Detection of non-melanoma skin cancer by in vivo fluorescence imaging with fluorocoxib A.

Non-melanoma skin cancer (NMSC) is the most common form of cancer in the US and its incidence is increasing. The current standard of care is visual inspection by physicians and/or dermatologists, followed by skin biopsy and pathologic confirmation. We have investigated the use of in vivo fluorescence imaging using fluorocoxib A as a molecular probe for early detection and assessment of skin tumors in mouse models of NMSC. Fluorocoxib A targets the cyclooxygenase-2 (COX-2) enzyme that is preferentially expressed by inflamed and tumor tissue, and therefore has potential to be an effective broadly active molecular biomarker for cancer detection. We tested the sensitivity of fluorocoxib A in a BCC allograft SCID hairless mouse model using a wide-field fluorescence imaging system. Subcutaneous allografts comprised of 1000 BCC cells were detectable above background. These BCC allograft mice were imaged over time and a linear correlation (R(2) = 0.8) between tumor volume and fluorocoxib A signal levels was observed. We also tested fluorocoxib A in a genetically engineered spontaneous BCC mouse model (Ptch1(+/-) K14-Cre-ER2 p53(fl/fl)), where sequential imaging of the same animals over time demonstrated that early, microscopic lesions (100 µm size) developed into visible macroscopic tumor masses over 11 to 17 days. Overall, for macroscopic tumors, the sensitivity was 88% and the specificity was 100%. For microscopic tumors, the sensitivity was 85% and specificity was 56%. These results demonstrate the potential of fluorocoxib A as an in vivo imaging agent for early detection, margin delineation and guided biopsies of NMSCs.

In vivo near-infrared dual-axis confocal microendoscopy in the human lower gastrointestinal tract.

Near-infrared confocal microendoscopy is a promising technique for deep in vivo imaging of tissues and can generate high-resolution cross-sectional images at the micron-scale. We demonstrate the use of a dual-axis confocal (DAC) near-infrared fluorescence microendoscope with a 5.5-mm outer diameter for obtaining clinical images of human colorectal mucosa. High-speed two-dimensional en face scanning was achieved through a microelectromechanical systems (MEMS) scanner while a micromotor was used for adjusting the axial focus. In vivo images of human patients are collected at 5 frames/sec with a field of view of 362×212 µm(2) and a maximum imaging depth of 140 µm. During routine endoscopy, indocyanine green (ICG) was topically applied a nonspecific optical contrasting agent to regions of the human colon. The DAC microendoscope was then used to obtain microanatomic images of the mucosa by detecting near-infrared fluorescence from ICG. These results suggest that DAC microendoscopy may have utility for visualizing the anatomical and, perhaps, functional changes associated with colorectal pathology for the early detection of colorectal cancer.

Optical design and imaging performance testing of a 9.6-mm diameter femtosecond laser microsurgery probe.

We present the optical design of a 9.6-mm diameter fiber-coupled probe for combined femtosecond laser microsurgery and nonlinear optical imaging. Towards enabling clinical use, we successfully reduced the dimensions of our earlier 18-mm microsurgery probe by half, while improving optical performance. We use analytical and computational models to optimize the miniaturized lens system for off-axis scanning aberrations. The optimization reveals that the optical system can be aberration-corrected using simple aspheric relay lenses to achieve diffraction-limited imaging resolution over a large field of view. Before moving forward with custom lenses, we have constructed the 9.6-mm probe using off-the-shelf spherical relay lenses and a 0.55 NA aspheric objective lens. In addition to reducing the diameter by nearly 50% and the total volume by 5 times, we also demonstrate improved lateral and axial resolutions of 1.27 µm and 13.5 µm, respectively, compared to 1.64 µm and 16.4 µm in our previous work. Using this probe, we can successfully image various tissue samples, such as rat tail tendon that required 2-3 × lower laser power than the current state-of-the-art. With further development, image-guided, femtosecond laser microsurgical probes such as this one can enable physicians to achieve the highest level of surgical precision anywhere inside the body.

In vivo imaging of human and mouse skin with a handheld dual-axis confocal fluorescence microscope.

Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC) microscope that is capable of in vivo fluorescence imaging of skin, using both mouse models and human skin. Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the efficacy of experimental molecular therapeutics.

Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope.

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery.

A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-of-focus light for high-contrast optical sectioning. A biaxial microelectromechanical system (MEMS) scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500x500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections.

In vivo brain imaging using a portable 2.9 g two-photon microscope based on a microelectromechanical systems scanning mirror.

We present a two-photon microscope that is approximately 2.9 g in mass and 2.0 x 1.9 x 1.1 cm(3) in size and based on a microelectromechanical systems (MEMS) laser-scanning mirror. The microscope has a focusing motor and a micro-optical assembly composed of four gradient refractive index lenses and a dichroic microprism. Fluorescence is captured without the detected emissions reflecting off the MEMS mirror, by use of separate optical fibers for fluorescence collection and delivery of ultrashort excitation pulses. Using this microscope we imaged neocortical microvasculature and tracked the flow of erythrocytes in live mice.

Three-dimensional in vivo imaging by a handheld dual-axes confocal microscope.

We present a handheld dual-axes confocal microscope that is based on a two-dimensional microelectromechanical systems (MEMS) scanner. It performs reflectance and fluorescence imaging at 488 nm wavelength, with three-dimensional imaging capability. The fully packaged microscope has a diameter of 10 mm and acquires images at 4 Hz frame rate with a maximum field of view of 400 microm x 260 microm. The transverse and axial resolutions of the handheld probe are 1.7 microm and 5.8 microm, respectively. Capability to perform real time small animal imaging is demonstrated in vivo in transgenic mice.

Miniaturized probe for femtosecond laser microsurgery and two-photon imaging.

Combined two-photon fluorescence microscopy and femtosecond laser microsurgery has many potential biomedical applications as a powerful "seek-and-treat" tool. Towards developing such a tool, we demonstrate a miniaturized probe which combines these techniques in a compact housing. The device is 10 x 15 x 40 mm(3) in size and uses an aircore photonic crystal fiber to deliver femtosecond laser pulses at 80 MHz repetition rate for imaging and 1 kHz for microsurgery. A fast two-axis microelectromechanical system scanning mirror is driven at resonance to produce Lissajous beam scanning at 10 frames per second. Field of view is 310 microm in diameter and the lateral and axial resolutions are 1.64 microm and 16.4 microm, respectively. Combined imaging and microsurgery is demonstrated using live cancer cells.

Miniature near-infrared dual-axes confocal microscope utilizing a two-dimensional microelectromechanical systems scanner.

The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5-7 microm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 x 100 microm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy.

Fiber-optic confocal microscope using a MEMS scanner and miniature objective lens.

We designed and constructed a single-fiber-optic confocal microscope (SFCM) with a microelectromechanical system (MEMS) scanner and a miniature objective lens. Axial and lateral resolution values for the system were experimentally measured to be 9.55 mum and 0.83 mum respectively, in good agreement with theoretical predictions. Reflectance images were acquired at a rate of 8 frames per second, over a 140 mum x 70 mum field-of-view. In anticipation of future applications in oral cancer detection, we imaged ex vivo and in vivo human oral tissue with the SFCM, demonstrating the ability of the system to resolve cellular detail.

Fast-scanning two-photon fluorescence imaging based on a microelectromechanical systems two- dimensional scanning mirror.

Towards overcoming the size limitations of conventional two-photon fluorescence microscopy, we introduce two-photon imaging based on microelectromechanical systems (MEMS) scanners. Single crystalline silicon scanning mirrors that are 0.75 mm x 0.75 mm in size and driven in two dimensions by microfabricated vertical comb electrostatic actuators can provide optical deflection angles through a range of approximately16 degrees . Using such scanners we demonstrated two-photon microscopy and microendoscopy with fast-axis acquisition rates up to 3.52 kHz.

Single fiber confocal microscope with a two-axis gimbaled MEMS scanner for cellular imaging.

We present a single fiber reflectance confocal microscope with a two dimensional MEMS gimbaled scanner. Achieved lateral and axial resolutions are 0.82 mum and 13 mum, respectively. The field of view is 140 x 100 mum at 8 frames/second. Images and videos of cell phantoms and tissue are presented with sub-cellular resolution.