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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics

| EXPLORING THE INTERFACE OF LIGHT AND BIOMEDICINE

  • Editor: Gregory W. Faris
  • Vol. 5, Iss. 7 — Apr. 26, 2010
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Photothermal and photoacoustic Raman cytometry in vitro and in vivo

Evgeny V. Shashkov, Ekaterina I. Galanzha, and Vladimir P. Zharov  »View Author Affiliations


Optics Express, Vol. 18, Issue 7, pp. 6929-6944 (2010)
http://dx.doi.org/10.1364/OE.18.006929


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Abstract

An integrated Raman-based cytometry was developed with photothermal (PT) and photoacoustic (PA) detection of Raman-induced thermal and acoustic signals in biological samples with Raman-active vibrational modes. The two-frequency, spatially and temporally overlapping pump–Stokes excitation in counterpropagating geometry was provided by a nanosecond tunable (420–2300 nm) optical parametric oscillator and a Raman shifter (639 nm) pumped by a double-pulsed Q-switched Nd:YAG laser using microscopic and fiberoptic delivery of laser radiation. The PA and PT Raman detection and imaging technique was tested in vitro with benzene, acetone, olive oil, carbon nanotubes, chylomicron phantom, and cancer cells, and in vivo in single adipocytes in mouse mesentery model. The integration of linear and nonlinear PA and PT Raman scanning and flow cytometry has the potential to enhance its chemical specificity and sensitivity including nanobubble-based amplification (up to 10- fold) for detection of absorbing and nonabsorbing targets that are important for both basic and clinically relevant studies of lymph and blood biochemistry, cancer, and fat distribution at the single-cell level.

© 2010 OSA

Introduction

Photothermal (PT) and photoacoustic (PA) methods employing nonradiative conversion of absorbed energy into heat and sound have successfully been used in spectroscopy, microscopy, analytical chemistry, biology, and medicine (e.g., [1

1. V. P. Zharov, and V. S. Letokhov, Laser Optoacoustic Spectroscopy (Springer-Verlag; Berlin Heidelberg 1986).

6

6. E. I. Galanzha, E. V. Shashkov, T. Kelly, J.-W. Kim, L. Yang, and V. P. Zharov, “In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells,” Nat. Nanotechnol. 4(12), 855–860 (2009). [CrossRef] [PubMed]

]). Conventional PA/PT spectroscopy in the visible and near-infrared (NIR) spectral ranges is based on optical mono-excitation of electronic and vibrational modes of natural chromophores (e.g., cytochromes, melanin, and hemoglobin) or synthetic absorbing micro- and nanoparticles as PA/PT contrast agents. In particular, we demonstrated the capability of linear PT and PA cytometry in scanning and flow modes to detect individual cells, bacteria, and nanoparticles in vitro and in vivo in blood and lymph flow at sensitivity thresholds that are unachievable with existing techniques [5

5. J. W. Kim, E. I. Galanzha, E. V. Shashkov, H. M. Moon, and V. P. Zharov, “Golden carbon nanotubes as multimodal photoacoustic and photothermal high-contrast molecular agents,” Nat. Nanotechnol. 4(10), 688–694 (2009). [CrossRef] [PubMed]

8

8. E. I. Galanzha, E. V. Shashkov, P. M. Spring, J. Y. Suen, and V. P. Zharov, “In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser,” Cancer Res. 69(20), 7926–7934 (2009). [CrossRef] [PubMed]

]. The selective detection in vivo of rare cells of interest (e.g., metastatic tumor cells) presents a challenge because of the complex biological absorption background. To spectrally identify fast flowing cells, we developed a time-resolved linear PA two-color cytometer, using two nanosecond laser pulses at selected wavelengths and delay times [6

6. E. I. Galanzha, E. V. Shashkov, T. Kelly, J.-W. Kim, L. Yang, and V. P. Zharov, “In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells,” Nat. Nanotechnol. 4(12), 855–860 (2009). [CrossRef] [PubMed]

8

8. E. I. Galanzha, E. V. Shashkov, P. M. Spring, J. Y. Suen, and V. P. Zharov, “In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser,” Cancer Res. 69(20), 7926–7934 (2009). [CrossRef] [PubMed]

]. These pulses were activated by the third (355 nm) and second (532 nm) harmonics of an Nd:YAG laser which pumped to an optical parametric oscillator (OPO) with a tunable spectral range of 420–2300 nm and a Raman shifter with a fixed wavelength of 639 nm, respectively. Here we show that this technique, after further modification, can provide a method of in vivo nonlinear PA and PT Raman cytometry with chemical specificity. This can be achieved by PT and PA detection of Raman-induced heat and sound in either the nonabsorbing or absorbing cells with Raman-active vibrational modes.

Historically, the nonlinear spectroscopic technique combining stimulated Raman scattering with acoustic detection, referred to as PA Raman spectroscopy (PARS), was first suggested in 1975 by Nechaev and Ponomarev [9

9. S. Nechaeiv and N. Ponomarev, “High-resolution Raman spectrometer,” Sov. J. Quantum Electron. 5, 72–76 (1975).

]. The technique was first employed in 1979 in gas by Barrett and Berry [10

10. J. J. Barrett and M. J. Berry, “Photoacoustic Raman spectroscopy (PARS) using cw laser sources,” Appl. Phys. Lett. 34(2), 144–147 (1979). [CrossRef]

] using two continuous-wave (CW) or nanosecond pulse lasers. Nonbiological liquids were studied by Patel and Tam using microsecond laser pulses [11

11. C. K. N. Patel and A. C. Tam, “Optoacoustic Raman gain spectroscopy of liquids,” Appl. Phys. Lett. 34(11), 760–763 (1979). [CrossRef]

,12

12. A. C. Tam, “Applications of photoacoustic sensing techniques,” Rev. Mod. Phys. 58(2), 381–431 (1986). [CrossRef]

]. Later application of PARS was focused mainly on gas analysis [e.g., 13

13. G. A. West, D. R. Siebert, and J. J. Barrett, “Gas phase photoacoustic Raman spectroscopy using pulsed laser excitation,” Appl. Phys. (Berl.) 51, 2823–2828 (1980). [CrossRef]

21

21. R. C. Sharma, “A novel demonstration of photoacoustic Raman spectroscopy with combined stimulated Raman pumping in H2 molecule,” Opt. Commun. 282(6), 1183–1185 (2009). [CrossRef]

]. In particular, we demonstrated the capability of PARS with counterpropagating geometry of Stokes and pump beams (i.e., pump positioned in the in forward direction and Stokes positioned in a backwards direction) increased the sensitivity and especially specificity of trace analysis in gaseous mixtures, using two nanosecond pulses from an Nd:YAG laser (second harmonic, 532 nm) and a tunable dye laser (545–630 nm) [16

16. A. M. Brodnikovskii, V.P. Zharov, and N.P. Koroteev, “Photoacoustic Raman spectroscopy of molecular gases,” Sov. J. Quantum Electron. •••, 2421–2430 (1985).

]. In these and other studies, nonlinear PARS techniques were used separately from conventional linear PA spectroscopy, and the laser energy level was relatively high that it could damage biological samples. Here we propose the integration of linear and nonlinear PA and PT Raman cytometry (PARC and PTRC, respectively) that may allow detection, with chemical specificity, both absorbing and weakly absorbing cells simultaneously at a laser energy level safe for biological tissues. We present a brief discussion of the theory underlying these techniques, optical scheme features, parameter testing with conventional nonbiological samples, and proof-of-concept, using normal and cancer cells in vitro and in vivo. The advantages, limitations, and further improvement of this new optical technology are also discussed.

Materials and methods

Principles of PT and PA Raman cytometry

Conventional linear PA and PT cytometry (which we will refer to as PA/PT until noted) is based on the direct detection of light absorption in a sample. Nonlinear PA/PT Raman technique is based on stimulated Raman scattering phenomena leading eventually to the generation of heat and sound (i.e., it is not necessary for light to be directly absorbed by the sample). Cells with Raman-active vibrational modes are simultaneously irradiated with two spatially and temporally overlapping laser pulses: the pump beam, with a relatively high frequency νP and energy EP, induces Raman scattering; and the Stokes (or signal) beam with a lower frequency νS and energy EP, acts as a wave at the Stokes frequency [Fig. 1(a)
Fig. 1 Method of PA and PT Raman cytometry with two-frequency excitation of the vibrational mode of the cells. (a) Level scheme of the Raman transition. (b) Schematic of cell irradiation. (c) Microscopic and fiber-based optical diagram. T, telescope; D, diaphragm; A, attenuator. (d) Double pumping of two-channel Nd:YAG laser. (e) Scheme details.
].

The Stokes photon energy level is lower than the pump photon level (i.e., hνS < hνP). If νP – νS is close to a Raman-active frequency (transition), νR of a cell (i.e., νR ≈νP – νS), during nonlinear interaction through the third-order nonlinear susceptibility, Raman gain occurs; that is, the pump beam is attenuated, and the Stokes beam is amplified, photon for photon [12

12. A. C. Tam, “Applications of photoacoustic sensing techniques,” Rev. Mod. Phys. 58(2), 381–431 (1986). [CrossRef]

]. In other words, the pump photon energy level induces transitions from a background state to a virtual state (which could also coincides with an actual state of the cell or other cells) and then to an excited state. The energy from an excited state can be converted through radiative relaxation to fluorescence or through nonradiative relaxation to translational energy via molecular collision, i.e., into heat and accompanying acoustic waves (i.e., as in conventional linear PA spectroscopy) that can be detected by the PT (e.g., thermolens) or PA techniques [Fig. 1(b)].

Sample preparation

For testing and calibration purposes, we used benzene, acetone (99.6% А929-4, Fisher Scientific), and olive oil (Extra Virgin, Cold Pressed Olive Oil, Pure, distributed by the KROGER CO., Cincinnati, Ohio 45202). Chylomicron phantoms measuring ~0.2 μm in diameter were prepared according to standard procedures [22

22. A. M. Sakashita, S. P. Bydlowski, D. A. F. Chamone, and R. C. Maranhão, “Plasma kinetics of an artificial emulsion resembling chylomicrons in patients with chronic lymphocytic leukemia,” Ann. Hematol. 79(12), 687–690 (2000). [CrossRef]

,23

23. Y. Park, W. J. Grellner, W. S. Harris, and J. M. Miles, “A new method for the study of chylomicron kinetics in vivo,” Am. J. Physiol. Endocrinol. Metab. 279(6), E1258–E1263 (2000). [PubMed]

]. Specifically, the mix, which consisted of vegetable oil (10%), bovine serum albumin (5%), and glycerol (2.5%) in phosphate-buffered saline (PBS) (87.5%), was subjected to sonic agitation for 5 minutes, ultracentrifuged at 40,000 rpm for 30 minutes, and then filtered with a 0.2-μm filter. The sample was kept at –20 °C.

Single-walled carbon nanotubes (CNTs; Carbon Nanotechnologies Inc., Houston, TX) were processed as described previously [5

5. J. W. Kim, E. I. Galanzha, E. V. Shashkov, H. M. Moon, and V. P. Zharov, “Golden carbon nanotubes as multimodal photoacoustic and photothermal high-contrast molecular agents,” Nat. Nanotechnol. 4(10), 688–694 (2009). [CrossRef] [PubMed]

,24

24. J.-W. Kim, E. V. Shashkov, E. I. Galanzha, N. Kotagiri, and V. P. Zharov, “Photothermal antimicrobial nanotherapy and nanodiagnostics with self-assembling carbon nanotube clusters,” Lasers Surg. Med. 39(7), 622–634 (2007). [CrossRef] [PubMed]

]. Their average length and diameter were 186 nm and 1.7 nm at a concentration of 22 μg/mL in PBS.

B16F10 mouse melanoma cells and WTY-1 human breast cancer cells (American Type Culture Collection, Manassas, VA) were cultured according to the vendor’s specifications (e.g., see [8

8. E. I. Galanzha, E. V. Shashkov, P. M. Spring, J. Y. Suen, and V. P. Zharov, “In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser,” Cancer Res. 69(20), 7926–7934 (2009). [CrossRef] [PubMed]

]). Viable cells were resuspended in PBS for all tests.

Animals

Nude nu/nu mice weighing 20–25 g (Harlan Sprague-Dawley, Indianapolis, IN) were used to obtain the PA and PT Raman signals from adipocytes in mouse mesentery in accordance with protocols approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee. After standard anesthesia by intraperitoneal injection of ketamine/xylazine (50 mg/10 mg/kg), mice were laparotomized by a small midabdominal incision, and the intestinal mesentery was placed on a customized, heated (37.7°C) microscope stage and suffused with warmed Ringer’s solution (37°C, pH 7.4) containing 1% albumin to prevent protein loss. In a conventional optical scheme, the mesentery was also submerged in an optical cuvette containing warmed PBS. The mesentery provides a minimally invasive but well-established in vivo model. No marked changes in tissue, microvessel morphology, or cell-flow dynamics were seen for at least 5 h of observation and over periods of repeated observation extending up to 2 months [25

25. E. I. Galanzha, V. V. Tuchin, and V. P. Zharov, “Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening,” World J. Gastroenterol. 13(2), 192–218 (2007). [PubMed]

]. Because of the thin, transparent mesenteric structure, this model represents a “gold standard” for providing first-step verification of this novel technology.

Experimental setup

PA waves were detected with an 3.5-MHz, 5.5-mm-diameter, ultrasound transducers (model 6528101, Imasonic Inc., Besançon, France; and a 2.25-MHz, 8-mm-diamater model V323-SM; Panametrics). Signals were then amplified (model 5660C; band, 2 MHz; gain, 60 dB; Panametrics) and recorded with a Tektronix TDS 3032B oscilloscope and a computer using standard and customized software. Ultrasound gel or warm water was used to provide acoustic and optical matching between the samples (e.g., microscopic slides) and the transducers.

PA methods were integrated with PT methods described previously [26

26. V. P. Zharov and D. O. Lapotko, “Photothermal imaging of nanoparticles and cells,” IEEE J. Sel. Top. Quantum Electron. 11(4), 733–751 (2005). [CrossRef]

]. The temperature rise associated with the energy transfer produced an expansion of the heated volume and a refractive index change. While in PT imaging (PTI) mode, laser (OPO)-induced temperature-dependent variations in the refractive index were visualized with the multiplex thermolens [33

33. V. P. Zharov, V. Galitovskiy, C. S. Lyle, and T. C. Chambers, “Superhigh-sensitivity photothermal monitoring of individual cell response to antitumor drug,” J. Biomed. Opt. 11(6), 064034 (2006). [CrossRef]

] technique using a second, collinear laser pulse from a Raman shifter (see above) at a tunable delay of 0–10 μs, and a CCD camera (AE-260E, Apogee Inc.) [26

26. V. P. Zharov and D. O. Lapotko, “Photothermal imaging of nanoparticles and cells,” IEEE J. Sel. Top. Quantum Electron. 11(4), 733–751 (2005). [CrossRef]

]. The pump and probe beams had stable, Gaussian intensity profiles and adjustable diameters in the ranges of 10–40 µm and 10–25 µm, respectively. Spatial resolution was determined by the microscope objective (e.g., 0.7 µm at 20 × , NA 0.4; 300 nm at 60 × , NA 1.25). While in PT thermolens mode, a laser (OPO)-induced refractive heterogeneity, called a thermolens effect, caused defocusing of a collinear He-Ne laser probe beam (stabilized He-Ne laser model 117A; wavelength, 632.8 nm; power, ~3 mW; Spectra-Physics Lasers Inc.) that led to a reduction in the beam’s intensity at its center detected by a photodiode (C5658, Hamamatsu Corp.; and PDA55, Thorlabs Inc.). All signals (PA, PTI, and PT thermolens) were normalized to the laser pulse energy and measured with a power meter [Fig. 1(c)].

Laser radiation was delivered to the samples with either a microscope or an optical fiber [Fig. 1(c)1(d)]. Pump and Stokes beams with the same horizontal polarization were combined by a dichroic mirror and focused by a microscope condenser onto 120-μm-thick slides holding liquid samples or cells and CNTs in suspension. We also used an outside microscope scheme with the pump and Stokes beams after the OPO and Raman shifter, through the use of additional mirrors, were focused by lenses L1 and L2 (focal length of both, ~6 cm) from opposite directions into the center of the 2 mm × 2 mm × 2 cm optical cuvette (3G10, Precision Cells Inc.) [Fig. 1(e)]. Spatial overlapping of the laser beams was achieved by use of a tiny hole in thin Al film produced by ablation with a high-energy pump beam delivered into the center of the cuvette. The hole was used to align the Stokes beam with the use of diaphragms D1 and D2 and telescopes T1 and T2 [Fig. 1(c)]. The transducer was partially immersed into the liquid inside the cuvette at a distance of ~5–6 mm from the focal volume of the laser beams. For PT thermolens detection, the probe beam from a CW He-Ne laser was focused into the focal volume of the pump and Stokes beams by lens L3; a PT thermolens signal was detected by focusing of the probe beam by lens L4 onto a photodetector with a pinhole [Fig. 1(e)]. The probe beam was directed perpendicularly to the pump and Stokes beams, thus allowing the highest spatial resolution (1–2 µm) to be achieved, as well as a reduction in the influence of background signals from scattered light.

Experimental results

PA Raman spectroscopy of benzene and acetone

To test and verify the PA/PT Raman setup, we first measured the PA Raman spectra (PARS) in acetone [Fig. 2(a)
Fig. 2 PA Raman signals in liquids obtained with parallel polarization of the pump and Stokes beams. (a) PA Raman spectra (PARS) of acetone obtained in a thin (120 μm) microscope slide by scanning of first beam in the visible spectral range (PARSVIS) and in the NIR range (PARSNIR) at a fixed second beam wavelength of 639 nm (15,649 cm–1). Dash curves show linear PA background during spectral scanning of first beam alone (i.e., without second beam at 639 nm). (b) PA Raman spectra of benzene obtained in a microscope slide at a fixed pump wavelength of 639 nm and spectral scanning of a Stokes beam in the NIR range. (c) PA signal amplitude as a function of delay time between the pump and Stokes beams for acetone. (d) PA Raman signal for acetone at delays of 0 (top, left) and 20 ns (top, right), compared to signals induced by the pump (bottom, left) and the Stokes (bottom, right) beams alone. Amplitude and time scale: 10 mV/div and 1 μs/div.
] and benzene [Fig. 2(b)], which have well characterized Raman spectra [10

10. J. J. Barrett and M. J. Berry, “Photoacoustic Raman spectroscopy (PARS) using cw laser sources,” Appl. Phys. Lett. 34(2), 144–147 (1979). [CrossRef]

12

12. A. C. Tam, “Applications of photoacoustic sensing techniques,” Rev. Mod. Phys. 58(2), 381–431 (1986). [CrossRef]

]. Acetone spectra were obtained by both tuning the frequency of the OPO, as a pump beam in the visible range from 19011 cm–1 (526 nm) to 18248 cm–1 (548 nm) at a fixed wavelength of the Stokes beam, 15,649 cm–1 (639 nm), and by tuning the frequency of the OPO as a Stokes beam in the NIR range from 12,048 cm–1 (830 nm) to 13,157 cm–1 (760 nm) at a fixed wavelength (15,649 cm–1) of the pump beam. PARS of benzene were obtained by tuning the frequency of the OPO as a Stokes beam in the NIR range only. Spectral data for each substance were acquired in 4 minutes and consisted of points separated by 12-30 cm–1. Figure 2(a) demonstrates that a nonlinear PA signal at two beam excitations are large enough above the linear PA background generated by a beam only. The spectral position of the peak for benzene in Fig. 2(b) in the range of νR 3040– 3070 cm–1 is approximately in line with available data on the Raman shift maximum νR ~3059 cm–1 [11

11. C. K. N. Patel and A. C. Tam, “Optoacoustic Raman gain spectroscopy of liquids,” Appl. Phys. Lett. 34(11), 760–763 (1979). [CrossRef]

]. However, we observed that the PA spectra width was greater than the Raman width (20 cm–1 [11

11. C. K. N. Patel and A. C. Tam, “Optoacoustic Raman gain spectroscopy of liquids,” Appl. Phys. Lett. 34(11), 760–763 (1979). [CrossRef]

], ), which can be explained by the limited spectral resolution of our setup (12–15 cm–1), and possibly an influence of linear background absorption as seen in the range of 2800–3200 cm–1.

PA Raman spectroscopy of olive oil

After evaluating the setup parameters with conventional samples with well characterized Raman spectra, we applied the PA/PT Raman technique to estimate the level of nonlinear Raman signals in olive oil droplets in water. In the first part of this experiment, the system was calibrated with microdroplets of pure oil in water. The linear signal amplitude profiles in olive oil (as in many other vegetable oils containing lipids), ranging from 2750 to 2950 cm–1, represent a superposition of several vibrational states of symmetric and asymmetric CH2 and CH3 stretching vibrations with the maximum at 2850–2885 cm–1 [27

27. R. M. El-Abassy, P. Donfak, and A. Materny, “Visible Raman spectroscopy for the discrimination of olive oils from different vegetable oils and the detection of adulteration,” J. Raman Spectrosc. •••, 2279 (2009).

,28

28. C. Heinrich, A. Hofer, A. Ritsch, C. Ciardi, S. Bernet, and M. Ritsch-Marte, “Selective imaging of saturated and unsaturated lipids by wide-field CARS-microscopy,” Opt. Express 16(4), 2699–2708 (2008). [CrossRef] [PubMed]

]. PARS were measured at two set frequencies of the pump and Stokes beams and at the same Raman shift in a tuning range between 3050 cm–1 and 2750 cm–1. Specifically, the pump wavelength νP was tuned in the visible range between 520 nm and 540 nm at a fixed νS of 639 nm, and the Stokes wavelength νS was tuned in the NIR range between 770 nm and 823 nm at a fixed νP of 639 nm. Two wavelengths set at a νP of 539 nm (18,534 cm–1) and a νS of 639 nm and a νP of 639 nm and a νS of 783 nm corresponded to a maximum Raman shift in lipid of νR ~2885 cm–1. As one can see from Fig. 3(a)
Fig. 3 PA Raman signals in olive droplets in a cuvette. (a) PARS at pump and Stokes beam frequencies in the visible and NIR ranges obtained by scanning at a pump beam wavelength tuned in the visible range and at a fixed Stokes wavelength and by scanning at a fixed pump beam wavelength and at a Stokes beam wavelength in the NIR range. (b) PA Raman signal amplitude as a function of the pump laser energy at 529 nm. (c,d) PA signal amplitude as function of delay time between pump and Stokes pulses with pump wavelengths in the visible (c) and the NIR ranges (d). (e) PT Raman signal at a νP of 539 nm and a νS of 639 nm at delays of 0 (left) and 20 ns (right). (f) Linear PA signals at delays of 2.5 μs (left), as well as with a 1-mm distance between pump and Stokes beams (right). Amplitude and time scales: 50 mV/div and 1 μs/div.
, both ranges of PA Raman spectra were similar, although in the NIR range lower background absorption was observed as a result of lower linear absorption at frequency νP and νR.

The measurement of PA signal amplitude at a changed laser pump energy of 529 nm (corresponding to the maximum Raman scattering spectra) revealed the linearity of PA signals in a broad laser energy range of a few microjoules to 30 μJ [Fig. 3(b)] and appearance of nonlinear effects at higher energy which can be associated with laser-induced overheating effects, accompanied by nano- and microbubble formation around local overheated zones (see details in [5

5. J. W. Kim, E. I. Galanzha, E. V. Shashkov, H. M. Moon, and V. P. Zharov, “Golden carbon nanotubes as multimodal photoacoustic and photothermal high-contrast molecular agents,” Nat. Nanotechnol. 4(10), 688–694 (2009). [CrossRef] [PubMed]

,8

8. E. I. Galanzha, E. V. Shashkov, P. M. Spring, J. Y. Suen, and V. P. Zharov, “In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser,” Cancer Res. 69(20), 7926–7934 (2009). [CrossRef] [PubMed]

]).

The data in Fig. 3(c)3(f) confirmed that the total linear signals with no time overlap of pump and Stokes beams (e.g., at a delay of 20 ns) are the sum of the signals induced by pump and Stokes beams alone. The energy level in most of the experiments with olive oil droplets was ~30 μJ at both wavelengths. The minimal diameter of a droplet detectable with a reasonable signal-to-noise ratio (≥ 2) is currently 500 nm.

PA Raman signal detection from carbon nanotubes

As demonstrated in the above experiments, the PARC/PTRC technique can detect nonlinear Raman effects in weakly absorbing (preferential application) samples. In the case of absorbing samples, the influence of background absorption can be excluded by subtracting nonlinear signal from linear signal components at different delay times between the pump and Stokes beams. To estimate this capability of integrated PA/PT techniques to detect nonlinear Raman signals in relatively strongly absorbing samples, we selected CNTs, which we have used as effective contrast agents in in vivo flow cytometry applications with both the NIR PT and Raman techniques [24

24. J.-W. Kim, E. V. Shashkov, E. I. Galanzha, N. Kotagiri, and V. P. Zharov, “Photothermal antimicrobial nanotherapy and nanodiagnostics with self-assembling carbon nanotube clusters,” Lasers Surg. Med. 39(7), 622–634 (2007). [CrossRef] [PubMed]

,29

29. A. S. Biris, E. I. Galanzha, Z. Li, M. Mahmood, Y. Xu, and V. P. Zharov, “In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues,” J. Biomed. Opt. 14(2), 021006 (2009). [CrossRef] [PubMed]

]. The Raman spectra of CNTs contain characteristic bands in two spectral regions: the so-called radial breathing modes (100–500 cm–1), which strongly depend on CNT diameter and chirality, and a high wave-number region (tangential and longitudinal modes) between 1200 and 3000 cm–1; the D band, positioned between 1250 and 1450 cm–1, is associated with vacancies and the presence of other carbonaceous impurities (amorphous carbon, glassy carbon, etc.) that destroy the graphitic symmetry [29

29. A. S. Biris, E. I. Galanzha, Z. Li, M. Mahmood, Y. Xu, and V. P. Zharov, “In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues,” J. Biomed. Opt. 14(2), 021006 (2009). [CrossRef] [PubMed]

]. Band G, lying between 1500 and 1600 cm–1 with a maximum at 1593 cm–1, corresponds to the splitting of the E2g stretching vibrational mode for graphite. This inherent Raman peak with the highest intensity was selected for our study.

We performed most measurements with CNTs, using protocols similar to those described above for other samples. As the laser energy increased at a frequency of 639 nm and a beam diameter of 20 µm, the four-phase PA signal behavior of CNTs was observed [Fig. 4(a)
Fig. 4 Linear and nonlinear PA/PT effects in CNTs. (a) PA signal as a function of laser energy. The insets show conventional (i.e., with one-frequency laser excitation) PT signals in linear (left) and nonlinear (right) modes associated with microbubble formation around overheated CNT clusters. (b) PA Raman signal amplitude as function delay between pump and Stokes beams. The inset shows PA signal at delays of 0 (left) and 30 ns (right). (c) PA Raman spectra at delays of 0 and 30 ns between the pump and Stokes beams.
]: (1) a gradual increase in linear PA signals in the range of 0.1–1 µJ; (2) nonlinear PA signal enhancement in the range of 1-10 µJ due to bubble formation around overheated CNT clusters accompanied by transitioning the linear positive PT thermolens signals to nonlinear signals with a negative component [Fig. 4(a), left top and right bottom inset, respectively; see other details in [6

6. E. I. Galanzha, E. V. Shashkov, T. Kelly, J.-W. Kim, L. Yang, and V. P. Zharov, “In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells,” Nat. Nanotechnol. 4(12), 855–860 (2009). [CrossRef] [PubMed]

,24

24. J.-W. Kim, E. V. Shashkov, E. I. Galanzha, N. Kotagiri, and V. P. Zharov, “Photothermal antimicrobial nanotherapy and nanodiagnostics with self-assembling carbon nanotube clusters,” Lasers Surg. Med. 39(7), 622–634 (2007). [CrossRef] [PubMed]

]); (3) slight saturation of signals in the range of 10-50 µJ; and (4) “secondary” nonlinear signal enhancement for energy >50 µJ, likely due to thermal explosion of CNTs accompanied by shock waves. To study Raman effects, we selected a relatively low laser energy level, in the range of 10–30 µJ, which nevertheless led to the bubble-formation phenomena. To exclude cumulative effects, each laser pulse irradiated new GNTs by mixing the solution after each laser pulse. Surprisingly, despite strong absorption by CNTs, we clearly observed nonlinear PA Raman signal components that were verified by a decreased signal amplitude as the length of the delay increased [Fig. 4(b)]. As a result, the PA Raman spectra of CNTs were obtained with the maximum that coincides with a Raman shift (1593 cm–1 [29

29. A. S. Biris, E. I. Galanzha, Z. Li, M. Mahmood, Y. Xu, and V. P. Zharov, “In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues,” J. Biomed. Opt. 14(2), 021006 (2009). [CrossRef] [PubMed]

], ) [Fig. 4(c)]. Repetition of these measurements at a delay of 15–30 ns led to decreases in spectral contrast that were likely determined predominantly by strong linear absorption by the CNTs.

Biological applications of PT/PA Raman technique: detection of cancer cells and chylomicron phantoms in vitro

We have obtained PA/PT Raman signals from biological samples including WTY-1 breast cancer cells, B16F10 mouse melanoma cells, and chylomicron phantom in suspension in vitro. The data for most samples were similar. Nonlinear components in PA signals were verified by decreasing the PA signal amplitude with a delay-time increase [Fig. 5(a)
Fig. 5 Linear and nonlinear PA effects in chylomicron phantoms and cells in vitro. (a) PA signal in breast cancer at delays of 0 (left) and 20 ns (right). (b,c) PA signals in 200-nm chylomicron phantoms at delays of 0 (left) and 20 ns (right) in the visible (b) and the NIR (c) spectral ranges.
5(c)]. In most measurements, maximum PA/PT Raman signal amplitudes were located at the Raman shift range of 2800–3000 cm–1, which was likely associated with the CH2 vibrational spectra of lipids with a maximum at 2885 cm–1 (corresponding to a pump wavelength of 530–540 nm and a Stokes of 639 nm) present in most cells [30

30. J. De Gelder, K. De Gussem, P. Vandenabeele, and L. Moens, “References database of Raman spectra of biological molecules,” J. Raman Spectrosc. 38(9), 1133–1147 (2007). [CrossRef]

]. In the case of chylomicron phantoms, PA Raman contrast was higher in the NIR excitation range [Fig. 5(c)] compared to the visible range [Fig. 5(b)], which can be explained by the lower linear NIR absorption background. In the case of melanoma cells, we also observed weak signals at a Raman shift of 1580 cm–1. This finding was in line with available data showing that the Raman spectra of melanin demonstrate a strong similarity to those of amorphous carbon, dominated by two peaks centered at 1550 cm–1 (1590 cm–1 in melanin) and 1350 cm–1 (1418 cm–1 in melanin) [31

31. V. Capozzi, G. Perna, A. Gallone, P. F. Biagi, P. Carmone, A. Fratello, G. Guida, P. Zanna, and R. Cicero, “Raman and optical spectroscopy of eumelanin films,” J. Mol. Struct. 744-747, 717–721 (2005). [CrossRef]

]. Recent theoretical models suggest that melanin is composed of much smaller oligomers condensed into nano-aggregates [31

31. V. Capozzi, G. Perna, A. Gallone, P. F. Biagi, P. Carmone, A. Fratello, G. Guida, P. Zanna, and R. Cicero, “Raman and optical spectroscopy of eumelanin films,” J. Mol. Struct. 744-747, 717–721 (2005). [CrossRef]

].

PT Raman imaging of adipocytes in vivo on the animal model

Discussion

Features of the PARC/PTRC technique

Comparison with existing Raman technique

Most existing Raman techniques, especially CARS spectroscopy, have received much attention as microscopic methods with spectroscopic resolution (e.g., [28

28. C. Heinrich, A. Hofer, A. Ritsch, C. Ciardi, S. Bernet, and M. Ritsch-Marte, “Selective imaging of saturated and unsaturated lipids by wide-field CARS-microscopy,” Opt. Express 16(4), 2699–2708 (2008). [CrossRef] [PubMed]

,32

32. T. T. Le, T. B. Huff, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of lipids in cancer metastasis,” BMC Cancer 9(1), 42 (2009). [CrossRef] [PubMed]

,34

34. C. Heinrich, C. Meusburger, S. Bernet, and M. Ritsch-Marte, “CARS miscoscopy in a wide-field geometry with nanosecond pulses,” J. Raman Spectrosc. 37(6), 675–679 (2006). [CrossRef]

,35

35. X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy,” J. Lipid Res. 44(11), 2202–2208 (2003). [CrossRef] [PubMed]

] and references there). As has been pointed out since its first application [10

10. J. J. Barrett and M. J. Berry, “Photoacoustic Raman spectroscopy (PARS) using cw laser sources,” Appl. Phys. Lett. 34(2), 144–147 (1979). [CrossRef]

12

12. A. C. Tam, “Applications of photoacoustic sensing techniques,” Rev. Mod. Phys. 58(2), 381–431 (1986). [CrossRef]

], the PARS and, as we have demonstrated, the PARC/PTRC method have several potential advantages over other stimulated Raman techniques. CARS spectroscopy requires a very stable laser to accurately measure the intensity of the Stokes beam; in contrast, the PARC technique is free of this requirement because it directly monitors the small amount of heat deposited in the sample. Because the PARC/PTRC technique does not depend on the nonresonant susceptibilities, it is more suitable for cytometry applications. Thus, the fundamental difference between PARC and CARS is that in PARC the energy deposited in a sample is detected directly rather than, as in CARS, through a change in output light intensity. The experimental apparatus needed for the PARC technique is simple (much less sophisticated than that for conventional Raman-gain measurements). Our setup uses only a standard microscopy component. Unlike in CARS microscopy, laser intensity control is not essential because possible intensity fluctuations do not reduce the detection capability or image quality. In addition, at the nanosecond scale, temporal overlapping of the 1-m-long light pulses in the sample plane is easily achieved. Ultrashort (pico- or femtosecond) laser pulses with high peak power are needed for efficient signal generation in CARS spectroscopy. The strong intensity at the focal center induced with the use of such laser pulses may cause photodamage of biological tissues, which can be avoided with PARC using nanosecond pulses with a relatively low energy output. The noise in PARC arises primarily from thermal fluctuations, and these can be reduced by controlling macroscopic medium parameters, unlike in other Raman processes in which noise arises from microscopic quantum fluctuation, which is harder to control [12

12. A. C. Tam, “Applications of photoacoustic sensing techniques,” Rev. Mod. Phys. 58(2), 381–431 (1986). [CrossRef]

].

Potential applications

In general, PARC/PTRC techniques offer several advantages: (1) phase matching of the pump and Stokes beams is not required for PARC/PTRC as it is in CARS [28

28. C. Heinrich, A. Hofer, A. Ritsch, C. Ciardi, S. Bernet, and M. Ritsch-Marte, “Selective imaging of saturated and unsaturated lipids by wide-field CARS-microscopy,” Opt. Express 16(4), 2699–2708 (2008). [CrossRef] [PubMed]

,32

32. T. T. Le, T. B. Huff, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of lipids in cancer metastasis,” BMC Cancer 9(1), 42 (2009). [CrossRef] [PubMed]

,34

34. C. Heinrich, C. Meusburger, S. Bernet, and M. Ritsch-Marte, “CARS miscoscopy in a wide-field geometry with nanosecond pulses,” J. Raman Spectrosc. 37(6), 675–679 (2006). [CrossRef]

,35

35. X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy,” J. Lipid Res. 44(11), 2202–2208 (2003). [CrossRef] [PubMed]

]; (2) the wavelengths of the pump and Stokes beams can be in the NIR range, in which biological samples are relatively transparent; and (3) adjusting the differences between the two beams allows tuning to different vibrational modes that are not viable for direct laser excitation (i.e., linear mode). The biomedical applications of PARC/PTRC can be similar to those of CARS spectroscopy, but with better sensitivity and a simpler schematic. Applications can also include new, unique tasks. As is clear from the above analysis, Eq. (1), and our preliminary results, the PARC technique can provide (1) Raman scattering spectroscopy (by a change in the wavelength of at least one laser beam), including potential measurement of Raman frequency, cross-section, and gain; (2) detection of nonabsorbing or weakly absorbing structures in the visible, NIR, and even infrared ranges by selection of the difference (vp – vst) coinciding with the vibrational transition of cell components; and (3) enhancement of sensitivity for detecting cells by the use of both linear and nonlinear signal components. In the second application, linear signals introduce an undesired background that can be minimized by the selection of frequencies of the pump and Stokes lasers in a range with the lowest resonance absorption. This will increase the sensitivity of detecting weakly absorbing cells, such as adipocytes or chylomicrons, in the presence of low background signal from surrounding tissue. In the third application, however, in order to increase both sensitivity and specificity, the frequencies of the pump and Stokes waves can provide strong linear PA signals due to resonance absorption.

In analogy to CARS spectroscopy, targets for the PARC/PTFC technique can be biological samples with lipid-rich, Raman-active fatty acid molecules, which generate strong Raman signals at the CH2 vibrational mode near 2840–2860 cm–1. Potential applications include label-free detection and imaging of lipid-rich metastatic cancer cells with chemical selectivity, differentiation between various vegetable oils at the signature of the weak = C-H stretching vibration [28

28. C. Heinrich, A. Hofer, A. Ritsch, C. Ciardi, S. Bernet, and M. Ritsch-Marte, “Selective imaging of saturated and unsaturated lipids by wide-field CARS-microscopy,” Opt. Express 16(4), 2699–2708 (2008). [CrossRef] [PubMed]

], studies of adipogenesis and metabolic lipid disorder, mapping of the distribution of dense cellular proteins (by tuning of the Raman shift to the amide I vibration at 1649 cm–1), and identification of saturated and unsaturated fatty acids in cells [28

28. C. Heinrich, A. Hofer, A. Ritsch, C. Ciardi, S. Bernet, and M. Ritsch-Marte, “Selective imaging of saturated and unsaturated lipids by wide-field CARS-microscopy,” Opt. Express 16(4), 2699–2708 (2008). [CrossRef] [PubMed]

]. A relatively new application, as we demonstrated here, may include detection and identification of individual cells in vivo in static and, potentially, flow conditions in blood and especially in lymph flow in the presence of a strongly scattering or autofluorescent background where spontaneous Raman scattering cannot be easily performed. Targets may include chylomicrons, lipid droplets (e.g., in mature adipocytes), liposomes, and various nanoparticles with strong Raman signals [29

29. A. S. Biris, E. I. Galanzha, Z. Li, M. Mahmood, Y. Xu, and V. P. Zharov, “In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues,” J. Biomed. Opt. 14(2), 021006 (2009). [CrossRef] [PubMed]

], including SERS (surface-enhanced Raman scattering)-active nanostructures. In analogy to application of CARS spectroscopy for study of fat distribution in single cells [32

32. T. T. Le, T. B. Huff, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of lipids in cancer metastasis,” BMC Cancer 9(1), 42 (2009). [CrossRef] [PubMed]

], we believe that our technique can achieve similar results in vivo with the potential advantage of being able to use a low laser energy level that is safe for biotissues.

Further improvements

The results that we have described were obtained under the non-ideal arrangement. It should be possible to significantly improve the signal-to-noise ratio with a better detection technique that would make detecting and identifying individual lipid drops, chylomicrons, or liposomes quite possible. Molecular targeting would also be possible with the use of functionalized SERS-active nanoparticles.

Quantitative analysis of a cell population in the microscopic scheme is limited by the relatively small field of view of less than a few hundred micrometers, which necessitates time-consuming scanning. Therefore, to increase the speed of cell analysis, the PARC/PTRC technique can be combined with another technique that we developed, PA/PT flow cytometry [6

6. E. I. Galanzha, E. V. Shashkov, T. Kelly, J.-W. Kim, L. Yang, and V. P. Zharov, “In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells,” Nat. Nanotechnol. 4(12), 855–860 (2009). [CrossRef] [PubMed]

8

8. E. I. Galanzha, E. V. Shashkov, P. M. Spring, J. Y. Suen, and V. P. Zharov, “In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser,” Cancer Res. 69(20), 7926–7934 (2009). [CrossRef] [PubMed]

], which benefits both cell detection speed with chemically selective cell analysis in a quantitative manner. Use of a higher pulse repetition rate laser [8

8. E. I. Galanzha, E. V. Shashkov, P. M. Spring, J. Y. Suen, and V. P. Zharov, “In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser,” Cancer Res. 69(20), 7926–7934 (2009). [CrossRef] [PubMed]

] (up to 0.5 MHz) with a stable pulse energy should substantially improve the detection limit by permitting more precise subtraction of linear PT/PA signals. As seen from the signal-to-noise ratio values in Figs. 56, we should easily be able to detect single cells in circulation and probably single chylomicrons, after further improvements.

Acknowledgements

This work was supported in part by the National Institute of Health grant nos R01EB000873, R01CA131164, R01 EB009230, and R21CA139373, the National Science Foundation grant nos DBI-0852737 and the Arkansas Biosciences Institute. We would like to thank S. Fergusson for his assistance with laser measurements. We also thank the Office of Grants and Scientific Publications for editorial assistance in the preparation of the manuscript.

References and links

1.

V. P. Zharov, and V. S. Letokhov, Laser Optoacoustic Spectroscopy (Springer-Verlag; Berlin Heidelberg 1986).

2.

V. P. Zharov, Laser optoacoustic spectroscopy in chromatography: in Laser Analytical Spectrochemistry, V. S. Letokhov, ed. (Boston, 1986), pp. 229–271.

3.

M. Xu and L. V. Wang, “Photoacoustic imaging in biomedicine,” Rev. Sci. Instrum. 77(4), 041101 (2006). [CrossRef]

4.

L. V. Wang, ed., Photoacoustic imaging and spectroscopy (CRC Press, 2009).

5.

J. W. Kim, E. I. Galanzha, E. V. Shashkov, H. M. Moon, and V. P. Zharov, “Golden carbon nanotubes as multimodal photoacoustic and photothermal high-contrast molecular agents,” Nat. Nanotechnol. 4(10), 688–694 (2009). [CrossRef] [PubMed]

6.

E. I. Galanzha, E. V. Shashkov, T. Kelly, J.-W. Kim, L. Yang, and V. P. Zharov, “In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells,” Nat. Nanotechnol. 4(12), 855–860 (2009). [CrossRef] [PubMed]

7.

V. P. Zharov, E. I. Galanzha, E. V. Shashkov, J.-W. Kim, N. G. Khlebtsov, and V. V. Tuchin, “Photoacoustic flow cytometry: principle and application for real-time detection of circulating single nanoparticles, pathogens, and contrast dyes in vivo,” J. Biomed. Opt. 12(5), 051503 (2007). [CrossRef] [PubMed]

8.

E. I. Galanzha, E. V. Shashkov, P. M. Spring, J. Y. Suen, and V. P. Zharov, “In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser,” Cancer Res. 69(20), 7926–7934 (2009). [CrossRef] [PubMed]

9.

S. Nechaeiv and N. Ponomarev, “High-resolution Raman spectrometer,” Sov. J. Quantum Electron. 5, 72–76 (1975).

10.

J. J. Barrett and M. J. Berry, “Photoacoustic Raman spectroscopy (PARS) using cw laser sources,” Appl. Phys. Lett. 34(2), 144–147 (1979). [CrossRef]

11.

C. K. N. Patel and A. C. Tam, “Optoacoustic Raman gain spectroscopy of liquids,” Appl. Phys. Lett. 34(11), 760–763 (1979). [CrossRef]

12.

A. C. Tam, “Applications of photoacoustic sensing techniques,” Rev. Mod. Phys. 58(2), 381–431 (1986). [CrossRef]

13.

G. A. West, D. R. Siebert, and J. J. Barrett, “Gas phase photoacoustic Raman spectroscopy using pulsed laser excitation,” Appl. Phys. (Berl.) 51, 2823–2828 (1980). [CrossRef]

14.

J. J. Barrett and D. F. Heller, “Theoretical analysis of photoacoustic Raman spectroscopy,” J. Opt. Soc. Am. 71(11), 1299–1308 (1981). [CrossRef]

15.

C. K. N. Patel and A. C. Tam, “Pulsed optoacoutstic spectroscopy of condensed matter,” Rev. Mod. Phys. 53(3), 517–550 (1981). [CrossRef]

16.

A. M. Brodnikovskii, V.P. Zharov, and N.P. Koroteev, “Photoacoustic Raman spectroscopy of molecular gases,” Sov. J. Quantum Electron. •••, 2421–2430 (1985).

17.

Y. Oki, N. Kawada, T. Ogawa, Y. Abe, and M. Maeda, “Y. Abe and M. Maeda, “Sensitive H 2 detection using a new technique of photoacoustic Raman spectroscopy,” Jpn. J. Appl. Phys. 36(Part 2, No. 9A/B), L1172–L1174 (1997). [CrossRef]

18.

K. Das, Y. Rostovtsev, K. Lehmann, and M. Scully, “Thermodynamic and noise considerations for the detection of microscopic particles in a gas by photoacoustic Raman spectroscopy,” Opt. Commun. 246(4-6), 551–559 (2005). [CrossRef]

19.

Y. Oki, S. Nakazono, Y. Nonaka, and M. Maeda, “Sensitive H2 detection by use of thermal-lens Raman spectroscopy without a tunable laser,” Opt. Lett. 25(14), 1040–1042 (2000). [CrossRef]

20.

Y. Oki, N. Kawada, Y. Abe, and M. Maeda, “Nonlinear Raman spectroscopy without tunable laser for sensitive gas detection in the atmosphere,” Opt. Commun. 161(1-3), 57–62 (1999). [CrossRef]

21.

R. C. Sharma, “A novel demonstration of photoacoustic Raman spectroscopy with combined stimulated Raman pumping in H2 molecule,” Opt. Commun. 282(6), 1183–1185 (2009). [CrossRef]

22.

A. M. Sakashita, S. P. Bydlowski, D. A. F. Chamone, and R. C. Maranhão, “Plasma kinetics of an artificial emulsion resembling chylomicrons in patients with chronic lymphocytic leukemia,” Ann. Hematol. 79(12), 687–690 (2000). [CrossRef]

23.

Y. Park, W. J. Grellner, W. S. Harris, and J. M. Miles, “A new method for the study of chylomicron kinetics in vivo,” Am. J. Physiol. Endocrinol. Metab. 279(6), E1258–E1263 (2000). [PubMed]

24.

J.-W. Kim, E. V. Shashkov, E. I. Galanzha, N. Kotagiri, and V. P. Zharov, “Photothermal antimicrobial nanotherapy and nanodiagnostics with self-assembling carbon nanotube clusters,” Lasers Surg. Med. 39(7), 622–634 (2007). [CrossRef] [PubMed]

25.

E. I. Galanzha, V. V. Tuchin, and V. P. Zharov, “Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening,” World J. Gastroenterol. 13(2), 192–218 (2007). [PubMed]

26.

V. P. Zharov and D. O. Lapotko, “Photothermal imaging of nanoparticles and cells,” IEEE J. Sel. Top. Quantum Electron. 11(4), 733–751 (2005). [CrossRef]

27.

R. M. El-Abassy, P. Donfak, and A. Materny, “Visible Raman spectroscopy for the discrimination of olive oils from different vegetable oils and the detection of adulteration,” J. Raman Spectrosc. •••, 2279 (2009).

28.

C. Heinrich, A. Hofer, A. Ritsch, C. Ciardi, S. Bernet, and M. Ritsch-Marte, “Selective imaging of saturated and unsaturated lipids by wide-field CARS-microscopy,” Opt. Express 16(4), 2699–2708 (2008). [CrossRef] [PubMed]

29.

A. S. Biris, E. I. Galanzha, Z. Li, M. Mahmood, Y. Xu, and V. P. Zharov, “In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues,” J. Biomed. Opt. 14(2), 021006 (2009). [CrossRef] [PubMed]

30.

J. De Gelder, K. De Gussem, P. Vandenabeele, and L. Moens, “References database of Raman spectra of biological molecules,” J. Raman Spectrosc. 38(9), 1133–1147 (2007). [CrossRef]

31.

V. Capozzi, G. Perna, A. Gallone, P. F. Biagi, P. Carmone, A. Fratello, G. Guida, P. Zanna, and R. Cicero, “Raman and optical spectroscopy of eumelanin films,” J. Mol. Struct. 744-747, 717–721 (2005). [CrossRef]

32.

T. T. Le, T. B. Huff, and J. X. Cheng, “Coherent anti-Stokes Raman scattering imaging of lipids in cancer metastasis,” BMC Cancer 9(1), 42 (2009). [CrossRef] [PubMed]

33.

V. P. Zharov, V. Galitovskiy, C. S. Lyle, and T. C. Chambers, “Superhigh-sensitivity photothermal monitoring of individual cell response to antitumor drug,” J. Biomed. Opt. 11(6), 064034 (2006). [CrossRef]

34.

C. Heinrich, C. Meusburger, S. Bernet, and M. Ritsch-Marte, “CARS miscoscopy in a wide-field geometry with nanosecond pulses,” J. Raman Spectrosc. 37(6), 675–679 (2006). [CrossRef]

35.

X. Nan, J. X. Cheng, and X. S. Xie, “Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy,” J. Lipid Res. 44(11), 2202–2208 (2003). [CrossRef] [PubMed]

OCIS Codes
(170.1530) Medical optics and biotechnology : Cell analysis
(190.4380) Nonlinear optics : Nonlinear optics, four-wave mixing
(300.6230) Spectroscopy : Spectroscopy, coherent anti-Stokes Raman scattering
(350.4990) Other areas of optics : Particles
(350.5340) Other areas of optics : Photothermal effects

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: January 6, 2010
Revised Manuscript: February 23, 2010
Manuscript Accepted: March 10, 2010
Published: March 19, 2010

Virtual Issues
Vol. 5, Iss. 7 Virtual Journal for Biomedical Optics

Citation
Evgeny V. Shashkov, Ekaterina I. Galanzha, and Vladimir P. Zharov, "Photothermal and photoacoustic Raman cytometry in vitro and in vivo," Opt. Express 18, 6929-6944 (2010)
http://www.opticsinfobase.org/vjbo/abstract.cfm?URI=oe-18-7-6929


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References

  1. V. P. Zharov, and V. S. Letokhov, Laser Optoacoustic Spectroscopy (Springer-Verlag; Berlin Heidelberg 1986).
  2. V. P. Zharov, Laser optoacoustic spectroscopy in chromatography: in Laser Analytical Spectrochemistry, V. S. Letokhov, ed. (Boston, 1986), pp. 229–271.
  3. M. Xu and L. V. Wang, “Photoacoustic imaging in biomedicine,” Rev. Sci. Instrum. 77(4), 041101 (2006). [CrossRef]
  4. L. V. Wang, ed., Photoacoustic imaging and spectroscopy (CRC Press, 2009).
  5. J. W. Kim, E. I. Galanzha, E. V. Shashkov, H. M. Moon, and V. P. Zharov, “Golden carbon nanotubes as multimodal photoacoustic and photothermal high-contrast molecular agents,” Nat. Nanotechnol. 4(10), 688–694 (2009). [CrossRef] [PubMed]
  6. E. I. Galanzha, E. V. Shashkov, T. Kelly, J.-W. Kim, L. Yang, and V. P. Zharov, “In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells,” Nat. Nanotechnol. 4(12), 855–860 (2009). [CrossRef] [PubMed]
  7. V. P. Zharov, E. I. Galanzha, E. V. Shashkov, J.-W. Kim, N. G. Khlebtsov, and V. V. Tuchin, “Photoacoustic flow cytometry: principle and application for real-time detection of circulating single nanoparticles, pathogens, and contrast dyes in vivo,” J. Biomed. Opt. 12(5), 051503 (2007). [CrossRef] [PubMed]
  8. E. I. Galanzha, E. V. Shashkov, P. M. Spring, J. Y. Suen, and V. P. Zharov, “In vivo, noninvasive, label-free detection and eradication of circulating metastatic melanoma cells using two-color photoacoustic flow cytometry with a diode laser,” Cancer Res. 69(20), 7926–7934 (2009). [CrossRef] [PubMed]
  9. S. Nechaeiv and N. Ponomarev, “High-resolution Raman spectrometer,” Sov. J. Quantum Electron. 5, 72–76 (1975).
  10. J. J. Barrett and M. J. Berry, “Photoacoustic Raman spectroscopy (PARS) using cw laser sources,” Appl. Phys. Lett. 34(2), 144–147 (1979). [CrossRef]
  11. C. K. N. Patel and A. C. Tam, “Optoacoustic Raman gain spectroscopy of liquids,” Appl. Phys. Lett. 34(11), 760–763 (1979). [CrossRef]
  12. A. C. Tam, “Applications of photoacoustic sensing techniques,” Rev. Mod. Phys. 58(2), 381–431 (1986). [CrossRef]
  13. G. A. West, D. R. Siebert, and J. J. Barrett, “Gas phase photoacoustic Raman spectroscopy using pulsed laser excitation,” Appl. Phys. (Berl.) 51, 2823–2828 (1980). [CrossRef]
  14. J. J. Barrett and D. F. Heller, “Theoretical analysis of photoacoustic Raman spectroscopy,” J. Opt. Soc. Am. 71(11), 1299–1308 (1981). [CrossRef]
  15. C. K. N. Patel and A. C. Tam, “Pulsed optoacoutstic spectroscopy of condensed matter,” Rev. Mod. Phys. 53(3), 517–550 (1981). [CrossRef]
  16. A. M. Brodnikovskii, V.P. Zharov, and N.P. Koroteev, “Photoacoustic Raman spectroscopy of molecular gases,” Sov. J. Quantum Electron. •••, 2421–2430 (1985).
  17. Y. Oki, N. Kawada, T. Ogawa, Y. Abe, and M. Maeda, “Y. Abe and M. Maeda, “Sensitive H 2 detection using a new technique of photoacoustic Raman spectroscopy,” Jpn. J. Appl. Phys. 36(Part 2, No. 9A/B), L1172–L1174 (1997). [CrossRef]
  18. K. Das, Y. Rostovtsev, K. Lehmann, and M. Scully, “Thermodynamic and noise considerations for the detection of microscopic particles in a gas by photoacoustic Raman spectroscopy,” Opt. Commun. 246(4-6), 551–559 (2005). [CrossRef]
  19. Y. Oki, S. Nakazono, Y. Nonaka, and M. Maeda, “Sensitive H2 detection by use of thermal-lens Raman spectroscopy without a tunable laser,” Opt. Lett. 25(14), 1040–1042 (2000). [CrossRef]
  20. Y. Oki, N. Kawada, Y. Abe, and M. Maeda, “Nonlinear Raman spectroscopy without tunable laser for sensitive gas detection in the atmosphere,” Opt. Commun. 161(1-3), 57–62 (1999). [CrossRef]
  21. R. C. Sharma, “A novel demonstration of photoacoustic Raman spectroscopy with combined stimulated Raman pumping in H2 molecule,” Opt. Commun. 282(6), 1183–1185 (2009). [CrossRef]
  22. A. M. Sakashita, S. P. Bydlowski, D. A. F. Chamone, and R. C. Maranhão, “Plasma kinetics of an artificial emulsion resembling chylomicrons in patients with chronic lymphocytic leukemia,” Ann. Hematol. 79(12), 687–690 (2000). [CrossRef]
  23. Y. Park, W. J. Grellner, W. S. Harris, and J. M. Miles, “A new method for the study of chylomicron kinetics in vivo,” Am. J. Physiol. Endocrinol. Metab. 279(6), E1258–E1263 (2000). [PubMed]
  24. J.-W. Kim, E. V. Shashkov, E. I. Galanzha, N. Kotagiri, and V. P. Zharov, “Photothermal antimicrobial nanotherapy and nanodiagnostics with self-assembling carbon nanotube clusters,” Lasers Surg. Med. 39(7), 622–634 (2007). [CrossRef] [PubMed]
  25. E. I. Galanzha, V. V. Tuchin, and V. P. Zharov, “Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening,” World J. Gastroenterol. 13(2), 192–218 (2007). [PubMed]
  26. V. P. Zharov and D. O. Lapotko, “Photothermal imaging of nanoparticles and cells,” IEEE J. Sel. Top. Quantum Electron. 11(4), 733–751 (2005). [CrossRef]
  27. R. M. El-Abassy, P. Donfak, and A. Materny, “Visible Raman spectroscopy for the discrimination of olive oils from different vegetable oils and the detection of adulteration,” J. Raman Spectrosc. •••, 2279 (2009).
  28. C. Heinrich, A. Hofer, A. Ritsch, C. Ciardi, S. Bernet, and M. Ritsch-Marte, “Selective imaging of saturated and unsaturated lipids by wide-field CARS-microscopy,” Opt. Express 16(4), 2699–2708 (2008). [CrossRef] [PubMed]
  29. A. S. Biris, E. I. Galanzha, Z. Li, M. Mahmood, Y. Xu, and V. P. Zharov, “In vivo Raman flow cytometry for real-time detection of carbon nanotube kinetics in lymph, blood, and tissues,” J. Biomed. Opt. 14(2), 021006 (2009). [CrossRef] [PubMed]
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  31. V. Capozzi, G. Perna, A. Gallone, P. F. Biagi, P. Carmone, A. Fratello, G. Guida, P. Zanna, and R. Cicero, “Raman and optical spectroscopy of eumelanin films,” J. Mol. Struct. 744-747, 717–721 (2005). [CrossRef]
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