David M. Huland

In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems

We characterize long (up to 285 mm) gradient index (GRIN) lens endoscope systems for multiphoton imaging. We fabricate a portable, rigid endoscope system suitable for imaging unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The portable device is capable of imaging a ~200 µm diameter field of view at 4 frames/s. The lateral and axial resolution in water is 0.85 µm and 7.4 µm respectively. In vivo images of unstained tissues in live, anesthetized rats using the portable device are presented. These results show great promise for GRIN endoscopy to be used clinically.

Identification of Spermatogenesis With Multiphoton Microscopy: An Evaluation in a Rodent Model

PURPOSE Microdissection testicular sperm extraction has replaced conventional testis biopsies for men with nonobstructive azoospermia and it has become first line treatment. The current problem is that the decision to retrieve tubules is based only on appearance and there is no guarantee that the tubules removed contain sperm. Multiphoton microscopy enables label-free immediate visualization of many biological processes in living tissue at subcellular resolution. MATERIALS AND METHODS We used multiphoton microscopy to study the different developmental stages of spermatogenesis using neonatal, pubertal and adult rat testes. We used a testis hypothermia plus ischemia model to study different testicular histopathologies with multiphoton microscopy. To assess the risk of photo damage DNA fragmentation in testis biopsies imaged at different intensities was assessed by TUNEL assay. RESULTS Multiphoton microscopy identified the stage of spermatogenesis in a seminiferous tubule in fresh tissue without using exogenous labels. We noted significant differences in fluorescence and spectroscopic characteristics between tubules with and without sperm. Sertolis-cell only tubules had abundant autofluorescence in the 420 to 490 and 550 to 650 nm wavelength ranges while tubules containing sperm had autofluorescence only in the 420 to 490 nm range. On DNA fragmentation assay sperm from tubules imaged by multiphoton microscopy had minimal DNA fragmentation at the laser intensities needed to distinguish tubules with and without sperm. CONCLUSIONS Multiphoton microscopy has the potential to facilitate real-time visualization of spermatogenesis in humans and aid in clinical applications, such as testicular sperm extraction for men with infertility.

Three-photon excited fluorescence imaging of unstained tissue using a GRIN lens endoscope

We present a compact and portable three-photon gradient index (GRIN) lens endoscope system suitable for imaging of unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The lateral and axial resolution in water is 1.0 μm and 9.5 μm, respectively. The ~200 μm diameter field of view is imaged at 2 frames/s using a fiber-based excitation source at 1040 nm. Ex vivo imaging is demonstrated with unstained mouse lung at 5.9 mW average power. These results demonstrate the feasibility of three-photon GRIN lens endoscopy for optical biopsy.

Sub‐surface, micrometer‐scale incisions produced in rodent cortex using tightly‐focused femtosecond laser pulses

Techniques that allow targeted, micrometer‐scale disruption in the depths of biological tissue, without affecting overlying structures or causing significant collateral damage, could potentially lead to new surgical procedures. We describe an optical technique to make sub‐surface incisions in in vivo rodent brain and characterize the relationship between the cut width and maximum depth of these optical transections as a function of laser energy.

Multiphoton gradient index endoscopy for evaluation of diseased human prostatic tissue ex vivo

Abstract. Multiphoton microscopy can instantly visualize cellular details in unstained tissues. Multiphoton probes with clinical potential have been developed. This study evaluates the suitability of multiphoton gradient index (GRIN) endoscopy as a diagnostic tool for prostatic tissue. A portable and compact multiphoton endoscope based on a 1-mm diameter, 8-cm length GRIN lens system probe was used. Fresh ex vivo samples were obtained from 14 radical prostatectomy patients and benign and malignant areas were imaged and correlated with subsequent H&E sections. Multiphoton GRIN endoscopy images of unfixed and unprocessed prostate tissue at a subcellular resolution are presented. We note several differences and identifying features of benign versus low-grade versus high-grade tumors and are able to identify periprostatic tissues such as adipocytes, periprostatic nerves, and blood vessels. Multiphoton GRIN endoscopy can be used to identify both benign and malignant lesions in ex vivo human prostate tissue and may be a valuable diagnostic tool for real-time visualization of suspicious areas of the prostate.

Multiphoton microscopy: Applications in urology and andrology

Multiphoton microscopy (MPM) enables real-time imaging of various cellular processes at submicron resolution. MPM is currently being used in neuroscience, oncology, and immunology. MPM has demonstrated promising results in urology. MPM has been used in the identification of spermatogenesis, evaluation of bladder cancer, and tissue identification in prostate cancer surgery. MPM has allowed the visualization of seminiferous tubules within the testis in a rat model and identified areas of spermatogenesis. MPM could potentially improve the efficacy of testicular sperm extraction. In bladder cancer evaluation, MPM has proven to be an effective imaging tool in identifying areas suspicious for malignancy. The imaging technology could be utilized in the future to provide urologists with an immediate impression of extracted bladder tissue, or as part of a cystoscopic device to evaluate the bladder in real time. Similarly, MPM has proven to be a useful imaging technique to evaluate prostate cancer. MPM could be utilized during a prostatectomy to help differentiate prostate from cavernous nerves that are closely adherent to the prostate. MPM uses a laser and safety studies will need to be performed prior to its utilization in the clinical setting.

A model-based acoustic reconstruction algorithm for deep tissue clinical photoacoustic imaging of cancerous tissues labeled with contrast agents (Conference Presentation)

Molecular photoacoustic imaging of targeted agents in vivo can be a valuable tool for biopsy guidance, tumor detection and delineation. Recently, our group has developed a prototype clinical, side looking, photoacoustic and ultrasonic system based on capacitive micro-machined ultrasound transducers. The system was used for imaging both fresh ex vivo pancreatic cancer samples labeled with functionalized IRDye-800 as well as in vivo trans-rectal imaging of prostate cancer patients with and without Indocyanine green contrast agent. Beamforming algorithms were used to provide real-time imaging albeit their low contrast to background ratio. To improve the quality images, presented here is a model-based acoustic reconstruction technique. The model is adapted to a sector scanning convention common in ultrasonography, compensates for the effects of non-ideal element directivity, impulse response and uneven responsivity as well as the ultrasound’s time dependent gain and the medium’s acoustic attenuation. Despite the low element count and the very limited viewing angle of the transducer, this technique was capable of reconstructing high-quality photoacoustic images both ex vivo and in vivo with a significant increase in contrast compared to the commonly used Universal back-projection algorithm. For ex vivo imaging, the results are also compared with fluorescence imaging showing a high degree of correlation. This study demonstrates the feasibility and the potential of the model based reconstruction approach for real-time visualization of contrast agents in vivo deep within the tissue, either intraoperatively or in routine imaging. Thus, such approach opens avenues for better cancer detection, diagnosis, treatment and monitoring.

Multiphoton GRIN Endoscope for Evaluation of Human Prostatic Tissue Ex Vivo

We characterize the diagnostic performance of a multiphoton GRIN endoscope using human prostate samples obtained from radical prostatectomy surgery. Ex vivo images of benign and tumor areas and images of peri-prostatic tissue are shown.

Intravital Multiphoton Endoscopy

When a patient has symptoms that could be indicative of cancer a clinical oncologist will perform a series of tests to reach an accurate diagnosis. The gold standard method for reaching a final diagnosis frequently involves locating and extracting tissue biopsies, which are then processed and studied at the cellular level by a pathologist. From this procedure a pathology report will be compiled that contains the diagnosis of the cancer type and grade. Deep penetration, low-resolution, imaging tests (e.g., magnetic resonance imaging MRI) scans, computed tomography (CT) scans, Ultrasound, etc.) can track the extent and spread of disease and serve as an initial screen for disease in tissues that are not easily accessed using conventional biopsy or optical imaging techniques (e.g. periphery lung, pancreas, small intestine). However, to date, these low resolution imaging tests have not been able to be used as stand alone diagnostic tests because they lack the ability to visualize disease at the cellular level and thus have been unable to match the sensitivity and specificity of diagnosis based upon biopsied tissue processed into histopathology slides. Finally, by compiling the data obtained from all these tests, the oncologist can determine the stage of the cancer (i.e., the severity or extent of the cancer) and use this information to provide the patient with a prognosis and course of treatment.

Three-Photon Excited Fluorescence Imaging of Unstained Tissue Using a GRIN Endoscope

We present a three-photon GRIN endoscope system capable of imaging a field of view of 200 μm diameter at 4 frames/s. Ex vivo images of unstained mouse lung are shown.