. The error bars correspond to variations in the fitted values               surements at 1270 nm showing the best fits ( 5 s) to 0:1, 1:3, 2:
between repeated experiments (N      3). The dotted lines show the              3 and 100:1 molar ratios. B: T and D vs molar ratio. The error bars
fits to Stern–Volmer relationships (for 1O2, kq  4.4    108 M 1 s 1              correspond to variations in the fitted values between repeated ex-
and for the PS triplet state kq 1.3    107 M 1 s 1).                            periments (N    3).
                                                                        Photochemistry and Photobiology, 2002, 75(4) 387

logical media. For [BSA]:[AlS4Pc] ratios between 1:3 and 1:
1, a three-parameter fit to Eq. (6) yielded poor results (av-
erage 2      18.9 per degree of freedom). A six-parameter fit
(A1, D1, T1 and A2, D2, T2), assuming two components with
different lifetimes, gave ( 2     2.8 per degree of freedom)
 D1    3.1    0.5 s, T1     2.4 0.5 s and D2 3.1 0.5
  s, T2    20     4 s. The value of A1 decreased monotoni-
cally with increasing BSA concentration, whereas A2 in-
creased. For [BSA]:[AlS4Pc]       1:3 and 1:1, A1/A2 was 0.9
   0.1 and 0.10 0.05, respectively. The lifetimes shown in
Fig. 4b are the weighted averages of the two components:
(A1· 1    A2· 2)/(A1    A2).
   Finally, when NaN3 was added to solutions of BSA and
PS, no signal was observed at 1270 nm after background
subtraction (data not shown). We interpret this to mean that
the long-lifetime signal was only caused by 1O2 lumines-
cence and not by possible PS triplet-state phosphorescence.

Cell suspension experiments

A statistically significant spectral peak was observed at 1270
nm in cell suspensions, as shown in Fig. 5a. However, de-
spite repeat washing of the cells before these measurements,
a small quantity of photosensitizer was always found in the
media by the time the luminescence measurements were
completed. Comparing the luminescence intensity from the
supernatant only (measured after the initial scan) with that
from media containing known photosensitizer concentra-
tions, this was approximately 0.25 M for cells incubated
                                                                Figure 5. 1O2 luminescence from suspensions of AML5 murine leu-
with 6 M AlS4Pc. Hence, the signal from cell suspensions        kemia cells. A: Time-integrated spectra ( 5 s) for (washed) sus-
represents 1O2 luminescence from both the cells and the me-     pensions incubated for 24 h with 6 or 3 M AlS4Pc. The error bars
dia.                                                            correspond to variations observed in repeated experiments. B: Time-
   Figure 5b shows the 1270 nm time-resolved curve for an       resolved measurements at 1270 nm for (washed) suspension of in-
AML5 cell suspension incubated with 6 M AlS4Pc, as well         cubated cells (6 M AlS4Pc) and for the supernatant from this sus-
                                                                pension with unincubated cells resuspended in it. The best-fit curves
as that for the supernatant with the same concentration of      shown are for T1      3.2   0.5 s, D1     3.2    0.5 s, T2     19
unincubated cells resuspended in it. For the former, a two-     3 s and D2      0.6     0.4 s (incubated cells), and T    3.2    0.2
component (six-parameter) fit gave T1        3.2    0.5 s, D1      s and D    3.3    0.2 s (supernatant     unincubated cells).
   3.2     0.5 s and T2      19     3 s, D2     0.6    0.4 s,
with a significantly lower 2 (2.3 per degree of freedom or
1.4 with outliers removed) than a one-component fit ( 2
316 or 16.6 per degree of freedom). For the resuspension of     and 85% from trace photosensitizer in the media. It should
unincubated cells a single component fitted well ( 2       3.5   be noted that spinning the cells to obtain the supernatant
or 1.5 per degree of freedom), giving T      3.3    0.2 s, D    probably caused additional photosensitizer leakage from the
   3.3    0.2 s, comparable to the values for AlS4Pc added      cells, so that the 15% is a lower limit for the true intracel-
to media (2.7      0.5 and 2.7    0.5 s, respectively) and to   lular fraction of the signal.
the second component in the cell suspension.                       In the second determination of the intracellular fraction,
   In order to determine the fraction of signal originating     the signal dropped to 19        3% immediately after adding
from intracellular photosensitizer, further experiments were    NaN3 to the washed cell suspension. After 7 and 15 min, the
performed on P388 cell suspensions incubated with 3 or 6        value decreased further to 10      3% and 2       3%, respec-
  M AlS4Pc. In the washed suspension, the 1270 nm signal,       tively. In comparison, the 1270 nm luminescence signal fell
after background subtraction, was 2.3        0.3 times higher   to approximately 1% of the original value in a solution of
with a photosensitizer concentration of 6 M than with 3         2.5 M AlS4Pc in cell-free media immediately upon addition
  M. When the supernatant was removed after spinning down       of the same concentration of NaN3, showing that the time
the cells and unincubated cells added to it, the 1270 nm        required for complete mixing of the NaN3 was negligible.
signal after background correction was reduced by 15 2%         We interpret these results to mean that 19 3% of the signal
and 16       2% for 5 and 2.5 g/mL AlS4Pc, respectively.        from the washed cell suspension was intracellular and that
(The time from the last wash to completing the first set of      some time was required for the NaN3 to diffuse into the cells
scans was 30 min and between the start of the first and          and quench this remaining signal. The values for the intra-
second measurements was 40 min.) We interpret this to           cellular fraction of the signal were therefore in good agree-
mean that only 15% of the 1O2 luminescence from the             ment between the two independent methods. Figure 6 sum-
incubated washed cell suspension originated from the cells      marizes these findings.
388 Mark Niedre et al.

Figure 6. Summary of the time-integrated 1O2 luminescence for
P388 cells in suspension under different conditions. The values are    Figure 8. In vivo time-resolved measurements at 1270 nm in the
normalized to that for the corresponding incubated cell suspensions    liver of a rat injected with 50 mg/kg AlS4Pc. Typical errors ( 1
in each case.                                                          standard deviation in photon counts) are shown. Also included is
                                                                       the best fit to a single exponential decay, corresponding to T 30
                                                                          5 s.

                                                                       Experiments in vivo
                                                                       As summarized in Fig. 7a,b, a 1270 nm peak was seen in
                                                                       all photosensitized tissues. Figure 7b shows the presubtract-
                                                                       ed signals from the control and sensitized animals as well
                                                                       as the signal measured at the two additional wavelengths.
                                                                       The normalized intensity was, on average, 21       3% higher
                                                                       in skin and 64      12% higher in liver for the sensitized an-
                                                                       imals compared with uninjected controls. At 1300 and 1330
                                                                       nm there was no significant difference between sensitized
                                                                       and control tissues. Hence, the spectral peak at 1270 nm
                                                                       appears to be caused by 1O2 luminescence from photosen-
                                                                       sitizer in the tissue. Because the measurements were made
                                                                       24 h after injection and the plasma half-life for AlS4Pc in
                                                                       rodents is 1.5 h (23), the photosensitizer should be fully
                                                                       tissue bound, rather than be in the circulation. The concen-
                                                                       tration of photosensitizer in tissue measured in postmortem
                                                                       samples was 9.5       0.3 in skin and 22.6   0.2 g/g in liver.
                                                                          The time-resolved 1270 nm luminescence was corrected
                                                                       for background by calculating:

                                                                              L 1270 (t)   1270 S
                                                                                                    S (t)
                                                                                                            [                    ]
                                                                                                                # 1270 SC (t) · dt
                                                                                                                # 1200 SC (t) · dt
                                                                                                                                   ·   1200 S
                                                                                                                                                S (t)   (7)

                                                                       where SS(t) and SC(t) are the delayed, time-integrated sig-
                                                                       nals at wavelength in sensitized and control animals, re-
                                                                       spectively. The rationale here is that the background signal
                                                                       at 1270 nm equals that at 1200 nm, scaled by the relative
                                                                       system detection efficiency and background luminescence
                                                                       contributions as represented by the ratio in the control ani-
                                                                       mals. Figure 8 shows an example of the corrected signal
                                                                       from liver. Points at 10 s time delay are highlighted, be-
Figure 7. In vivo time-integrated spectra ( 10 s) in rats injected     cause these early points appear to have a significant contri-
with 50 mg/kg AlS4Pc. A: Delayed luminescence measured in sen-
sitized animals normalized to the spectrum in uninjected controls.     bution from fluorescence, as evidenced by analysis of their
The peak values plotted, in individual animals, are 0.18 0.03, 0.23    spectral composition and kinetics. This was likely because
    0.06 and 0.7     0.1. The error bars correspond to 1 standard      of the tissue surface being at 45 to the laser beam, such that
deviation in the point-to-point signal variations in each animal. B:   some specularly reflected excitation light entered the detec-
Precorrected delayed luminescence spectra (with extra filters at 1250
and 1285 nm), measured on the skin of a sensitized rat and of an
                                                                       tion optics, generating secondary fluorescence. This was
unsensitized rat. The error bars correspond to point-to-point signal   confirmed by checking the signal from a cuvette filled with
variations in each animal.                                             water at an angle of 45 to the incident light. Because the
                                                                               Photochemistry and Photobiology, 2002, 75(4) 389

Table 2. Summary of AlS4Pc triplet state ( T) and 1O2 ( D) life-        observed (Fig. 7), so that we conclude that this hydration
times (microseconds) obtained in solution, cell suspensions and in      artifact does not occur and that the 1270 nm peak seen in
                                                                        vivo is the true 1O2 luminescence.
                                                                           In simple solutions, it was observed that the PS triplet
                                             T                D
                                                                        state lifetime was longer in water than in methanol. This
H2O                                    2.4       0.3    3.0       0.3   effect has been observed previously (12) and has been attri-
MeOH                                   0.7       0.3   10.2       0.5   buted to higher solubility of oxygen in methanol than in
H2O   BSA ( 2:1 molar ratio)
  Component 1                          2.4       0.5    3.1       0.5
                                                                           As BSA was added to solutions of AlS4Pc in water, T
  Component 2                          20        4      3.1       0.5   increased from 2.4 0.3 s in the self-aggregated, unbound
Cell suspension
                                                                        state (with no BSA) to 20       2 s for the likely completely
  Component 1                          3.2       0.5    3.2       0.5
                                                                        bound state ([BSA]/[AlS4Pc] 2). For molar ratios between
  Component 2                          19        3      0.6       0.4   1:3 and 1:1, curve fitting indicated two distinct sets of ki-
Resuspension of supernatant            3.2       0.2    3.3       0.2   netics, corresponding approximately to unbound and bound
  with unincubated cells                                                values for T. As the concentration of BSA increased, the
In vivo (liver)                         30       5      0.03–0.17       signal from the bound (long T) and unbound (short T) frac-
In vivo (skin)                          26       5      0.04–0.18       tions monotonically increased and decreased, respectively.
                                                                        Hence, the measurement system appears to be sensitive
                                                                        enough to distinguish between two components in a sample
                                                                        with different photophysical kinetics that are at least quali-
initial rising part of the time-resolved curves was thus ob-
                                                                        tatively indicative of the microenvironment. Ideally, this
scured, Eq. (6) could not be fitted reliably. Instead, a single
                                                                        would be verified by independently measuring T using a
exponential decay was fitted to the data above 10 s, giving
                                                                        pulsed transient absorption spectroscopy system such as that
a time constant of 30      5 s. This can be attributed to the
                                                                        used by Aveline et al. (22). We are currently planning to
PS triplet state (i.e. T     30    5 s), because it is known
                                                                        add this capability to our experimental system.
that T k D in vivo. Similarly, a PS triplet-state lifetime of
                                                                           In the cell suspension experiments, it was critical to dem-
26     5 s was measured in the skin.
                                                                        onstrate that the observed 1O2 luminescence originated from
   Table 2 summarizes the various AlS4Pc triplet state and
1O lifetimes obtained in these experiments in solution, cells           intracellular photosensitizer in the cells, not in the media.
                                                                        This has been a potential artifact in other published studies
and tissue.
                                                                        in vitro that have reported positive results (9,10). The fact
                                                                        that only some 15–19% of the signal appeared to come from
                                                                        the cells, even using a long photosensitizer incubation time
As in our earlier attempt to measure 1O2 luminescence in                (to allow intracellular binding) and measuring as soon as
vitro and in vivo (6), the luminescence spectrum was sam-               possible after multiple washing, reinforces this point. The
pled here using discrete bandpass filters rather than a mono-            interpretation of this fraction as truly intracellular in origin
chromator, in order to achieve the highest possible signal-to           is supported by the consistency between the resuspension
noise-ratio. Although this limited the spectral information,            experiments and the change in signal upon adding NaN3 to
the 1O2 luminescence signal should be unambiguous, be-                  quench the 1O2 in the media. The kinetics of the 1O2 lumi-
cause it comprises a single peak at around 1270 nm with a               nescence also supports this conclusion. Thus, in resuspen-
full width at half maximum (FWHM) of 30 nm (24) that                    sion of the supernatant with unincubated cells, T and D
is adequately sampled by the 3 filters at 1200, 1270 and 1300            were the same as for solutions of AlS4Pc in media alone,
nm. The 1200 nm and 1300 nm filters also allow subtraction               but both were substantially different in the incubated cell
of the background tissue and photosensitizer fluorescence.               suspensions. Specifically in the latter, there appeared to be
The 1250 nm and 1285 nm filters were added in later ex-                  two distinct sets of kinetics. One set ( T1      3.2     0.5 s,
periments to provide further spectral confirmation of the                 D1     3.2     0.5 s) was comparable to AlS4Pc in media,
1270 nm peak. The agreement in simple solutions between                 whereas the other was significantly different, with increased
our measured photosensitizer triplet state and 1O2 lifetimes            triplet lifetime ( T2    19     3 s) consistent with protein
and the published values, and the observed spectral peak at             binding in the cellular environment and decreased 1O2 life-
1270 nm, support the interpretation of the signal as 1O2 lu-            time ( D2     0.6   0.4 s) consistent with increased quench-
minescence, as does the elimination of the 1270 nm signal               ing of 1O2. Other authors have reported similar results of
by NaN3.                                                                increased T in cells (22,26,27). For example, Aveline et al.
   The extra filter was added at 1330 nm to cross-check for              (22) showed that, for benzoporphyrin derivative in pellets of
a possible differential light absorption artifact in vivo that          P388D1 and NBT-II cells, T increased to approximately 23
could arise as follows. Water has a local absorption maxi-                s (again, T in solution was lower but was not explicitly
mum at 1190 nm and a minimum at around 1270 nm (25).                    stated), whereas Truscott et al. (26) obtained a value of 7.7
Hence, if the PDT treatment caused an increase in tissue                  s using transient absorption spectroscopy in fibroblast sus-
hydration, this could give rise to an artifactual increase in           pensions incubated with hematoporphyrin derivative com-
the 1270 nm signal relative to the 1200 nm signal. However,             pared with 2.4 s in water (6). Again, an experiment to
because the water absorption increases from 1300 to 1330                independently measure T would be valuable to verify these
nm, such altered hydration would also produce a signal at               results.
1330 nm that is less than that at 1300 nm. This was not                    Although we recognize that only a small number of ani-
390 Mark Niedre et al.

mals were used for the in vivo experiments, a statistically        signal in vivo in the presence of photosensitizer: e.g. for the
significant peak at 1270 nm was observed in all cases and           data of Fig. 7B the signal at 1200 nm was about four times
in both tissues (P      0.005 for liver and 0.05 for skin by       higher in the sensitized animal than in the uninjected control.
Student’s t-test) for sensitized animals compared with con-        However, the 1270 nm peak is only seen in the former. It is
trols. As would be expected from the much higher AlS4Pc            unlikely, but possible, that the triplet-state phosphorescence
uptake in liver (4), the 1O2 signal was stronger than that from    also happens to have a peak emission at 1270 nm. (This can
skin ( approximately three-fold) but was also more variable        be investigated in the future using other photosensitizers,
from point to point, probably because of breathing move-           because the triplet-state phosphorescence spectrum should
ment during scanning.                                              be photosensitizer-specific.) Furthermore, we showed that
   Because of the distortion in the early part of the signal,      the PS triplet state phosphorescence was negligible for
determining D in vivo by directly fitting Eq. (3) to the time-      AlS4Pc in BSA solutions and therefore would probably be
resolved data was not accurate, as discussed previously.           small also in vivo. At a recent conference where we pre-
However, it could be determined using the total number of          sented this work (30), Hirano et al. also reported successful
photon counts observed, as follows. The reliable part of the       measurement of 1O2 luminescence spectra in a murine tumor
time-resolved data ( 10 s) was fitted to a single exponen-          model injected with 25 mg/kg Photofrin or ATX-S10Na(a)
tial, and the area under this curve from 0 to 80 s was taken       using the same type of PMT, which supports our conclusion
as the total 1O2 counts: average values      4.3    103 and 2.0    that this detector does enable measurement of the 1270 nm
   103 (after background subtraction) over 1200 laser pulses       1O luminescence emission in vivo (31).
for liver and skin, respectively. A cuvette containing 2.5 M          In conclusion, utilizing this new PMT technology has pro-
AlS4Pc was then positioned in a geometry similar to that           vided compelling evidence that it is possible to detect and
used in vivo, i.e. with the face at 45 degrees to the excitation   quantify 1O2 luminescence from intracellular photosensitizer
light, and the signal measured. As in the aforementioned           in cell suspension in H2O-based media and from tissues in
estimate of the system sensitivity, it was calculated that each    vivo. Previous attempts to detect 1O2 in cells have either
photon count represented (4.9         0.1)   103 molecules of      required the use of D2O to increase the lifetime to detectable
1O undergoing radiative decay in the cuvette. Hence, we            levels and increase transmission at 1270 nm (9–11) or have
calculate that approximately 2.1        107 and 9.8     106 1O2    not definitively distinguished intra- and extracellular photo-
molecules underwent radiative decay in liver and skin, re-         sensitizer (9,10). For the latter, even with repeated washing
spectively. Using these data along with Eq. (5), D in vivo         of cells after prolonged incubation, the trace quantities of
could be calculated using the following additional values:         photosensitizer in the external media can contribute a large
photosensitizer uptake      22.6    0.2 g/mg in liver and 9.5      fraction of the detected luminescence signal, because of the
   0.3 g/mg in skin, effective irradiation volume equivalent       much greater fractional volume ( 98%) and longer 1O2 life-
to a sphere of 3 mm radius (based on tissue optical prop-          time. It should be noted that this effect may be less signifi-
erties) and 1O2 quantum yield ( D) 0.38. The 1O2 radiative         cant with non–water soluble photosensitizers that are not
lifetime ( R) is unknown in vivo, but was assumed to be close      photodynamically activated in aqueous environments be-
to its value of 5.55 s in water (20). This gives D          0.17   cause of self-aggregation. Nevertheless, very careful design
  s in liver and 0.18 s in skin. (The concentration of 1O2         of in vitro studies is required, for example in experiments
produced in vivo was also calculated using Eq. (5) and these       aimed at determining the relationship between 1O2 genera-
values for D, giving an average concentration of 1O2 im-           tion and photobiological damage in cells.
mediately after the laser pulse of approximately 4        1 nM        In vivo, the conditions used here were aimed at maximiz-
in the liver and 2     1 nM in the skin.)                          ing the 1O2 luminescence signal and are not necessarily gen-
   It should be noted that this calculation of D is sensitive      erally applicable. For example, the measurements were per-
to the value of R used. For example, it has been shown (20)        formed on well-vascularized and therefore well oxygenated
that R can be estimated using the refractive index (n) of the      tissues, and the OPO laser used to generate the luminescence
solvent. In tissue, typical values for n are 1.37–1.45 (28),       signal did not have high enough average power to deliver a
which yields an estimate of the radiative lifetime of approx-      therapeutic light dose in a reasonable treatment time. Hence,
imately 1.5 s. This reduces D to 0.04 s in liver and 0.05          in order to apply this instrumentation to correlate in vivo 1O2
  s in skin. The range of values for D agrees with estimates       generation with the PDT response, a separate PDT irradia-
on the upper limit of 0.2 s by Baker and Kanofsky (10),            tion light source, with switching between this and the OPO
the range 0.01–0.04 s by Moan and Berg (13) and our                during the experiments, or a high repetition-rate and high
previous upper estimate of 0.5 s (6). To our knowledge,            average power laser will be required. Both options are cur-
no measurement for triplet-state lifetimes in vivo has been        rently being explored, together with improvements in the
published for any photosensitizer. However, on the basis of        optical collection efficiency by increasing the numerical ap-
the increase found in cell suspensions and protein-rich en-        erture of the detection system. The problem of secondary
vironments compared with simple solutions, it would be ex-         fluorescence from specular reflection will also be addressed
pected to increase in vivo as observed.                            in the next system by adapting the beam–tissue–detector ge-
   Gorman and Rogers noted that it would be difficult to            ometry, using low fluorescence components or by using po-
differentiate 1O2 luminescence from PS triplet-state phos-         larizing optics or both. With these modifications, we intend
phorescence, given the strongly reduced 1O2 lifetime in vivo       to start applying this technology to determining the contri-
(29). However, this did not take into account the possibility      bution of 1O2 to the photobiological effect of PDT in a va-
of spectrally resolving the NIR emission, as done here. Cer-       riety of in vitro and in vivo models, for example to test the
tainly, we observe an increase in the overall background NIR       hypothesis that photochemical depletion of molecular oxy-
                                                                                  Photochemistry and Photobiology, 2002, 75(4) 391

gen in tissue at high fluence rates is responsible for reduced                 detection of singlet molecular oxygen: a Stern–Volmer quench-
photodynamic efficacy because of decreased 1O2 generation                      ing experiment with sodium azide. Photochem. Photobiol. 45,
(32). At the same time, we will evaluate the utility of 1O2             16.   Patterson, M. S., B. C. Wilson and R. Graff (1990) In vivo tests
luminescence monitoring as a PDT dosimetry tool in com-                       of the concept of photodynamic threshold dose in normal rat
parison with other direct and indirect dosimetry methods (8).                 liver photosensitized by aluminum chlorosulphonated phthalo-
If these studies are encouraging, we then intend to develop                   cyanine. Photochem. Photobiol. 51, 343–349.
                                                                        17.   Lilge, L., C. O’Carroll and B. C. Wilson (1997) A solubilization
this system for clinical use during PDT treatments.                           technique for photosensitizer quantification in ex vivo tissue
Acknowledgements This work was supported by the Canadian Can-                 samples. J. Photochem. Photobiol. B39, 229–235.
cer Society under a grant from the National Cancer Institute of Can-    18.   Krasnovsky Jr., A. A. (1981) Quantum yield of photosensitized
ada. The authors wish to thank Hamamatsu Corp., Japan, and in                 luminescence and radiative lifetime of singlet (1 g) molecular
particular Dr. Ken Kaufmann, Hamamatsu, NJ, for providing the                 oxygen in solution. Chem. Phys. Lett. 81, 443–445.
PMT system. Dr. Richard Hill, OCI, provided the AML5 cell line          19.   Fernandez, J. M., M. D. Bilgin and L. I. Grossweiner (1997)
and QLT Phototherapeutics, Vancouver, BC, Canada, kindly sup-                 Singlet oxygen generation by photodynamic agents. J. Photo-
plied Photofrin. The assistance of Kathryn Adams, Kristen Camp-               chem. Photobiol. B37, 131–140.
bell, Jason Forbes, Dr. Brian McIlroy, Andrea Molckovsky, Poupak        20.   Poulsen, T. D., P. R. Ogilby and K. V. Mikkelsen (1998) Sol-
Pournazari and Dr. Robert Weersink in performing these experi-                vent effects on the O2(a1 g)-O2(X3 g ) radiative transition: com-
ments is also gratefully acknowledged.                                        ments regarding charge-transfer interactions. J. Phys. Chem. A.
                                                                              102, 9829–9832.
                                                                        21.   Foley, M. S. C., A. Beeby, A. W. Parker, S. M. Bishop and D.
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