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Melanoma is one of the most serious skin cancers in the world. As circulating tumor cells have been proved to be an important marker of early metastasis of cancer, the detection of circulating tumor cells of melanoma is of great significance for early diagnosis and the monitoring of tumor progression. In vivo photoacoustic flow cytometry (PAFC) is constructed to achieve real-time and non-invasive detection of circulating melanoma cells in vivo. However, as the photoacoustic signals acquired in the detection process are disturbed by various kinds of noise, it is difficult to accurately distinguish the photoacoustic signals of background and circulating tumor cells by the traditional triple mean square deviation method. Therefore, a photoacoustic signal classification method is proposed based on convolutional neural network, which can greatly improve the accuracy of detection. Features of signals are extracted by the convolutional neural network to distinguish photoacoustic signals of melanoma cells and background. We construct a convolutional neural network based on one-dimensional input signals. For training the classifier, a large number of samples are selected. The accuracy rate in the test set can reach 95%. Besides, a neural network is built based on VGG16 model and transfer learning, and the trained classifier can realize the accuracy of 98% in the test set. Experiments show that the method of photoacoustic signal classification based on convolutional neural network greatly improves the accuracy of signal classification, and realizes the rapid and accurate analysis of a large number of data.

Melanoma, developing from melanocytes, is the deadliest type of malignant skin tumors in the world. Due to high light absorption of melanin, rare circulating melanoma cells, as an endogenous marker for metastasis at the early stage, can be quantitatively detected in small superficial vessels of mouse ears by in vivo photoacoustic flow cytometry (PAFC). Before clinical application, the capability of promising PAFC platform should be verified and optimized by mouse vessels, which are similar in size and depth to human vessels. In the current study, compared with optical resolution PAFC (OR-PAFC), we build acoustic resolution PAFC (AR-PAFC) using focused ultrasonic transducer and 1064 nm laser with lower pulse rate, leading to higher detection depth and lower laser power density in mouse models. Besides, based on laser frequency doubling and high absorption coefficient of hemoglobin at 532nm wavelength, the blood vessels can be positioned by lowcost navigation system rather than the expensive system of two coupled lasers or charged coupled device with depth limitation. We confirm that AR-PAFC can be applied to noninvasive label-free counting of circulating melanoma cells in mouse tail veins, and validated by in vitro assays using phantom models, which simulates the scattering and absorption coefficients of living tissue. These results show that AR-PAFC platform has great potential for preoperative diagnosis and postoperative evaluation of melanoma patients.

Melanoma is a malignant tumor whose circulating tumor cell (CTC) count has been shown as a prognostic marker for metastasis development. Therefore detection of circulating melanoma cells plays an important role in monitoring tumor metastasis and prevention after diagnosis. In Vivo Photoacoustic Flow Cytometry (PAFC) is established here to achieve in vivo melanoma inspection, meanwhile guarantees non-invasive and real-time detection.Accurate tumor cell detection is of great significance to achieve highly specific diagnosis and avoid unnecessary medical tests.However, the amount of data detected by PAFC is large and original photoacoustic signal is mixed with various noises.The traditional triple mean square deviation method has lower accuracy and consumes a lot of time in data processing. Here, a classification approach in photoacoustic is proposed, which could discriminate signals and noises based on features extracted from photoacoustic waves compared to normal cells using Support Vector Machines algorithm. Due to similar shape and structure of cells, the photoacoustic signals usually have similar vibration mode. By analyzing the correlations and the signal features in time domain and frequency domain, we finally choose the continuity, amplitude, and photoacoustic waveform pulse width as the features to characterize the signal.More than 600,000 samples were selected as the training set (normalized in advance), and a classifier with a precision of 85% accuracy to filter out the photoacoustic signals rapidly was trained by the support vector machine method.The classifier introduced here has proved to optimize the signal acquisition and reduce signal processing time, realizing real-time detection and real-time analysis in PAFC.

Alzheimer's disease (AD) is a chronic neurodegenerative disease. It is generally believed that there are some connections between AD and amyloid protein plaques in the brain. The typical symptoms of AD are memory and spatial learning difficulties, language disorders, loss of motivation and behavioral issues. Currently, the main therapeutic method is pharmacotherapy, which temporarily relieves symptoms, and yet brings with some side effects. Near infrared (NIR) light therapy has been studied in a range of single and multiple irradiation protocols in previous studies and was found beneficial for neuropathology. In our research, we demonstrated the effect of NIR light on AD through transgenic mouse model. We designed an experimental apparatus consisted of a box with a LED array emitting NIR light inside. After the treatment, we assessed the effects of infrared light by testing cognitive performance of mice in Morris water maze, and detecting plaque load by immunofluorescence analysis. Our results show that NIR therapy is able to attenuate the Aβ burden and cognitive deficits in the mouse model. It might provide a novel and safe way to treat AD.

Malignant melanoma, developing from melanocytes, is a kind of high metastatic tumor. Circulating melanoma cells, as a marker for metastasis development, are found in blood or lymphatic system at the early stage. Thus, quantitative detection of circulating melanoma cells has great significance to diagnose carcinoma and monitor tumor metastasis. In contrast to in vitro detection methods and in vivo fluorescence-based flow cytometry (IVFC), the in vivo photoacoustic flow cytometry (PAFC) utilizes melanoma cells’ predominant optical absorption in the near-infrared range over other absorbers to receive the photoacoustic (PA) signals without fluorescent dye labeling in a non-invasive way. The sensitivity of the PAFC system was verified by in vitro and in vivo experiments. Besides, we solves the technical problem that blood vessels cannot be positioned in the process of in vivo detection by designing the laser signal positioning navigation system using frequency doubling technique. PAFC provides a new tool for in vivo, label-free, and noninvasive detection of circulating tumor cells (CTCs) and has strong practicality and favorable clinical prospects.

Melanoma, developing from melanocytes, is the most serious type of skin cancer. Circulating melanoma cells, the prognosis marker for metastasis, are present in the circulation at the early stage. Thus, quantitative detection of rare circulating melanoma cells is essential for monitoring tumor metastasis and prognosis evaluation. Compared with in vitro assays, in vivo flow cytometry is able to identify circulating tumor cells without drawing blood. Here, we built in vivo photoacoustic flow cytometry based on the high absorption coefficient of melanoma cells, which is applied to labelfree counting of circulating melanoma cells in tumor-bearing mice.

Melanoma is a kind of a malignant tumor of melanocytes with the properties of high mortality and high metastasis rate. The circulating melanoma cells with the high content of melanin can be detected by light absorption to diagnose and treat cancer at an early stage. Compared with conventional detection methods such as in vivo flow cytometry (IVFC) based on fluorescence, the in vivo photoacoustic flow cytometry (PAFC) utilizes melanin cells as biomarkers to collect the photoacoustic (PA) signals without toxic fluorescent dyes labeling in a non-invasive way. The information of target tumor cells is helpful for data analysis and cell counting. However, the raw signals in PAFC system contain numerous noises such as environmental noise, device noise and in vivo motion noise. Conventional denoising algorithms such as wavelet denoising (WD) method and means filter (MF) method are based on the local information to extract the data of clinical interest, which remove the subtle feature and leave many noises. To address the above questions, the nonlocal means (NLM) method based on nonlocal data has been proposed to suppress the noise in PA signals. Extensive experiments on in vivo PA signals from the mice with the injection of B16F10 cells in caudal vein have been conducted. All the results indicate that the NLM method has superior noise reduction performance and subtle information reservation.

Melanoma is known as a malignant tumor of melanocytes, which usually appear in the blood circulation at the metastasis stage of cancer. Thus the detection of circulating melanoma cells is useful for early diagnosis and therapy of cancer. Here we have developed an in vivo photoacoustic flow cytometry (PAFC) based on the photoacoustic effect to detect melanoma cells. However, the raw signals we obtain from the target cells contain noises such as environmental sonic noises and electronic noises. Therefore we apply correlation comparison and feature separation methods to the detection and verification of the in vivo signals. Due to similar shape and structure of cells, the photoacoustic signals usually have similar vibration mode. By analyzing the correlations and the signal features in time domain and frequency domain, we are able to provide a method for separating photoacoustic signals generated by target cells from background noises. The method introduced here has proved to optimize the signal acquisition and signal processing, which can improve the detection accuracy in PAFC.

It is generally believed that there are some connections between Alzheimer’s disease and amyloid protein plaques in the brain. The typical symptoms of Alzheimer’s disease are memory loss, language disorders, mood swings, loss of motivation and behavioral issues. Currently, the main therapeutic method is pharmacotherapy, which may temporarily reduce symptoms, but has many side effects. Infrared light therapy has been studied in a range of single and multiple irradiation protocols in previous studies and was found beneficial for neuropathology. In our research we have studied the effect of infrared light on Alzheimer’s disease through transgenic mouse model. We designed an experimental apparatus for treating mice, which primarily included a therapeutic box and a LED array, which emitted infrared light. After the treatment, we assessed the effects of infrared light by performing two tests: cognitive performance of mice in Morris water maze, and plaque load by immunofluorescence analysis. Immunofluorescence analysis was based on measuring the quantity of plaques in mouse brain slices. Our results show that infrared therapy is able to improve cognitive performance in the mouse model. It might provide a novel and safe way to treat Alzheimer’s disease.

In recent years, the in vivo flow cytometry (IVFC) has been a useful technology in detecting and quantifying the circulating cells dynamically in living animals, especially in the research related to the cell tracking and the cancer metastasis. In practice, however, the unstable background signals caused by the experiment animals’ respiratory movement, limb movement and photo-bleaching of tissues’ auto-fluorescence exist in many IVFC data, which could affect the accuracy of cell counting results in the following post-processing procedure, making the IVFC signals less available. Here we developed a signal processing method that could effectively correct the unstable background signals by using methods combining interpolating, fitting, automatic segmenting and wavelet-based denoising. Compared with the previously used non-correction methods, i.e., the “line-gating” method or the automatic threshold method, this method showed a higher accuracy and efficiency in counting cell numbers of IVFC signals, as well as demonstrating a better statistic results in the Pearson’s correlation coefficient R2 and the mean-squared error (MSE).

Melanoma is a malignant tumor of melanocytes. Melanoma cells have high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC), which is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. We have developed in vitro experiments to prove the ability of PAFC system of detecting photoacoustic signals from melanoma cells. For in vivo experiments, we have constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells, B16F10 with subcutaneous injection. PA signals are detected in the blood vessels of mouse ears in vivo. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The processing methods have a great potential for analyzing signals accurately and rapidly. By counting circulating melanoma cells termly, we obtain the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation.

Alzheimer's disease (AD) is an extensive neurodegenerative disease. It is generally believed that there are some connections between AD and amyloid protein plaques in the brain. AD is a chronic disease that usually starts slowly and gets worse over time. The typical symptoms are memory loss, language disorders, mood swings and behavioral issues. Gradual losses of somatic functions eventually lead patients to death. Currently, the main therapeutic method is pharmacotherapy, which may temporarily reduce symptoms, but has many side effects. No current treatment can reverse AD's deterioration. Infrared (IR) light therapy has been studied in a range of single and multiple irradiation protocols in previous studies and was found beneficial for neuropathology. In our research, we have verified the effect of infrared light on AD through Alzheimer's disease mouse model. This transgenic mouse model is made by co-injecting two vectors encoding mutant amyloid precursor protein (APP) and mutant presenilin-1 (PSEN1). We designed an experimental apparatus for treating mice, which primarily includes a therapeutic box and a LED array, which emits infrared light. After the treatment, we assessed the effects of infrared light by testing cognitive performance of the mice in Morris water maze. Our results show that infra-red therapy is able to improve cognitive performance in the mouse model. It might provide a novel and safe way to treat Alzheimer's disease.

At early stage of cancer, a small number of circulating tumor cells (CTCs) appear in the blood circulation. Thus, early detection of malignant circulating tumor cells has great significance for timely treatment to reduce the cancer death rate. We have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of CTCs and record the signals from target cells. Information of target cells which is helpful to the early therapy would be obtained through analyzing and processing the signals. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The PAFC technique can detect signals from circulating tumor cells or other particles. The processing methods have a great potential for analyzing signals accurately and rapidly.

Melanoma is a malignant tumor of melanocytes. Circulating melanoma cell has high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC). PAFC is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. In our research, we developed in vitro experiments to prove the ability of PAFC system of detecting PA signals from melanoma cells. For in vivo experiments, we constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells B16F10 with subcutaneous injection. PA signals were detected in the blood vessels of mouse ears in vivo. By counting circulating melanoma cells termly, we obtained the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation. Our PAFC system is an efficient tool to monitor melanoma metastases, cancer recurrence and therapeutic efficacy.

Cells sense and respond to chemical stimuli on their environment via signal transduction pathways, complex networks of proteins whose interactions transmit chemical information. This work describes an implementation of image informatics, imaging-based methodologies for studying signal transduction networks. The methodology developed focuses on studying signal transduction networks in cells that interact with 3D matrices. It utilizes shRNA-based knock down of network components, 3D high-content imaging of cells inside the matrix by spectral multi-photon microscopy, and single-cell quantification using features that describe both cell morphology and cell-matrix adhesion pattern. The methodology is applied in a pilot study of TGFβ signaling via the SMAD pathway in fibroblasts cultured inside porous collagen-GAG scaffolds, biomaterials similar to the ones used clinically to induce skin regeneration. Preliminary results suggest that knocking down all rSMAD components affects fibroblast response to TGFβ1 and TGFβ3 isoforms in different ways, and suggest a potential role for SMAD1 and SMAD5 in regulating TGFβ isoform response. These preliminary results need to be verified with proteomic results that can provide solid evidence about the particular role of individual components of the SMAD pathway.

Melanoma, a malignant tumor of melanocytes, is the most serious type of skin cancer in the world. It accounts for about 80% of deaths of all skin cancer. For cancer detection, circulating tumor cells (CTCs) serve as a marker for metastasis development, cancer recurrence, and therapeutic efficacy. Melanoma tumor cells have high content of melanin, which has high light absorption and can serve as endogenous biomarker for CTC detection without labeling. Here, we have developed an in vivo photoacoustic flow cytometry (PAFC) to monitor the metastatic process of melanoma cancer by counting CTCs of melanoma tumor bearing mice in vivo. To test in vivo PAFC’s capability of detecting melanoma cancer, we have constructed a melanoma tumor model by subcutaneous inoculation of highly metastatic murine melanoma cancer cells, B16F10. In order to effectively distinguish the targeting PA signals from background noise, we have used the algorithm of Wavelet denoising method to reduce the background noise. The in vivo flow cytometry (IVFC) has shown a great potential for detecting circulating tumor cells quantitatively in the blood stream. Compared with fluorescence-based in vivo flow cytometry (IVFC), PAFC technique can be used for in vivo, label-free, and noninvasive detection of circulating tumor cells (CTCs).

To view the individual cells and ambient microvasculature simultaneously will be helpful to study tumor angiogenesis and microenvironments. To achieve this, two molecular contrast mechanisms were exploited simultaneously by integrating two imaging modalities, confocal fluorescence microscopy (CFM) and photoacoustic microscopy (PAM). These share the same scanning optical path and laser source. The induced photoacoustic (PA) signal was detected by a highly sensitive needle hydrophone; while the back-traveling fluorescent photons emitted from the same sample were collected by an avalanche photodetector. Experiments on ex vivo rat bladders were conducted. The CFM image depicted the shape and size of the individual cells successfully. Besides large polygonal umbrella cells, some intracellular components can also be discerned. With the CFM image presenting morphologic cellular information in the bladder wall, the PAM image provides the complementary information, based on the endogenous optical absorption contrast, of the microvascular distribution inside the bladder wall, from large vessels to capillaries. Such multimodal imaging provides the opportunity to realize both histological assay and characterization of microvasculature using one imaging setup. This approach offers the possibility of comprehensive diagnosis of cancer in vivo.

It has been studied that a potential marker to obtain prognostic information about bladder cancer is tumor neoangiogenesis, which can be quantified by morphometric characteristics such as microvascular density. Photoacoustic microscopy (PAM) can render sensitive three-dimensional (3D) mapping of microvasculature, providing promise to evaluate the neoangiogenesis that is closely related to the diagnosis of bladder cancer. To ensure good image quality, it is desired to acquire bladder PAM images from its inside via the urethra, like conventional cystoscope. Previously, we demonstrated all-optical PAM systems using polymer microring resonators to detect photoacoustic signals and galvanometer mirrors for laser scanning. In this work, we build a miniature PAM system using a microelectromechanical systems (MEMS) scanning mirror, demonstrating a prototype of an endoscopic PAM head capable of high imaging quality of the bladder. The system has high resolutions of 17.5 μm in lateral direction and 19 μm in the axial direction at a distance of 5.4 mm. Images of printed grids and the 3D structure of microvasculature in animal bladders ex vivo by the system are demonstrated.

Macrophages appear to be directly involved in cancer progression and metastasis. However, the role of macrophages in influencing tumor metastasis has not been fully understood. Here, we have used an emerging technique, namely in vivo flow cytometry (IVFC) to study the depletion kinetics of circulating prostate cancer cells in mice and how depletion of macrophages by the liposome-encapsulated clodronate affects the depletion kinetics. Our results show different depletion kinetics of PC-3 prostate cancer cells between macrophage-deficient group and the control group. The number of circulating tumor cells (CTCs) in macrophage-deficient group decreases in a slower manner compared to the control mice group. The differences in depletion kinetics indicate that the absence of macrophages might facilitate the stay of prostate tumor cells in circulation. We speculate that macrophages might be able to arrest, phagocytose and digest PC-3 cancer cells. Therefore, the phagocytosis may mainly contribute to the differences in depletion kinetics. The developed methods here would be useful to study the relationship between macrophages and cancer metastasis in small animal tumor model.

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, with approximately 1,000,000 cases reported every year. The fate of circulating tumor cells (CTCs) is an important determinant of metastasis and recurrence, which lead to most deaths in HCC. Therefore, quantification of CTCs proves to be an emerging tool for diagnosing, stratifying and monitoring patients with metastatic diseases. In vivo flow cytometry (IVFC) has the capability to monitor the dynamics of fluorescently labeled CTCs continuously and non-invasively. Here, we combine IVFC technique and a GFP-transfected HCC orthotopic metastatic tumor model to monitor CTC dynamics. Our IVFC has ~1.8-fold higher sensitivity than whole blood analysis by conventional flow cytometry. We find out a significant difference of CTC dynamics between orthotopic and subcutaneous (s.c.) tumor models. We also investigate whether liver resection promotes or restricts hematogenous metastasis in advanced HCC. Our result shows that the number of CTCs and early metastases decreases after the resection. CTC dynamics is correlated with tumor growth in our orthotopic tumor model. The number and size of distant metastases correspond to CTC dynamics. The novel IVFC technique combined with orthotopic tumor models might provide insights to tumor hematogenous metastasis and guidance to cancer therapy.

Metastasis is a very complicated multi-step process and accounts for the low survival rate of the cancerous patients. To metastasize, the malignant cells must detach from the primary tumor and migrate to secondary sites in the body through either blood or lymph circulation. Macrophages appear to be directly involved in tumor progression and metastasis. However, the role of macrophages in affecting cancer metastasis has not been fully elucidated. Here, we have utilized an emerging technique, namely in vivo flow cytometry (IVFC) to study the depletion kinetics of circulating prostate cancer cells in mice and how depletion of macrophages by the liposome-encapsulated clodronate affects the depletion kinetics. Our results show different depletion kinetics of PC-3 cells between macrophage-deficient group and the control group. The number of circulating tumor cells (CTCs) in macrophage-deficient group decreases in a slower manner compared to the control mice group. The differences in depletion kinetics indicate that the absence of macrophages facilitates the stay of prostate cancer cells in circulation. We speculate that macrophages might be able to arrest, phagocytose and digest PC-3 cells. Therefore, the phagocytosis may mainly contribute to the depletion kinetic differences. The developed methods here would be useful to study the relationship between macrophages and tumor metastasis in small animal cancer model.

Prostate cancer is the most common malignancy in American men and the second leading cause of deaths from cancer, after lung cancer. The tumor usually grows slowly and remains confined to the gland for many years. As the cancer advances, however, it can metastasize throughout other areas of the body, such as the bones, lungs, and liver. Surgical resection, hormonal therapy, chemotherapy and radiation therapy are the foundation of current prostate cancer therapies. Treatments for prostate cause both short- and long-term side effects that may be difficult to accept. Molecular mechanisms of prostate cancer metastasis need to be understood better and new therapies must be developed to selectively target to unique characteristics of cancer cell growth and metastasis. We have developed the "in vivo microscopy" to study the mechanisms that govern prostate cancer cell spread through the microenvironment in vivo in real-time confocal near-infrared fluorescence imaging. A recently developed "in vivo flow cytometer" and optical imaging are used to assess prostate cancer cell spreading and the circulation kinetics of prostate cancer cells. We have measured the depletion kinetics of cancer cells with different metastatic potential. Interestingly, more invasive PC-3 prostate cancer cells are depleted faster from the circulation than LNCaP cells.

Liver cancer is one of the most common malignancies in the world, with approximately 1,000,000 cases reported every year. Hepatocellular carcinoma may metastasize to lung, bones, kidney, and many other organs. Surgical resection, liver transplantation, chemotherapy and radiation therapy are the foundation of current HCC therapies. However the outcomes are poor: the survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed. A recently developed "in vivo flow cytometer" combined with real-time confocal fluorescence imaging are used to assess spreading and the circulation kinetics of liver tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labeled cells in vivo in real time without extracting blood sample. We have measured the depletion kinetics of two related human HCC cell lines, high-metastatic HCCLM3 cells and low-metastatic HepG2 cells, which were from the same origin and obtained by repetitive screenings in mice. >60% HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison

Zebrafish have become a powerful vertebrate model organism for drug discovery, cancer and stem cell research. A recently developed transparent adult zebrafish using double pigmentation mutant, called casper, provide unparalleled imaging power in in vivo longitudinal analysis of biological processes at an anatomic resolution not readily achievable in murine or other systems. In this paper we introduce an optical method for simultaneous visualization and cell quantification, which combines the laser scanning confocal microscopy (LSCM) and the in vivo flow cytometry (IVFC). The system is designed specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish casper, under physiological conditions in the same fish over time. The confocal imaging part in this system serves the dual purposes of imaging fish tissue microstructure and a 3D navigation tool to locate a suitable vessel for circulating cell counting. The multi-color, multi-channel instrument allows the detection of multiple cell populations or different tissues or organs simultaneously. We demonstrate initial testing of this novel instrument by imaging vasculature and tracking circulating cells in CD41: GFP/Gata1: DsRed transgenic casper fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. Circulating fluorescent cell incidents were recorded and counted repeatedly over time and in different types of vessels. Great application opportunities in cancer and stem cell researches are discussed.

Prostate cancer is the most common malignancy in American men and the second leading cause of deaths from cancer, after lung cancer. The tumor usually grows slowly and remains confined to the gland for many years. During this time, the tumor produces little or no symptoms or outward signs. As the cancer advances, however, it can metastasize throughout other areas of the body, such as the bones, lungs, and liver. Surgical resection, hormonal therapy, chemotherapy and radiation therapy are the foundation of current prostate cancer therapies. Treatments for prostate cause both short- and long-term side effects that may be difficult to accept. Molecular mechanisms of prostate cancer metastasis need to be understood better and new therapies must be developed to selectively target to unique characteristics of cancer cell growth and metastasis. We have developed the "in vivo microscopy" to study the mechanisms that govern prostate cancer cell spread through the microenvironment in vivo in real-time confocal near-infrared fluorescence imaging. A recently developed "in vivo flow cytometer" and optical imaging are used to assess prostate cancer cell spreading and the circulation kinetics of prostate cancer cells. A real- time quantitative monitoring of circulating prostate cancer cells by the in vivo flow cytometer will be useful to assess the effectiveness of the potential therapeutic interventions.

Prostate cancer is the most common malignancy in American men and the second leading cause of deaths from cancer, after lung cancer. The tumor usually grows slowly and remains confined to the gland for many years. During this time, the tumor produces little or no symptoms or outward signs. As the cancer advances, however, it can metastasize throughout other areas of the body, such as the bones, lungs, and liver. Surgical resection, hormonal therapy, chemotherapy and radiation therapy are the foundation of current prostate cancer therapies. Treatments for prostate cause both short- and long-term side effects that may be difficult to accept. Molecular mechanisms of prostate cancer metastasis need to be understood better and new therapies must be developed to selectively target to unique characteristics of cancer cell growth and metastasis. We have developed the "in vivo microscopy" to study the mechanisms that govern prostate cancer cell spread through the microenvironment in vivo in real-time confocal nearinfrared fluorescence imaging. A recently developed "in vivo flow cytometer" and optical imaging are used to assess prostate cancer cell spreading and the circulation kinetics of prostate cancer cells. A real- time quantitative monitoring of circulating prostate cancer cells by the in vivo flow cytometer will be useful to assess the effectiveness of the potential therapeutic interventions.

A recently developed "in vivo flow cytometer" and optical imaging are used to assess liver tumor cell spreading and the circulation kinetics of liver tumor cells. The in vivo flow cytometer has the capability to detect and quantify the number and flow characteristics of fluorescently labeled cells in vivo continuously. The depletion rate of circulating tumor cells provides insights in early cancer metastasis. It is useful to understand the molecular mechanisms of liver tumor metastasis. A real-time quantitative monitoring of circulating liver tumor cells by the in vivo flow cytometer will help assess the effectiveness of the potential therapeutic interventions.

Primary liver cancer (hepatocellular carcinoma, or HCC) is associated with liver cirrhosis 60–80% of the time. Liver cancer is one of the most common malignancies in the world, with approximately 1,000,000 cases reported every year. About 80% of people with primary liver cancer are male. Although two-thirds of people have advanced liver disease when they seek medical help, one third of the patients have cancer that has not progressed beyond the liver. HCC may metastasize to the lung, bones, kidney, and many other organs. Surgical resection, liver transplantation, chemotherapy and radiation therapy are the foundation of current HCC therapies. However the outcomes are poor:the survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed to selectively target to unique characteristics of HCC cell growth and metastasis. We have developed the “in vivo microscopy” to study the mechanisms that govern liver tumor cell spread through the microenvironment in vivo with real-time confocal near-infrared fluorescence imaging. A recently developed “in vivo flow cytometer” and optical imaging are used to assess liver tumor cell spreading and the circulation kinetics of liver tumor cells. A real- time quantitative monitoring of circulating liver tumor cells by the in vivo flow cytometer will be useful to assess the effectiveness of the potential therapeutic interventions.

Prostate cancer is the most common malignancy in American men and the second leading cause of deaths from cancer, after lung cancer. The tumor usually grows slowly and remains confined to the gland for many years. During this time, the tumor produces little or no symptoms or outward signs. As the cancer advances, however, it can metastasize throughout other areas of the body, such as the bones, lungs, and liver. Surgical resection, hormonal therapy, chemotherapy and radiation therapy are the foundation of current prostate cancer therapies. Treatments for prostate cause both short- and long-term side effects that may be difficult to accept. Molecular mechanisms of prostate cancer metastasis need to be understood better and new therapies must be developed to selectively target to unique characteristics of cancer cell growth and metastasis. We have developed the "in vivo microscopy" to study the mechanisms that govern prostate cancer cell spread through the microenvironment in vivo in real-time confocal nearinfrared fluorescence imaging. A recently developed "in vivo flow cytometer" and optical imaging are used to assess prostate cancer cell spreading and the circulation kinetics of prostate cancer cells. A real- time quantitative monitoring of circulating prostate cancer cells by the in vivo flow cytometer will be useful to assess the effectiveness of the potential therapeutic interventions.

Liver cancer is one of the most common malignancies in the world, with approximately 1,000,000 cases reported every year. This ranges from 15,000 cases in the United States to more than a 250,000 in China. About 80% of people with primary liver cancer are male. Although two-thirds of people have advanced liver disease when they seek medical help, one third of the patients have cancer that has not progressed beyond the liver. Primary liver cancer (hepatocellular carcinoma, or HCC) is associated with liver cirrhosis 60-80% of the time. HCC may metastasize to the lung, bones, kidney, and many other organs. Surgical resection, liver transplantation, chemotherapy and radiation therapy are the foundation of current HCC therapies. However the outcomes are poor-the survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed to selectively target to unique characteristics of HCC cell growth and metastasis. We have developed the "in vivo microscopy" to study the mechanisms that govern liver tumor cell spread through the microenvironment in vivo in real-time confocal near-infrared fluorescence imaging. A recently developed "in vivo flow cytometer" and optical imaging are used to assess liver tumor cell spreading and the circulation kinetics of liver tumor cells. A real-time quantitative monitoring of circulating liver tumor cells by the in vivo flow cytometer will be useful to assess the effectiveness of the potential therapeutic interventions.

Poly(ethylene glycol)-coated Au nanocages have been evaluated as a potential near-infrared (NIR) contrast agent for photoacoustic tomography (PAT). Previously, Au nanoshells were found to be an effective NIR contrast agent for PAT; however, Au nanocages, with their more compact sizes (100 nm for Au nanoshells) and larger optical absorption cross-sections, should be better suited for in vivo applications. In this study, we tested Au nanocages as a contrast agent for PAT. The result suggests that Au nanocages are promising contrast agents for our applications. We also present PAT results when novel, dye-containing nanoparticles are used as contrast agents.

Chemotherapies currently constitute one main venue of cancer treatment. For a large number of adult and elderly patients, however, treatment options are poor. These patients may suffer from disease that is resistant to conventional chemotherapy or may not be candidates for curative therapies because of advanced age or poor medical conditions. To control disease in these patients, new therapies must be developed that are selectively targeted to unique characteristics of tumor cell growth and metastasis. A reliable early evaluation and prediction of response to the chemotherapy is critical to its success. Chemotherapies induce apoptosis in tumor cells and a portion of such apoptotic cancer cells may be present in the circulation. However, the fate of circulating tumor cells is difficult to assess with conventional methods that require blood sampling. We report the in situ measurement of circulating apoptotic cells in live animals using in vivo flow cytometry, a novel method that enables real-time detection and quantification of circulating cells without blood extraction. Apoptotic cells are rapidly cleared from the circulation with a half-life of ~10 minutes. Real-time monitoring of circulating apoptotic cells can be useful for detecting early changes in disease processes, as well as for monitoring response to therapeutic intervention.

Cytotoxic chemotherapy agents are the foundation of current leukemia therapy. For a large number of adult and elderly patients, however, treatment options are poor. These patients may suffer from disease that is resistant to conventional chemotherapy or may not be candidates for curative therapies because of advanced age or poor medical conditions. To control disease in these patients, new therapies must be developed that are selectively targeted to unique characteristics of leukemic cell growth and metastasis. A large body of elegant work in the field of immunology has demonstrated the mechanisms whereby leukocytes traffic to specific sites within the body. Vascular cell adhesion molecules and chemicalattractants combine to direct white blood cells to appropriate environments. Although it has been hypothesized that leukemic white blood cells home to hematopoietic organs using mechanisms similar to those of their benign leukocyte counterparts, detailed study of leukemic cell transit through bone marrow has yet to be undertaken. We develop the "in vivo microscopy" to study the mechanisms that govern leukemic cell spread through the bone marrow microenvironment in vivo in real-time confocal infrared fluorescence imaging. A recently developed "in vivo flow cytometer" and optical imaging are used to assess leukemic cell spreading and the circulation kinetics of leukemic cells. A real- time quantitative monitoring of circulating leukemic cells by the in vivo flow cytometer will be useful to assess the effectiveness of the potential therapeutic interventions.

Lymphocytes are the central players in the human adaptive immune response. In the body, individual T helper lymphocytes need to be activated first by physical contact with antigen- presenting cells (APC). T-cell contact with APCs initiated an activation cascade, which includes an increase in T-cell intracellular calcium, leading to T-cell proliferation, differentiation and lymphokine production. Calcium imaging are combined with optical manipulation to investigate the physical properties of T-cell activation. We study cell-cell contact requirements for T-cell activation using optical tweezers to control the orientation of T-cell/APC pairs and fluorescence microscopy to measure the subsequent T-cell intracellular calcium level [(Ca2+)i] response. APCs or beads coated with antibodies to the T-cell receptor (TCR) are trapped with a near-infrared titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling. T cells which are presented with antigen at the leading edge have a higher probability of responding and a shorter latency of response than those contacting APCs or beads with their trailing end. Alterations in antibody density and bead size are used to determine the spatial requirements for T cell activation and the minimum number of receptors which must be engaged in order to transmit a positive signal.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

T-cell contact with antigen-presenting cells (APC) initiates an activation cascade which includes an increase in T-cell intracellular calcium [(Ca2+)i] and leads to T-cell proliferation and differentiation. Although T-cell/APC physical contact is required for an immune response, little is known about the patterns of cellular interaction and their relation to activation. We have combined fluorescence spectroscopy and imaging with optical manipulation to investigate the contact requirements for T-cell activation, using optical tweezers to control the orientation of T- cell/APC pairs and fluorescence microscopy to measure the subsequent (Ca2+)i response, detected as an emission shift from the combination of fura-red and oregon- green, two cytoplasmic (Ca2+) indicators. APCs or beads coated with antibodies to the T-cell receptor (TCR) are trapped with a near-infrared titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling (2-5 micrometers /min). T cells contacted with antigen- presenting cells or antibody-coated beads entered a dynamic and reproducible program in the first 10 - 20 mins, including (Ca2+)i increase, changes in shape and motility, engulfment, and stable contact. T cells presented with antigen at the leading edge had a higher probability of responding (85%) and a shorter latency of response (50 secs) than those contacting APCs or beads with their trailing end (APCs: 30%, 150 secs; beads: 6%, 300 secs). Alterations in antibody density, quantified by FACS analysis, and bead size were used to determine the spatial requirements for T cell activation and the minimum number of receptors which must be engaged in order to transmit a positive signal. Preliminary data show that T cell responses [response percentage, latency and (Ca2+)i pattern] depend on both antibody density and bead size.© (1998) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

In the body, individual T helper lymphocytes need to be EtiVated first by physical contact with antigen-presentingcells (APC). T-cell contact with APCs initiates an activation cascade which includes an increase in T-cell intracellular calciumand leads to T-cell proliferation and differentiation. We have combined fluorescence spectroscopy and imaging with opticalmanipulation to investigate the physical properties of T-cell activation. We study cell-cell contact requirements for T-celiactivation using optical tweezers to conirol the orientation of T-celI/APC pairs and fluorescence microscopy to measure thesubsequent T-cell intracellular calcium level ([Cai1) response. APCs or beads coated with antibodies to the T-cell receptortrapped with a near-infrared titanium-sapphire laser and pled at different locations along the T-cell, which has a poliwizedappearance defined by the shape and direction of crawling. T cells which are presented with antigen at the leading edge have ahigher probability of responding and a shorter latency of response than those contacting APCs or beads with their trailing end.Alterations in antibody density and bead size are used to determine the spatial requirements for T cell activation and theminimum number of receptors which must be engaged in order to transmit a positive signal.Keywords: Optical laser trapping, fluorescence detection, optical manipulation, T-cell activation, T cell receptor, calciumimagin antibody-coated bead

T-cell contact with antigen-presenting B cells initiates an activation cascade which includes an increase in T-cell intracellular calcium and leads to T-cell proliferation and differentiation. We studied cell-cell contact requirements for T-cell activation using an optical trap to control the orientation of T-cell/B-cell pairs and fluorescence microscopy to measure subsequent T-cell(Ca2+)i response. B cells or beads coated with antibodies to the T- cell receptor are trapped with a titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling. T-cell (Ca2+)i is detected as an emission shift from the combination of fura-red and oregon- green, two cytoplasmic (Ca2+)i indicators. T- cells which are presented antigen at the leading edge have a higher probability of responding and a shorter latency of response than those contacting B-cells or beads with their trailing end.© (1998) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Optical laser trapping microscopy has emerged as a powerful tool not only for the optical manipulation of cells and macromolecules, but also for the study of cellular physiological responses via force transduction and fluorescence imaging. We describe here the most recent results from our laboratory in the use and application of laser trapping microscopy to a variety of studies at the cellular and molecular levels. Fluorescence spectroscopy and imaging have been successfully combined with optical micromanipulation. A single near-infrared laser beam is used for two-photon fluorescence excitation and micromanipulation of trapped biological specimens. Cell viability is observed and monitored with a Nd:YAG laser ((lambda) equals 1064 nm) and an Al:GaAs diode laser ((lambda) equals 809 nm). Traps and conventional fluorescence imaging are also used simultaneously to examine T-cell activation dynamics.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.