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1.IntroductionOptical biopsy—the use of native tissue fluorescence—for detection of cancer has been an area of investigation for over two decades.1, 2, 3 The bases behind the operation of optical biopsy is that the onset of carcinogenesis results in structural changes (thickening of the mucosa layer, increased vascularity) and molecular changes (increased nucleic acids, alterations in protein structure, increased cell metabolism) that modify the spectroscopic properties of tissue and, thereby, create unique optical signatures that can be used to detect malignant and premalignant tissues. In the UV and blue spectral regions, the major native tissue fluorophores are tryptophan (trp), collagen, elastin, reduced nicotinamide adenine dinucleotide (NADH), and flavins. Optical biopsy has been demonstrated to be an accurate, real-time tool for distinguishing normal tissues from malignant and premalignant tissues. Prior investigations have demonstrated that there are several ratio-based algorithms that can distinguish malignant tissue from normal with high sensitivity and specificity.3, 4, 5 The wavelength combinations used in those ratios isolated the contributions from different pairs of tissue fluorophores, one of which was trp. The ratio of emission (with excitation) has been shown to be a useful ratio to identify malignant tissues from many different organ sites.4, 6 However, when the ratio was applied to ex vivo human breast tissue, the presence of adipose tissue required that multiple wavelength ratio pairs be evaluated to successfully distinguish adipose tissue from malignant tissue by fluorescence.7 Identification of additional spectroscopic fingerprints to distinguish adipose and normal glandular tissues from malignant breast tissue can significantly enhance optical biopsy of breast tissues and improve the efficacy of either fine needle aspiration or micro-endoscopic ductal examination. For excitation between 250 and , trp is the predominant native fluorophore in tissues. The trp fluorescence and phosphorescence properties, such as Stokes shift and quantum efficiency, are strongly dependent on the local environment and protein structure.8 Therefore, trp emission is expected to differ in different types of tissue environments. The trp Stokes shift is highly dependent on solvent polarity, with a more polar solvent producing a greater Stokes shift, i.e., red-shifted fluorescence.9, 10, 11 In proteins with multiple trp residues, the emission spectrum is the sum of each residue’s individual spectrum. Trp phosphoresces in the range from . At room temperature, phosphorescence from trp solution is difficult to observe due to quenching by dissolved oxygen and other quenchers.12 In complex proteins, aromatic amino acid residues can be buried within the protein structure, and thus have limited exposure to potential quenching molecules. In such proteins, trp phosphorescence may be observed at room temperature.12, 13, 14, 15, 16 In the absence of quenching, the phosphorescence lifetime of trp is at , and between at room temperature.12, 17 Proximity to potential quenchers and rigidity of the protein structure can also affect the trp phosphorescence with lifetime changes of 3 to 4 orders of magnitude.17, 18 Protein folding hinders the quenching of phosphorescence for internal trp residues.12 In proteins, trp phosphorescence is enhanced19 at both high and low pH compared to neutral pH. Phosphorescence lifetime of trp residues is sensitive to protein conformational changes.20 These properties makes room temperature trp phosphorescence an effective tool to investigate protein structure.12, 21 Breast cancer causes changes in local environment and proteins which may affect trp phosphorescence. Breast cancer changes the pH (from neutral or slightly alkaline in normal cells to slightly acidic in cancer cells) as well as changes in the respiratory mechanisms of cells.22 Cancer cells consume less oxygen than normal cells, becoming anaerobic and utilizing glucose to produce lactic acid, which decreases pH. This decrease in pH and changes in the respiration mechanism can affect protein structure with subsequent changes in phosphorescence. Thus phosphorescence may be an additional spectroscopic signature to distinguish malignant tissue from benign.23 Although phosphorescence is much weaker than fluorescence, the longer lifetime of phosphorescence (milliseconds to seconds) as compared to fluorescence enables the two signals to be separated by using a pulsed excitation and time-gated photodetection. In this paper, for the first time, the phosphorescence and combined phosphorescence and fluorescence spectra from ex vivo human breast tissues were measured and differences in the ratio of trp phosphorescence to fluorescence intensities were observed between malignant tissue and normal glandular and adipose tissues. Ratio maps were generated from the tissue spectra that designated regions of tumor from normal glandular and adipose tissue. This paper proposes a new optical approach to detect cancer extending the fluorescence approach.1, 2, 3 This paper also reports the first use of AlGaN alloy UV LEDs to excite native tissue fluorophores. The use of UV LEDs instead of lamps can significantly advance the development of compact optical biopsy instrumentation. The narrower bandwidth of LEDs compared to xenon or mercury lamps can reduce stray light leakage. LEDs are more efficient at coupling light into optical fibers than lamps. Their small size and lower current requirements can reduce the size of instrumentation for use in miniature photonic devices.24 2.Materials and Methods2.1.Experimental SetupTwo experimental setups were used to acquire the spectra from trp powder and tissues. One setup used a CD-Scan (Mediscience Technology Corp, Cherry Hill, New Jersey). The CD-Scan is a modified LS50B (Perkin Elmer, Shelton, Connecticut) fluorescence spectrophotometer. The CD-Scan uses a pulsed xenon excitation lamp and gated photomultiplier tube (PMT) detectors. The delay between the lamp pulse and the detector gate is computer selectable, enabling collection of either fluorescence (zero delay between lamp pulse and detector gate) or phosphorescence (delays greater than the lamp pulse width). The illumination area of the CD-Scan is a function of the excitation monochromator slit width. For the phosphorescence measurements, the excitation monochrometer resolution was set to , giving an excitation area of approximately . The scan rate was . The photodetector gate delay can be varied in steps. The second experimental setup shown in Fig. 1 was used to generate ratio maps from the breast tissue specimens. The excitation source was an AlGaN alloy UV LED with emission centered at with of average power. A narrow-band (NB) interference filter ( FWHM) was used to block the longer wavelength fluorescence emitted by the LED. The emission was collected through a optical fiber inserted inside a metal jacket. The collection region was approximately in diameter. The metal jacket is similar in size to a stereotactic needle such as the type used for fine needle aspiration (FNA) procedures on human breast and the collection fiber can easily be integrated into an FNA system. The digital output of the CCD is connected to a PC via a USB (universal serial bus) connection. The integration time of the CCD is computer controllable and was set to so that both fluorescence and phosphorescence were detected simultaneously. The spectrograph exhibited relatively more efficiency in the visible region than in the UV, compared to the CD-Scan. Thus, after normalizing the spectra to the trp fluorescence peak , the trp phosphorescence normalized “counts” were higher with the spectrograph setup than with the CD-Scan. Specimens were mounted on a two-axis translation stage, enabling spectra to be acquired at different locations on the samples. 2.2.Tissue SpecimensFresh surgical specimens of cancerous and normal breast tissues were acquired under Institutional Review Board approval, from a tissue bank within of surgery. The normal specimens, acquired from subjects undergoing breast reduction surgery, had clearly defined regions of normal glandular and adipose tissue. The malignant specimens were acquired from patients undergoing lumpectomies. These specimens were from the margins of the tumor and were a mix of malignant and normal tissues. Specimens were stored at and were not subjected to any additional processing prior to measuring the spectra. Specimens were cut into pieces ranging from and placed in a quartz cuvette, prior to mounting on the translation stage. Using the spectrograph setup, the spectra were acquired in a grid pattern with a spacing so as to cover most of the specimen. Each spectrum was integrated for . Intensity ratio maps were generated from the individual spectra. Five cancerous and six normal breast tissue specimens were scanned for a total of locations on the tissue specimens. After completion of a sequence of scans with the spectrograph apparatus, the fluorescence and phosphorescence were measured with the CD-Scan. In fluorescence mode, the photodetector gate was synchronized with the excitation lamp pulse (zero time delay). The much higher efficiency of fluorescence compared to phosphorescence meant that the signal was dominated by the fluorescence. In phosphorescence mode, the photodetector gate delay was varied from with respect to the xenon lamp pulse. 3.Results and Discussion3.1.Trp PowderFigure 2 shows the room-temperature phosphorescence spectra of D-L trp powder excited at 282, 300, 380, and and acquired with the CD-Scan. Each spectrum shown in Fig. 2 is the sum of seven spectra, acquired with delays of in steps. The gate width was , thus each spectrum shown is the integrated intensity from . Although it is well known that the trp excitation maximum is at , and that trp does not have significant absorption at wavelengths longer than , the phosphorescence intensity with excitation was weaker than with excitation. For the longer wavelength excitations (380 and ), the phosphorescence was blue shifted compared to shorter wavelength excitation (282 and ). For 380- and excitation, trp exhibits two phosphorescence peaks at 480 and . For excitation at 282 and , only the phosphorescence peak at was observed. Both the mechanism responsible for the shift in phosphorescence and the reason for the more intense phosphorescence at excitation is not known. It may be due to dimers or trimers present in the trp powder. Fig. 2Phosphorescence spectra of D-L tryptophan powder for different excitation wavelengths. Spectra are integrated from 0.5- to delay with respect to excitation lamp pulse. ![]() Figures 3a and 3b show a 3-D representation of trp phosphorescence plotted as a function of wavelength and photodetector gate delay for and , respectively. The emission intensity at is plotted in Figs. 3c and 3d as a function of gate delay for and , respectively. For , the lifetime is , and for , the lifetime is . 3.2.Phosphorescence from ex vivo Human Breast TissuesAlthough the phosphorescence intensity in trp powder was greater for excitation at than for excitation at , tissues contain several fluorophores (NADH, collagen, and flavins) whose emission wavelengths can overlap with the trp phosphorescence. Therefore, the breast tissue specimens were excited at to reduce the contribution from these fluorophores. The main types of tissue types found in human breast are glandular and adipose. Figure 4 shows the phosphorescence from ex vivo human normal and malignant breast tissues acquired with the CD-Scan. The plots shown in Fig. 4 are each the integration of seven spectra acquired with gate delays of with intervals. The normal tissues exhibited phosphorescence emission from . Fluorescence can be also observed at —most likely due to a long “tail” on the lamp emission. The normal tissues showed greater fluorescence and phosphorescence than the malignant tissues, which had almost no detectable phosphorescence. Fig. 4Phosphorescence from normal and malignant glandular breast tissues. Signals were integrated for detector gate delays of . ![]() Typical spectra, excited at , from cancerous and normal glandular and adipose breast tissues are presented in Fig. 5 . These spectra were acquired with the spectrograph system. The spectra have been normalized to unit maxima. The integration time enabled collection of both fluorescence and phosphorescence, and both types of emissions can be observed in Fig. 5. The CCD detector sensitivity is weaker in the UV than the visible, thus, the trp “counts” for fluorescence and phosphorescence do not differ as much as they would have if corrected for spectral response of the detector. Referring to Fig. 5, it can be observed that the adipose tissue with a fluorescence peak at has a smaller Stokes shift than either the normal glandular or cancerous tissue. This may be a result of lower water content in adipose tissue. A broad trp phosphorescence band from is observed in both the normal glandular and adipose tissues, which is not present in the cancerous tissues. The presence of trp phosphorescence in normal tissue but not in cancerous tissues suggests that the ratio of trp phosphorescence to fluorescence may be able to distinguish malignant breast tissue from normal glandular or adipose tissue. This difference can be quantified by computing the ratio of emission intensity at (trp fluorescence) to the intensity at (trp phosphorescence). Fig. 5Combined fluorescence and phosphorescence spectra of normal adipose and glandular breast and cancerous breast tissue. Excitation at . ![]() A map of the ratio and positions of fluorescence peaks for a normal glandular specimen are shown in Figs. 6a and 6b , respectively. Spectra were acquired at 50 locations on a specimen. The average value of the ratio is and the average fluorescence peak wavelength is . Figures 7a and 7b plot the ratio and fluorescence peak position for an adipose specimen, respectively. This specimen was also scanned at a total of 50 locations. The ratio average value is and the fluorescence peak is at . The larger standard deviation in the Stokes shift of the glandular tissues reflects the greater variation in local protein environment in the glandular tissue, a result of its more complicated structure compared to fat tissue. The ratio for the adipose specimen is a factor of 5 larger than for the glandular tissue, indicating the relatively weaker phosphorescence from fat. The Stokes shift for the adipose tissue is less than for the glandular tissue, indicating that trp residue in fat tissues have less solvent (water) exposure than in glandular tissues. The ratio was calculated at 431 locations on malignant breast tissues and 455 locations on normal glandular and adipose tissues. These ratio values are plotted in Fig. 8 . For the normal specimens (glandular and adipose) the ratio is and for malignant specimens, the ratio is . Using a cutoff ratio value of 12, 421 of the 431 malignant and 446 of the 455 nonmalignant locations are correctly identified for a sensitivity of 97.6% and a specificity of 98%. Figure 9a shows a map of the ratio from a malignant breast tissue specimen. The specimen consists of a malignant tumor located on the right side of the figure, adipose tissue in the upper left, and regions of normal glandular tissue in the middle left. The malignant regions distinctly show a higher ratio [darker regions in Fig. 9a] than the adipose or normal glandular tissue. Figure 9b plots the location of the emission peak from the same specimen. The areas of adipose tissue had a smaller Stokes shift, i.e., shorter wavelength fluorescence [lighter regions in Fig. 9b] than glandular tissue. 4.ConclusionsThis newly developed optical technique for cancer detection, based on phosphorescence and fluorescence spectroscopy, is fast, minimally invasive, and nondestructive. This technique may be applicable for either in vivo or ex vivo tissue analysis. The ratio, with excitation at , provides an excellent fingerprint for cancer detection in ex vivo tissues. The use of ratios, as markers for malignancy, enables comparison of data under different illumination conditions and with different surface structure. An ratio of 0.5 to 5 corresponds to normal glandular breast tissue, a ratio of 5 to 12 corresponds the normal adipose breast tissue and a ratio higher than 12 indicates cancerous breast tissue. The Stokes shift information gives additional data for validation of the analysis. Since trp is present in practically every cell in the body, these fluorescence and phosphorescence fingerprints may be applicable in the detection of cancer at many different organ sites. Further research will be needed to confirm that the techniques applied here will provide accurate in vivo tissue diagnostic information. This paper demonstrated that LEDs emitting in the UV can be used to excite native tissue fluorescence, which can advance the development of miniature instrumentation for optical biopsy. AcknowledgmentsThis work was funded in part by grants from the New York State Office Science, Technology and Academic Research (NYSTAR), and Mediscience Technology Corp. ReferencesR. Alfano,
D. Tata,
J. Cordero,
P. Tomashefsky,
F. Longo, and
M. Alfano,
“Laser induced fluorescence spectroscopy from native cancerous and normal tissue,”
IEEE J. Quantum Electron., 20
(12), 1507
–1511
(1984). https://doi.org/10.1109/JQE.1984.1072322 0018-9197 Google Scholar
R. Alfano,
G. Tang,
A. Pradhan,
W. Lam,
D. Choy, and
E. Opher,
“Fluorescence spectra from cancerous and normal human breast and lung tissues,”
IEEE J. Quantum Electron., 23
(10), 1806
–1811
(1987). https://doi.org/10.1109/JQE.1987.1073234 0018-9197 Google Scholar
R. R. Alfano,
B. B. Das,
J. Cleary,
R. Prudente, and
E. J. Celmer,
“Light sheds light on cancer—distinguishing malignant tumors from benign tissues and tumors,”
Bull. N. Y. Acad. Med., 67
(2), 143
–150
(1991). 0028-7091 Google Scholar
B. B. Das,
W. S. Glassman,
R. R. Alfano,
J. Cleary,
R. Prudente,
E. J. Celmer, and
S. Lubicz,
“UV-fluorescence spectroscopic technique in the diagnosis of breast, ovarian, uterus, and cervix cancer,”
Proc. SPIE, 1427 368
–373
(1991). 0277-786X Google Scholar
A. Katz,
H. E. Savage,
S. P. Schantz,
S. A. McCormick, and
R. R. Alfano,
“Noninvasive native fluorescence imaging of head and neck tumors,”
Technol. Cancer Res. Treat., 1
(1), 9
–15
(2002). 1533-0346 Google Scholar
W. S. Glassman,
C. H. Liu,
G. C. Tang,
S. Lubicz, and
R. R. Alfano,
“Ultraviolet excited fluorescence spectra from non-malignant and malignant tissues of the gynecological tract,”
Lasers Life Sci., 5
(1–2), 49
–58
(1992). 0886-0467 Google Scholar
Y. Yang,
A. Katz,
E. J. Celmer,
M. Zurawska-Szczepaniak, and
R. R. Alfano,
“Optical spectroscopy of benign and malignant breast tissues,”
Lasers Life Sci., 7
(2), 115
–127
(1996). 0886-0467 Google Scholar
J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic, New York
(1999). Google Scholar
G. Weber,
“Fluorescence-polarization spectrum and electronic-energy transfer in tyrosine, tryptophan and related compounds,”
Biochem. J., 75 335
–345
(1960). 0264-6021 Google Scholar
B. Valeur and
G. Weber,
“Resolution of the fluorescence excitation spectrum of indole into the and excitation bands,”
Photochem. Photobiol., 25 441
–444
(1977). 0031-8655 Google Scholar
P. R. Callis,
“ and transitions of tryptophan: applications of theory and experimental observations to fluorescence of proteins,”
Methods Enzymol., 278 113
–150
(1997). 0076-6879 Google Scholar
M. L. Saviotti and
W. C. Galley,
“Room temperature phosphorescence and the dynamic aspects of protein structure,”
Proc. Natl. Acad. Sci. U.S.A., 71
(10), 4154
–4158
(1974). 0027-8424 Google Scholar
Y. Kai and
K. Imakubo,
“Temperature dependence of the phosphorescence lifetimes of heterogeneous tryptophan residues in globular proteins between 293 and ,”
Photochem. Photobiol., 29 261
–265
(1979). 0031-8655 Google Scholar
G. B. Strambini,
P. Cioni, and
R. A. Felicioli,
“Characterization of tryptophan environments in glutamate dehydrogenases from temperature-dependent phosphorescence,”
Biochemistry, 26
(16), 4968
–4975
(1987). 0006-2960 Google Scholar
J. M. Vanderkooi,
D. B. Calhoun, and
S. W. Englander,
“On the prevalence of room-temperature protein phosphorescence,”
Science, 236
(4801), 568
–569
(1987). 0036-8075 Google Scholar
D. B. Calhoun,
S. W. Englander,
W. W. Wright, and
J. M. Vanderkooi,
“Quenching of room temperature protein phosphorescence by added small molecules,”
Biochemistry, 27
(22), 8466
–8474
(1988). 0006-2960 Google Scholar
G. B. Strambini and
M. Gonnelli,
“The indole nucleus triplet-state lifetime and its dependence on solvent microviscosity,”
Chem. Phys. Lett., 115
(2), 196
–200
(1985). 0009-2614 Google Scholar
V. Subramaniam,
A. Gafni, and
D. G. Steel,
“Time-resolved tryptophan phosphorescence spectroscopy: a sensitive probe of protein folding and structure,”
IEEE J. Sel. Top. Quantum Electron., 2
(4), 1107
–1114
(1996). https://doi.org/10.1109/2944.577341 1077-260X Google Scholar
T. Truong,
R. Bersohn,
P. Brumer,
C. K. Luk, and
T. Tao,
“Effect of pH on the phosphorescence of tryptophan, tyrosine, and proteins,”
J. Biol. Chem., 242
(12), 2979
–2985
(1967). 0021-9258 Google Scholar
E. G. Strambini and
G. B. Strambini,
“Tryptophan phosphorescence as a monitor of protein conformation in molecular films,”
Biosens. Bioelectron., 15
(9–10), 483
–490
(2000). https://doi.org/10.1016/S0956-5663(00)00086-5 0956-5663 Google Scholar
J. A. Schauerte,
D. G. Steel, and
A. Gafni,
“Time-resolved room temperature tryptophan phosphorescence in proteins,”
Methods Enzymol., 278 49
–71
(1997). 0076-6879 Google Scholar
A. K. Brewer,
“The high pH therapy for cancer tests on mice and humans,”
Pharmacol., Biochem. Behav., 21 1
–5
(1984). 0091-3057 Google Scholar
A. Alimova,
A. Katz,
V. Sriramoju,
Y. Budansky,
A. A. Bykov,
R. Zeylikovich and
R. R. Alfano,
“Hybrid native phosphorescence and fluorescence spectroscopy for cancer detection,”
Proc. SPIE, 6091 22
–25
(2006). 0277-786X Google Scholar
L. Wang,
G. Zhang,
J. C. Luo,
F. Zeng,
Q. Z. Wang,
S. A. Alfano,
A. Katz,
M. Zevallos, and
R. R. Alfano,
“Wireless spectroscopic compact photonic explorer for diagnostic optical imaging,”
Biomed. Microdevices, 7
(2), 111
–115
(2005). 1387-2176 Google Scholar
|