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In JoVE (1)
Other Publications (22)
- Lasers in Surgery and Medicine
- Physics in Medicine and Biology
- Applied Optics
- Lasers in Surgery and Medicine
- The Journal of Investigative Dermatology
- Journal of Biomedical Optics
- Lasers in Surgery and Medicine
- Journal of Biomedical Optics
- Lasers in Surgery and Medicine
- The Journal of Investigative Dermatology. Symposium Proceedings / the Society for Investigative Dermatology, Inc. [and] European Society for Dermatological Research
- Optics Letters
- Physics in Medicine and Biology
- Physics in Medicine and Biology
- Optics Express
- Journal of Biomechanical Engineering
- Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
- Journal of Biomedical Optics
- Lasers in Surgery and Medicine
- Annals of Biomedical Engineering
- Journal of Biomedical Nanotechnology
- Pharmaceutical Research
- Lasers in Surgery and Medicine
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Articles by John A. Viator in JoVE
JoVE Bioengineering
كشف وعزل خلايا الميلانوما تعميم استخدام Flowmetry الضوئي
وضعنا عداد الكريات تدفق باستخدام الموجات فوق الصوتية الليزر التي يسببها للكشف عن تعميم خلايا سرطان الجلد كمؤشر مبكر من المرض المنتشر.
Other articles by John A. Viator on PubMed
Clinical Testing of a Photoacoustic Probe for Port Wine Stain Depth Determination
Successful laser treatment of port wine stain (PWS) birthmarks requires knowledge of lesion geometry. Laser parameters, such as pulse duration, wavelength, and radiant exposure, and other treatment parameters, such as cryogen spurt duration, need to be optimized according to epidermal melanin content and lesion depth. We designed, constructed, and clinically tested a photoacoustic probe for PWS depth determination.
Spectra from 2.5-15 Microm of Tissue Phantom Materials, Optical Clearing Agents and Ex Vivo Human Skin: Implications for Depth Profiling of Human Skin
Infrared measurements have been used to profile or image biological tissue, including human skin. Usually, analysis of such measurements has assumed that infrared absorption is due to water and collagen. Such an assumption may be reasonable for soft tissue, but introduction of exogenous agents into skin or the measurement of tissue phantoms has raised the question of their infrared absorption spectrum. We used Fourier transform infrared spectroscopy in attenuated total reflection mode to measure the infrared absorption spectra, in the range of 2-15 microm, of water, polyacrylamide, Intralipid, collagen gels, four hyperosmotic clearing agents (glycerol, 1,3-butylene glycol, trimethylolpropane, Topicare), and ex vivo human stratum corneum and dermis. The absorption spectra of the phantom materials were similar to that of water, although additional structure was noted in the range of 6-10 microm. The absorption spectra of the clearing agents were more complex, with molecular absorption bands dominating between 6 and 12 microm. Dermis was similar to water, with collagen structure evident in the 6-10 microm range. Stratum corneum had a significantly lower absorption than dermis due to a lower content of water. These results suggest that the assumption of water-dominated absorption in the 2.5-6 microm range is valid. At longer wavelengths, clearing agent absorption spectra differ significantly from the water spectrum. This spectral information can be used in pulsed photothermal radiometry or utilized in the interpretation of reconstructions in which a constant mu(ir) is used. In such cases, overestimating mu(ir) will underestimate chromophore depth and vice versa, although the effect is dependent on actual chromophore depth.
In Vivo Port-wine Stain Depth Determination with a Photoacoustic Probe
We have designed a photoacoustic probe for port-wine stain (PWS) depth measurements consisting of optical fibers for laser light delivery and a piezoelectric element for acoustic detection. We characterized the capabilities and limitations of the probe for profiling PWS skin. The probe induced and measured photoacoustic waves in acrylamide tissue phantoms and PWS skin in vivo. The optical properties of the phantoms were chosen to mimic those of PWS skin. We denoised acoustic waves using spline wavelet transforms, then deconvolved with the impulse response of the probe to yield initial subsurface pressure distributions in phantoms and PWS skin. Using the phantoms, we determined that the limit in resolving epidermal and PWS layers was less than 70 microm. In addition, we used the phantoms to determine that the maximum epidermal melanin concentration that allowed detection of PWS was between 13 and 20%. In vivo measurements of PWS skin with different epidermal melanin concentrations correlated with the phantoms. Thus the photoacoustic probe can be used to determine PWS depth for most patients receiving laser therapy.
Increase of Dermal Blood Volume Fraction Reduces the Threshold for Laser-induced Purpura: Implications for Port Wine Stain Laser Treatment
The average success rate in achieving total blanching of port wine stain (PWS) lesions treated with laser-induced selective photothermolysis is below 25%, even after multiple treatments. This is because smaller diameter (5-20 microm) PWS blood vessels are difficult to destroy with selective photothermolysis since the volumetric heat generated by absorption of laser light is insufficient to adequately heat the entire vessel wall. The aim of this study was to investigate a potential technique for more efficient photocoagulation of small diameter PWS blood vessels in PWS that respond poorly to selective photothermolysis.
A Comparative Study of Photoacoustic and Reflectance Methods for Determination of Epidermal Melanin Content
Although epidermal melanin content has been quantified non-invasively using visible reflectance spectroscopy (VRS), there is currently no way to determine melanin distribution in the epidermis. We have developed a photoacoustic probe that uses a Q-switched, frequency-doubled Nd:YAG (neodymium, yttrium, aluminum, garnet) laser operating at 532 nm to generate acoustic pulses in skin in vivo. The probe contained a piezoelectric element that detected photoacoustic waves that were then analyzed for epidermal melanin content using a photoacoustic melanin index (PAMI). Melanin content was compared between results of photoacoustics and VRS. Spectra from human skin were fitted to a model based on diffusion theory that included parameters for epidermal thickness, melanin content, hair color and density, and dermal blood content. Ten human subjects with skin phototypes I-VI were tested using the photoacoustic probe and VRS. A plot of PAMI v. VRS showed a good linear fit with r2=0.85. Photoacoustic and VRS measurements are shown for a human subject with vitiligo, indicating that melanin was almost completely absent. We present preliminary modeling for photoacoustic probe design and analysis necessary for depth profiling of epidermal melanin.
Accurate Measurement of Blood Vessel Depth in Port Wine Stained Human Skin in Vivo Using Pulsed Photothermal Radiometry
We report on application of pulsed photothermal radiometry (PPTR) to determine the depth of port wine stain (PWS) blood vessels in human skin. When blood vessels are deep in the PWS skin (>100 microm), conventional PPTR depth profiling can be used to determine PWS depth with sufficient accuracy. When blood vessels are close or partially overlap the epidermal melanin layer, a modified PPTR technique using two-wavelength (585 and 600 nm) excitation is a superior method to determine PWS depth. A direct difference approach in which PWS depth is determined from a weighted difference of temperature profiles reconstructed independently from two-wavelength excitation is demonstrated to be appropriate for a wider range of PWS patients with various blood volume fractions, blood vessel sizes, and depth distribution. The most superficial PWS depths determined in vivo by PPTR are in good agreement with those measured using optical Doppler tomography (ODT).
Influence of Laser Wavelength and Pulse Duration on Gas Bubble Formation in Blood Filled Glass Capillaries
Hypervascular skin lesions (HVSL) are treated with medical lasers characterized by a variety of parameters such as wavelength lambda, pulse duration t(p), and radiant exposure E that can be adjusted for different pathology and blood vessel size. Treatment parameters have been optimized assuming constant optical properties of blood during laser photocoagulation. However, recent studies suggest that this assumption may not always be true. Our objective was to quantify thermally induced changes in blood that occur during irradiation using standard laser parameters.
Determination of Human Skin Optical Properties from Spectrophotometric Measurements Based on Optimization by Genetic Algorithms
We present an initial study on applying genetic algorithms (GA) to retrieve human skin optical properties using visual reflectance spectroscopy (VRS). A three-layered skin model consisting of 13 parameters is first used to simulate skin and, through an analytical model based on optical diffusion theory, we study their independent effects on the reflectance spectra. Based on a preliminary analysis, nine skin parameters are chosen to be fitted by GA. The fitting procedure is applied first on simulated reflectance spectra with added white noise, and then on measured spectra from normal and port wine stain (PWS) human skin. A normalized residue of less than 0.005 is achieved for simulated spectra. In the case of measured spectra from human skin, the normalized residue is less than 0.01. Comparisons between applying GA and manual iteration (MI) fitting show that GA performed much better than the MI fitting method and can easily distinguish melanin concentrations for different skin types. Furthermore, the GA approach can lead to a reasonable understanding of the blood volume fraction and other skin properties, provided that the applicability of the diffusion approximation is satisfied.
Relationship Between Damaged Fraction and Reflected Spectra of Denaturing Tissues
During thermal therapy of tissue, such as induced by microwave heating, the initiation of denaturation should be monitored for proper thermal dosage. Additionally, denaturation should be confined to the pathologic volume, while preserving surrounding healthy tissue. The relationship between the damaged fraction and reflected spectra of denaturing tissues was investigated for a variation of the temperature of the tissues.
Imaging Melanoma in a Murine Model Using Reflectance-mode Confocal Scanning Laser Microscopy and Polarized Light Imaging
The light-scattering properties of cutaneous tissues provide optical contrast for imaging the presence and depth of pigmented melanoma in a highly pigmented murine model, the C57/B6 mouse. Early lesions are difficult to identify when viewing black lesions on a black mouse. Two methods were used to image early lesions in this model. (1) A reflectance-mode confocal scanning laser microscope (rCSLM) was built to provide horizontal images (x-y at depth z) and transverse images (x-z at position y) non-invasively in the living mouse. (2) A polarized light imaging (PLI) camera was built using a linearly polarized white light source that viewed the skin through an analyzing linear polarizer oriented either parallel or perpendicular to the illumination's polarization to yield two images, "PAR" and "PER," respectively. The difference image, PAR-PER, eliminated multiply scattered light and yielded an image of the superficial but subsurface tissues based only on photons scattered once or a few times so as to retain their polarization. rCSLM could image melanoma lesions developing below the epidermis. PLI could distinguish superficial from deeper melanoma lesions because the melanin of the superficial lesions attenuated the PAR-PER image, whereas deeper lesions failed to attenuate the PAR-PER image.
Photoacoustic Detection of Metastatic Melanoma Cells in the Human Circulatory System
Detection of disseminating tumor cells among patients suffering from various types and stages of cancer can function as an early warning system, alerting the physician of the metastatic spread or recurrence of the disease. Early detection of such cells can result in preventative treatment of the disease, while late stage detection can serve as an indicator of the effectiveness of chemotherapeutics. The prognostic value of exposing disseminating tumor cells poses an urgent need for an efficient, accurate screening method for metastatic cells. We propose a system for the detection of metastatic circulating tumor cells based on the thermoelastic properties of melanoma. The method employs photoacoustic excitation coupled with a detection system capable of determining the presence of disseminating cells within the circulatory system in vitro. Detection trials consisting of tissue phantoms and a human melanoma cell line resulted in a detection threshold of the order of ten individual cells, thus validating the effectiveness of the proposed mechanism. Results imply the potential to assay simple blood draws, from healthy and metastatic patients, for the presence of cancerous melanoma providing an unprecedented method for routine cancer screening.
Photoacoustic Discrimination of Viable and Thermally Coagulated Blood Using a Two-wavelength Method for Burn Injury Monitoring
Discriminating viable from thermally coagulated blood in a burn wound can be used to profile burn depth, thus aiding the removal of necrotic tissue. In this study, we used a two-wavelength photoacoustic imaging method to discriminate coagulated and non-coagulated blood in a dermal burn phantom. Differences in the optical absorption spectra of coagulated and non-coagulated blood produce different values of the ratio of peak photoacoustic amplitude at 543 and 633 nm. The absorption values obtained from spectroscopic measurements indicate that the ratio of photoacoustic pressure for 543 and 633 nm for non-coagulated blood was 15.7:1 and 1.6:1 for coagulated blood. Using planar blood layers, we found the photoacoustic ratios to be 13.5:1 and 1.6:1, respectively. Using the differences in the ratios of coagulated and non-coagulated blood, we propose a scheme using statistical classification analysis to identify the different blood samples. Based upon these distinctly different ratios, we identified the planar blood samples with an error rate of 0%. Using a burn phantom with cylindrical vessels containing coagulated and non-coagulated blood, we achieved an error rate of 11.4%. These results have shown that photoacoustic imaging could prove to be a valuable tool in the diagnosis of burns.
Automated Wavelet Denoising of Photoacoustic Signals for Circulating Melanoma Cell Detection and Burn Image Reconstruction
Photoacoustic image reconstruction may involve hundreds of point measurements, each of which contributes unique information about the subsurface absorbing structures under study. For backprojection imaging, two or more point measurements of photoacoustic waves induced by irradiating a biological sample with laser light are used to produce an image of the acoustic source. Each of these measurements must undergo some signal processing, such as denoising or system deconvolution. In order to process the numerous signals, we have developed an automated wavelet algorithm for denoising signals. We appeal to the discrete wavelet transform for denoising photoacoustic signals generated in a dilute melanoma cell suspension and in thermally coagulated blood. We used 5, 9, 45 and 270 melanoma cells in the laser beam path as test concentrations. For the burn phantom, we used coagulated blood in 1.6 mm silicon tube submerged in Intralipid. Although these two targets were chosen as typical applications for photoacoustic detection and imaging, they are of independent interest. The denoising employs level-independent universal thresholding. In order to accommodate nonradix-2 signals, we considered a maximal overlap discrete wavelet transform (MODWT). For the lower melanoma cell concentrations, as the signal-to-noise ratio approached 1, denoising allowed better peak finding. For coagulated blood, the signals were denoised to yield a clean photoacoustic resulting in an improvement of 22% in the reconstructed image. The entire signal processing technique was automated so that minimal user intervention was needed to reconstruct the images. Such an algorithm may be used for image reconstruction and signal extraction for applications such as burn depth imaging, depth profiling of vascular lesions in skin and the detection of single cancer cells in blood samples.
PDMS Embedded Opto-fluidic Microring Resonator Lasers
Opto-fluidic ring resonator (OFRR) dye lasers are embedded in low index polydimethylsiloxane (PDMS) to achieve enhanced portability, mechanical stability, and potential integration with conventional soft lithography based microfluidics for development of micro total analysis systems. The OFRR retains high Q-factors (> 10(6)) and exhibits low lasing threshold (<1 microJ/mm(2)). Fiber prisms and tapered optical fibers are used to directionally couple out the laser emission. At 2.2 microJ/mm(2) pump intensity, the laser output from the fiber prism is 80 nW, corresponding to 50% power extraction efficiency. A microarray structure of parallel OFRRs is also demonstrated, allowing simultaneous multi-color emissions.
Photoacoustic Detection of Melanoma Micrometastasis in Sentinel Lymph Nodes
Melanoma is the deadliest form of skin cancer and has the fastest growth rate of all cancer types. Proper staging of melanoma is required for clinical management. One method of staging melanoma is performed by taking a sentinel node biopsy, in which the first node in the lymphatic drainage path of the primary lesion is removed and tested for the presence of melanoma cells. Current standard of care typically involves taking fewer than ten histologic sections of the node out of the hundreds of possible sections available in the tissue. We have developed a photoacoustic method that probes the entire intact node. We acquired a lymph node from a healthy canine subject. We cultured a malignant human melanoma cell line HS 936. Approximately 1 x 10(6) cells were separated and injected into the lymph node. We also had a healthy lymph node in which no melanoma cells were implanted. We used a tunable laser system set at 532 nm to irradiate the lymph nodes. Three piezoelectric acoustic detectors were positioned near the lymph node to detect photoacoustic pulses generated within the lymph nodes. We also acquired lymph nodes from pigs and repeated the experiments with increased amplification and improved sensors. We detected photoacoustic responses from a lymph node with as few as 500 melanoma cells injected into the tissue, while normal lymph nodes showed no response. Photoacoustic generation can be used to detect melanoma micrometastasis in sentinel lymph nodes. This detection can be used to guide further histologic study of the node, increasing the accuracy of the sentinel lymph node biopsy.
Detection of Circulating Melanoma Cells in Human Blood Using Photoacoustic Flowmetry
Detection of circulating tumor cells (CTC's) in human blood and lymph systems has the potential to aid clinical decision making in the treatment of cancer. The presence of CTC's may signify the onset of metastasis, indicate relapse, or may be used to monitor disease progression. A photoacoustic flowmetry system was designed and tested for detecting circulating melanoma cells (CMC's) by exploiting the broadband absorption spectrum of melanin within CMC's. The device was tested on cultured melanoma cells in saline suspension and in a Stage IV melanoma patient. The device showed a detection threshold of a single melanotic melanoma cell from culture. Transient photoacoustic events were detected in a sample derived from a Stage IV melanoma patient that corresponded to particles passing through the laser beam path, indicating the presence of single melanoma cells in the human circulatory system.
Photoacoustic Discrimination of Vascular and Pigmented Lesions Using Classical and Bayesian Methods
Discrimination of pigmented and vascular lesions in skin can be difficult due to factors such as size, subungual location, and the nature of lesions containing both melanin and vascularity. Misdiagnosis may lead to precancerous or cancerous lesions not receiving proper medical care. To aid in the rapid and accurate diagnosis of such pathologies, we develop a photoacoustic system to determine the nature of skin lesions in vivo. By irradiating skin with two laser wavelengths, 422 and 530 nm, we induce photoacoustic responses, and the relative response at these two wavelengths indicates whether the lesion is pigmented or vascular. This response is due to the distinct absorption spectrum of melanin and hemoglobin. In particular, pigmented lesions have ratios of photoacoustic amplitudes of approximately 1.4 to 1 at the two wavelengths, while vascular lesions have ratios of about 4.0 to 1. Furthermore, we consider two statistical methods for conducting classification of lesions: standard multivariate analysis classification techniques and a Bayesian-model-based approach. We study 15 human subjects with eight vascular and seven pigmented lesions. Using the classical method, we achieve a perfect classification rate, while the Bayesian approach has an error rate of 20%.
Detection of Melanoma Cells in Vitro Using an Optical Detector of Photoacoustic Waves
Circulating tumor cells have been shown to correlate positively with metastatic disease state in patients with advanced cancer. We have demonstrated the ability to detect melanoma cells in a flow system by generating and detecting photoacoustic waves in melanoma cells. This method is similar to flow cytometry, although using photoacoustics rather than fluorescence. Previously, we used piezoelectric films as our acoustic sensors. However, such films have indicated false-positive signals due to unwanted direct interactions between photons from the high laser fluence in the flow system and the film itself. We have adapted an optical detection scheme that obviates the need for piezoelectric films.
Plasma Membrane Integrity and Survival of Melanoma Cells After Nanosecond Laser Pulses
Circulating tumor cells (CTCs) photoacoustic detection systems can aid clinical decision-making in the treatment of cancer. Interaction of melanin within melanoma cells with nanosecond laser pulses generates photoacoustic waves that make its detection possible. This study aims at: (1) determining melanoma cell survival after laser pulses of 6 ns at λ = 355 and 532 nm; (2) comparing the potential enhancement in the photoacoustic signal using λ = 355 nm in contrast with λ = 532 nm; (3) determining the critical laser fluence at which melanin begins to leak out from melanoma cells; and (4) developing a time-resolved imaging (TRI) system to study the intracellular interactions and their effect on the plasma membrane integrity. Monolayers of melanoma cells were grown on tissue culture-treated clusters and irradiated with up to 1.0 J/cm². Surviving cells were stained with trypan blue and counted using a hemacytometer. The phosphate buffered saline absorbance was measured with a nanodrop spectrophotometer to detect melanin leakage from the melanoma cells post-laser irradiation. Photoacoustic signal magnitude was studied at both wavelengths using piezoelectric sensors. TRI with 6 ns resolution was used to image plasma membrane damage. Cell survival decreased proportionally with increasing laser fluence for both wavelengths, although the decrease is more pronounced for 355 nm radiation than for 532 nm. It was found that melanin leaks from cells equally for both wavelengths. No significant difference in photoacoustic signal was found between wavelengths. TRI showed clear damage to plasma membrane due to laser-induced bubble formation.
Gold Nanoparticle Mediated Detection of Prostate Cancer Cells Using Photoacoustic Flowmetry with Optical Reflectance
The presence of circulating tumor cells in the bloodstream has been correlated with disease state in cancer patients. While we have successfully exploited melanin, the natural light absorber in melanoma cells, to induce photoacoustic waves for tumor cell detection, non-pigmented tumor cells do not have sufficient optical contrast for such a method. For example, breast, prostate and lung cancers lack intrinsic pigmentation and thus do not generate photoacoustic waves. In order to induce optical contrast in non-pigmented cancer cells, we have attached gold nanoparticles to a prostate cancer cell line. This optical absorption will enable us to detect such cells in a photoacoustic flowmeter designed to find circulating tumor cells in blood samples. We tested a prostate cancer cell line, PC-3, by tagging them with gold nanoparticles. We determined the photoacoustic response over the wavelengths 470-570 nm to identify the absorption peak. We then determined the response from serial dilutions of PC-3 cells suspended in saline. Finally, we showed photoacoustic response from PC-3 cells suspended among white blood cells in the flow meter to demonstrate our ability to detect single cells under flow.
An Effective Strategy for the Synthesis of Biocompatible Gold Nanoparticles Using Cinnamon Phytochemicals for Phantom CT Imaging and Photoacoustic Detection of Cancerous Cells
The purpose of the present study was to explore the utilization of cinnamon-coated gold nanoparticles (Cin-AuNPs) as CT/optical contrast-enhancement agents for detection of cancer cells.
Enhanced Photoacoustic Detection of Melanoma Cells Using Gold Nanoparticles
We tagged melanoma cells with gold nanoparticles to show their viability for increasing sensitivity in a photoacoustic detection system. Ultimately, this study models the detection of circulating tumor cells, which are an important prognostic factor in the progress of melanoma.
