Photoacoustic spectroscopy is a powerful optical biopsy technique that enables rapid tumor diagnosis in situ. It
has also been reported that photoacoustic spectroscopy can be used to diagnose pre-malignant tissue based on the
chemical differences between healthy and pre-malignant tissues. Since the acoustic signals obtained from tissues in these
analyses suffer from minimum damping, photoacoustic spectroscopy can be highly sensitive. This paper focuses on the
characterization of a novel multiphoton excited photoacoustic methodology for margining of malignant and pre-malignant
The two-photon excitation process in tissues using nanosecond laser pulses produces ultrasonic signals that
transmit through tissue with minimal attenuation. Additionally, the two-photon excitation process is highly localized
since only ballistic photons contribute to the excitation process; thereby eliminating potential absorption events in tissue
not of interest (i.e., along the beam path) and increasing the spatial resolution of the diagnostic technique to that
achievable via optics. This work characterizes the two-photon excitation process for photoacoustic signal measurements
on a model dye. Using gelatin phantoms to mimic real tissues, tissue penetration studies were performed, revealing
chemical species as deep as 1.3 cm in the tissue can easily be detected using this methodology. Furthermore, the
resolution of this multiphoton excitation process was determined to be as great as 50 μm (near cellular level resolution).
Due to the narrow vibrational bandwidths and unique molecular fingerprints, Raman spectroscopy can be an
information rich transduction technique for chemical imaging. Dynamic systems are often difficult to measure using
spontaneous Raman due to the relatively weak scattering cross-sections. Using a Raman enhancement mechanism such
as surface enhanced Raman scattering (SERS), exposure times can be reduced to a reasonable level for dynamic
imaging, due to the increased Raman signal intensity.
This paper will discuss the development of a novel SERS substrate, fabricated on the tips of fiber-optic imaging
bundles, which can be integrated into a multispectral imaging system for non-scanning chemical imaging. These
substrates are fabricated by mechanically tapering a polished fiber optic imaging bundle consisting of 30,000 individual
elements; producing 100-nm or smaller diameter core elements on the distal tip. Chemical etching with hydrofluoric
acid creates uniform cladding spikes onto which a SERS active metal is vacuum deposited, forming the SERS active
surface. By varying the size of the silver islands deposited on the cladding peaks active, surface plasmons can be tuned
to various excitation frequencies. The surface of these tapered fiber optic probes will be evaluated by analysis of the
SERS signal, location and shape of the active surface plasmons. The cross talk between the fiber elements will also be
Chemical imaging not only provides structural and spatial information about a sample but also chemical information
about the sample. Raman spectroscopy can be a powerful transduction mechanism for chemical imaging due to the
narrow vibrational bandwidths and unique spectral fingerprints. Unfortunately, Raman cross-sections are extremely
weak (~10-30 cm-2), often necessitating long exposure times, making dynamic chemical imaging impractical, particularly
for high-resolution images. By utilizing a Raman enhancement technique such as surface enhanced Raman
spectroscopy (SERS), the effective scattering cross-sections are increased making practical imaging times feasible.
This paper will discuss the fabrication, characterization, and demonstration of a novel SERS substrate and instrumental
system for non-scanning SERS chemical imaging with sub-diffraction limited spatial resolution. These substrates are
fabricated by chemically etching a polished fiber optic imaging bundle consisting of 30,000, hexagonally packed, 4-
micron diameter elements. The chemical etching process creates uniform array of cladding spikes onto which a SERS
active metal is vacuum deposited, forming the SERS active surface. By varying the size of the silver islands deposited
on the cladding peaks active surface plasmons can be tuned to various excitation frequencies. SERS signals measured
both on and off the plasmon absorption band demonstrate that these SERS fiber bundles can be tuned for various
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.