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
]). 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
]. 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
]. 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
]. The technique was first employed in 1979 in gas by Barrett and Berry [10
] using two continuous-wave (CW) or nanosecond pulse lasers. Nonbiological liquids were studied by Patel and Tam using microsecond laser pulses [11
]. Later application of PARS was focused mainly on gas analysis [e.g., 13
]. 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
]. 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 viv
o. The advantages, limitations, and further improvement of this new optical technology are also discussed.