Detection and capture of breast cancer cells with photoacoustic flow cytometry.

According to the Centers for Disease Control and Prevention, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis­the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems­significantly worsens the prognosis of any breast cancer patient. A technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser is used to interrogate thousands of blood cells with one pulse as they flow through the beam path. Cells that are optically absorbing, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to enhance optical absorption. After which, the PA cytometry device is calibrated to demonstrate the ability to detect single cells. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25 to 45 breast cancer cells per 1 mL of blood. An in vitro PA flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy but also it can be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

Evanescent Field Based Photoacoustics: Optical Property Evaluation at Surfaces.

Here, we present a protocol to estimate material and surface optical properties using the photoacoustic effect combined with total internal reflection. Optical property evaluation of thin films and the surfaces of bulk materials is an important step in understanding new optical material systems and their applications. The method presented can estimate thickness, refractive index, and use absorptive properties of materials for detection. This metrology system uses evanescent field-based photoacoustics (EFPA), a field of research based upon the interaction of an evanescent field with the photoacoustic effect. This interaction and its resulting family of techniques allow the technique to probe optical properties within a few hundred nanometers of the sample surface. This optical near field allows for the highly accurate estimation of material properties on the same scale as the field itself such as refractive index and film thickness. With the use of EFPA and its sub techniques such as total internal reflection photoacoustic spectroscopy (TIRPAS) and optical tunneling photoacoustic spectroscopy (OTPAS), it is possible to evaluate a material at the nanoscale in a consolidated instrument without the need for many instruments and experiments that may be cost prohibitive.

Capture and Isolation of Circulating Melanoma Cells Using Photoacoustic Flowmetry.

Circulating tumor cells (CTCs) are those cells that separate from a solid tumor and spread through the blood or lymphatic systems. While there are many open questions concerning the biology of CTCs, there is mounting evidence that some of these cells go on to create secondary tumors in distant organs, thus enabling metastatic disease. Detection of CTCs may have clinical impact by providing prognostic information. Furthermore, molecular and genetic analysis of CTCs may enable cancer biologists to answer questions about the metastatic process, such as whether these cells undergo epithelial-mesenchymal transition. Using a photoacoustic flowmeter, in which we induce ultrasonic responses from circulating melanoma cells (CMCs), we identify, capture, and isolate these cells for further analysis.

Photoacoustic measurement of refractive index of dye solutions and myoglobin for biosensing applications.

Current methods of determining the refractive index of chemicals and materials, such as ellipsometry and reflectometry, are limited by their inability to analyze highly absorbing or highly transparent materials, as well as the required prior knowledge of the sample thickness and estimated refractive index. Here, we present a method of determining the refractive index of solutions using the photoacoustic effect. We show that a photoacoustic refractometer can analyze highly absorbing dye samples to within 0.006 refractive index units of a handheld optical refractometer. Further, we use myoglobin, an early non-invasive biomarker for malignant hyperthermia, as a proof of concept that this technique is applicable for use as a medical diagnostic. Comparison of the speed, cost, simplicity, and accuracy of the techniques shows that this photoacoustic method is well-suited for optically complex systems.

Photoacoustic spectroscopy of ß-hematin.

Malaria affects over 200 million individuals annually, resulting in 800,000 fatalities. Current tests use blood smears and can only detect the disease when 0.1-1% of blood cells are infected. We are investigating the use of photoacoustic flowmetry to sense as few as one infected cell among 10 million or more normal blood cells, thus diagnosing infection before patients become symptomatic. Photoacoustic flowmetry is similar to conventional flow cytometry, except that rare cells are targeted by nanosecond laser pulses to induce ultrasonic responses. This system has been used to detect single melanoma cells in 10 ml of blood. Our objective is to apply photoacoustic flowmetry to detection of the malaria pigment hemozoin, which is a byproduct of parasite-digested hemoglobin in the blood. However, hemozoin is difficult to purify in quantities greater than a milligram, so a synthetic analog, known as ß-hematin was derived from porcine haemin. The specific purpose of this study is to establish the efficacy of using ß-hematin, rather than hemozoin, for photoacoustic measurements. We characterized ß-hematin using UV-vis spectroscopy, TEM, and FTIR, then tested the effects of laser irradiation on the synthetic product. We finally determined its absorption spectrum using photoacoustic excitation. UV-vis spectroscopy verified that ß-hematin was distinctly different from its precursor. TEM analysis confirmed its previously established nanorod shape, and comparison of the FTIR results with published spectroscopy data showed that our product had the distinctive absorbance peaks at 1661 and 1206 cm(-1). Also, our research indicated that prolonged irradiation dramatically alters the physical and optical properties of the ß-hematin, resulting in increased absorption at shorter wavelengths. Nevertheless, the photoacoustic absorption spectrum mimicked that generated by UV-vis spectroscopy, which confirms the accuracy of the photoacoustic method and strongly suggests that photoacoustic flowmetry may be used as a tool for diagnosis of malaria infection.

Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow.

Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are unable to detect early onset of metastatic disease. Patients must wait until macroscopic secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and travel through the blood or lymph system can provide data for diagnosing and monitoring metastatic disease. By irradiating enriched blood samples spiked with cultured melanoma cells with nanosecond duration laser light, we induced photoacoustic responses in the pigmented cells. Thus, we can detect and enumerate melanoma cells in blood samples to demonstrate a paradigm for a photoacoustic flow cytometer. Furthermore, we capture the melanoma cells using microfluidic two phase flow, a technique that separates a continuous flow into alternating microslugs of air and blood cell suspension. Each slug of blood cells is tested for the presence of melanoma. Slugs that are positive for melanoma, indicated by photoacoustic waves, are separated from the cytometer for further purification and isolation of the melanoma cell. In this paper, we evaluate the two phase photoacoustic flow cytometer for its ability to detect and capture metastatic melanoma cells in blood.

Total internal reflection photoacoustic spectroscopy for the detection of ß-hematin.

Evanescent field sensing methods are currently used to detect many different types of disease markers and biologically important chemicals such as the HER2 breast cancer receptor. Hinoue et al. used Total Internal Reflection Photoacoustic Spectroscopy (TIRPAS) as a method of using the evanescent field to detect an optically opaque dye at a sample interface. Although their methods were successful at detecting dyes, the results at that time did not show a very practical spectroscopic technique, which was due to the less than typical sensitivity of TIRPAS as a spectroscopy modality given the low power (≈ 1 to 2 W) lasers being used. Contrarily, we have used an Nd:YAG laser with a five nanosecond pulse that gives peak power of 1 MW coupled with the TIRPAS system to increase the sensitivity of this technique for biological material sensing. All efforts were focused on the eventual detection of the optically absorbing material, hemozoin, which is created as a byproduct of a malarial infection in blood. We used an optically analogous material, ß-hematin, to determine the potential for detection in the TIRPAS system. In addition, four properties which control the sensitivity were investigated to increase understanding about the sensor's function as a biosensing method.

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.

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.

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.