Two of our own laboratory's instrumentation developments comprise much of our core research and development (R&D) program. These are Multiphoton Microscopy (MPM, Denk et al, 1990) and Fluorescence Correlation Spectroscopy (FCS, Magde et al, 1972). Multiphoton microscopy was first invented, developed and guided into commercial reality in this laboratory using NCRR funding, which demonstrates the implementation of one of the primary goals of a Biomedical Technology Resource Center. The explosive growth of MPM has been driven primarily by the overwhelming advantages of multiphoton excitation (MPE) in meeting demands for functional imaging deep in living tissues. Today, MPM is a successful technique with an essentail niche in which it excels: live cell and tissue imaging, and more recently in vivo or intra-vital imaging in live animals. Our broad goals for this research funding period range from instrument and laser development to MPM applications research and development. The following outline lists the core R&D sections.
Project I - Technology Development for Improved Intravital Multiphoton Microscopy
The goal of this project is to improve multiphoton imaging for applications requiring high resolution imaging in tissue and live animals, an area where MPM has become the preeminent imaging tool surpassing all other available forms of microscopy. In this project we (1) outline the design, construction and optimization of the next generation multiphoton imaging system engineered to advance the field of multiphoton microscopy and meet the challenges of our core and collaborative research, (2) we investigate the factors that limit deep MPM imaging and develop new technologies to overcome the these limitations, and (3) develop new fluorescent labeling techniques and probes specifically designed for tissue and live animal imaging applications.
Aim 1: Multiphoton microscopy hardware and software development
Aim 2: Strategies for deeper imaging in tissue
Aim 3: Multiphoton probe developments for intravital imaging
Project II - Development of Novel Femtosecond Light Sources
We are now developing new fiber and fiber laser technologies that will not only significantly increase the accessibility of femtosecond sources to biomedical researchers but also expand the spectral windows of biomedical imaging. The proposed research program consists of two components: (1) Developing turn-key, wavelength tunable, all-fiber, energetic femtosecond sources at the wavelength range of 1060 to 1300 nm, outside the spectral range that is currently provided by the femtosecond Ti:S laser; and (2) developing a novel optical fiber platform that will enable the existing femtosecond Ti:S laser to efficiently reach wavelength between 1060 and 1300 nm. The program involves close collaboration between Cornell University and our industrial partner OFS Laboratories, with design input from collaborating biomedical researchers.
Aim 1: Design of a novel HOM fiber platform
Aim 2: An all-fiber femtosecond source with a tuning range of 1060 to 1300 nm
Aim 3: Femtosecond wavelength conversion from the Ti:S laser to wavelengths between 1060 and 1300 nm
Project III - Endoscopic and Clinical Imaging
This project is focused on developing instrumentation and infrastructure for clinical microscopy, fiber-delivered laparoscopic and endoscopic imaging devices for research and clinical use.
Aim 1: Fiber delivered imaging instrumentation
Aim 2: Characterization of intrinsic tissue signals and other contrast sources for clinical use
Project IV - Technology Development For Studies of Cellular Regulation
In this project we develop instrumentation and methods for our collaborative experiments in the area of intracellular and extracellular cell signaling, cellular regulation and in vivo and in vitro analysis of protein aggregation. We refine our applications of fluorescence correlation spectroscopy (FCS) and other single molecule methods for studies of protein-protein interactions, protein-nucleic acid interactions and intracellular diffusion. Quantitative fluorescence applications in cells has always been an active area for our Resource, and we anticipate many new projects requiring these techniques with the relocation of DRBIO to the new life sciences building (Weill Hall) at Cornell, which also houses the Weilll Institute for Cell and Molecular Biology (WICMB), directed by Scott Emr. The WICMB will become an important new source of collaborators that drive our technology development in the areas such as FRET, FLIM, FCS and high resolution optical imaging methods for use in cells.
Aim 1: Improved instrumentation for fluorescence measurements in cells
Aim 2: Studies of protein aggregation and folding
Aim 3: Improving second harmonic based membrane potential imaging