Multiphoton Instrumentation for Translational Assays from Human Tissue Biopsies

Krummel, Matthew

Abstract

We are requesting funds to purchase a state-of-the-art commercial multiphoton microscope specifically configured and situated to accommodate a portfolio of translational imaging approaches, largely dedicated to pursuing studies of cell-cell dynamics in human normal and diseased biopsy tissues and organ slices. The instrument will be specifically configured and certified for human tissues/BSL-2 and will be located in the UCSF Parnassus Heights Biological Imaging Development Center's (BIDC) development space, adjacent to the UCSF hospital. This application represents a collaboration of expertise from clinical and basic scientists, as described in the proposal. This will provided much-needed capacity through a facility well-versed in the art and will facilitate the transition of the technology into promising areas that are currently marginalized by instrument availability and configuration. The instrument will be primarily used by a core of 11 NIH funded investigators and their laboratories which facilitate 15 NIH-funded R- P- or U- level projects. The principal type of experiment for which this microscope system will be used include multidimensional timelapse imaging of cells in living tissues, typically revealed with fluorochrome conjugated non-stimulatory antibodies or protein conjugates, directly applied to tissue biopsies or to redundant transplant tissues. It will also accommodate imaging of a wide variety of fluorescent proteins in comparative mouse models. Located in the BIDC and utilized approximately 80% by the investigators on this proposal, it will also be generally accessible by the larger campus community served by this facility. The system we are requesting, a Zeiss 710LM was chosen amongst A-B trials comparing our custom built microscopes, whose design has proven compatible with deep tissue/high sensitivity data collection, with the commercial offerings. The microscope will be equipped with an environmental chamber as well as modules for on-stage organ culture, a motorized stage, and high-efficiency PMTs for high resolution and high sensitivity imaging. Dipping water immersion objectives are requested for the optimal collection of data from live cells or tissues in a 3D environment at the highest achievable optical resolution. A combination of high-sensitivity PMTs and 32-element 'spectral'PMT detection arrays is requested to permit imaging of all commonly used dye combinations and/or fluorescent protein variants but also to extend local success using multichannel autofluorescence background subtraction methods to enhance weaker signals. The public userbase at UCSF has two highly functional custom-built multiphoton instruments and two older commercial adaptations. However, the functional instruments are oversubscribed due to the high volume and long timelapses of the typical experiments being run. In addition, 'translational'imaging requires a microscope which is both specifically certified for human studies and which has sufficient capacity to accommodate the short lead-times of human tissue samples.