Multiphoton excitation of exogenous dyes and endogenous biochemical species has been used extensively for tissue
diagnosis by fluorescence spectroscopy. Unfortunately, the majority of endogenous biochemical chromophores have
low quantum yields, less than 0.2, therefore determining two-photon cross sections of weakly luminescencing molecules
is difficult using two-photon fluorescence spectroscopy. Accurate determination of two-photon cross sections of these
biochemicals could provide insight into fluorescence signal reduction caused by the absorption of excitation energy by
non-target intracellular species.
Non-resonant multiphoton photoacoustic spectroscopy (NMPPAS) is a novel technique we have developed for
condensed matter measurements that has the potential for accurately determining two-photon absorption cross-sections
of chemicals with small or non-existant fluorescence quantum yields. In this technique, near infrared light is used to
generate an ultrasonic signal following a non-resonant two-photon excitation process. This ultrasonic wave is directly
related to the non-radative relaxation of the chromophore of interest and is measured using a contact piezoelectric
ultrasonic transducer. The signal from the ultrasonic transducer can then be used to calculate two-photon absorption
cross sections. This paper will describe the validation of this technique by measuring the two-photon absorption cross-
sections of well characterized chromophores such as rhodamine B and coumarin 1 in solution as well as riboflavin in a
gelatin tissue phantom.
Brain cancer affects approximately 16,500 people a year and individuals diagnosed with glioblastoma multiforme have an average life expectancy of less than 12-18 months after diagnosis. A portable fiber-optic probe capable of distinguishing between healthy and tumor tissues, with a high degree of spatial resolution, deep within a sample would be a valuable tool for tumor diagnosis and margining. A novel technique that combines 1-2 cm penetration depths with cellular level spatial resolution to chemically distinguish cancerous from non-cancerous tissues is non-resonant multiphoton photoacoustic spectroscopy (NMPPAS). This technique focuses pulsed near infrared light into a sample, creating a two-photon excitation event, and measures the resulting non-radiative decay as an ultrasonic signal. This paper discusses the optimization of a portable fiber-optic NMPPAS probe capable of delivering nanosecond laser pulses from 740nm-1100nm to a series of lens, which focus the light into the sample. The resulting ultrasonic signal is measured using a polyvinylidene fluoride based piezoelectric detector. The two-photon excitation efficiency of the portable NMPPAS probe system has been evaluated by measuring the two-photon excitation and emission spectra of common fluorescent dyes such as rhodamine B and fluorescein. In addition, this paper also demonstrates the diagnostic potential of this technique for tumor detection and margining without the need for acquisition of an entire spectrum.
In this paper we describe the development of a novel fiber optic probe for subsurface tumor diagnostics, based on non-resonant multiphoton photoacoustic spectroscopy (NMPPAS). In this technique, endogenous biomarkers present in tissues are irradiated in the near infrared, using a tunable high-power laser. The resulting multiphoton excitation events are detected as an acoustic (i.e. ultrasonic) signal, using an ultrasonic piezoelectric transducer. The signal from the piezoelectric transducer is then corrected for laser power fluctuations by normalizing the NMPPAS signal at each wavelength with the laser intensity recorded, from an optical diode. By scanning the laser excitation over the appropriate wavelength range for the tissue of interest, absorption differences between normal and tumor tissues can be measured and analyzed. The fiber optic probe was characterized and optimized for transmission efficiency as well as its time dependent response to high power laser pulses. The focusing optics were optimized and a piezoelectric transducer film detector chosen based on its sensitivity in the ultrasonic frequency range of interest. Using this probe system NMPPAS measurements were performed on several common fluorescent dyes including rhodamine 6G as well as well-characterized biomarkers like tryptophan. Furthermore, the technique was further successfully applied to the differentiation of tumorous and healthy human brain tissues.
We have developed a surface enhanced Raman scattering (SERS) based nanoimaging probe capable of chemical imaging with nanometer scale spatial resolution. Using this SERS-nanoimaging probe it is possible to image individual chemical components within sub-cellular environments. The probe consists of a tapered coherent fiber optic imaging bundle that has been coated with a roughened layer of metal, providing a SERS active substrate. The fiber optic bundle is tapered using a specially programmed micropipette puller, allowing precise control over the probe tip's diameter, and thus the resolution of images. Tapered bundles having individual fiber elements ranging from 100-800 nanometers on the tapered end and 4 micrometers in diameter on the proximal end have been investigated. Through modification of the fibers' tapered tips, generation of nanoscale imaging with inherent image magnification and short pass filtering effects is possible. Following tapering of the fiber optic bundles, the fiber probes are spin-coated with alumina particles and coated with silver to provide a reproducible SERS active surface. Characterization of the response of these SERS nanoimaging probes has been evaluated using common SERS active chemical species (e.g., benzoic acid, brilliant cresyl blue, etc.) and application of these nanoimaging sensors to biological systems is discussed.
In this paper we describe the development of a novel non-invasive multispectral imaging technique, multiphoton photoacoustic spectroscopy (MPPAS). In this technique, a tunable high-power laser is used to perform multiphoton excitation events, which are then detected as an acoustic (i.e. ultrasonic) signal, using a commercial ultrasonic piezoelectric (PZT) transducer. The signal from the PZT is then corrected for laser power fluctuations, resulting in normalized MPPAS signal intensity. Because MPPAS relies on non-radiative relaxation of the absorbing species, unlike single or multiphoton fluorescence spectroscopies, it is capable of monitoring non-fluorescent species. In addition, since the majority of the energy imparted to most molecules upon the absorption of light is released through non-radiative pathways, sensitive measurements of even fluorescent molecules, can be performed. A test solution of aqueous rhodamine 6G was used to obtain the MPPAS spectrum. It matches well with that of the steady state absorbance of the same solution. In addition, concentration dependent studies of rhodamine 6G have shown that the technique is even sensitive to nanomolar concentrations and below.