We request funds to purchase a multiphoton laser scanning microscope (Zeiss LSM 710 NLO) as a replacement for our outdated BioRad MRC-1024 MP which we have had since 1999. Studies using this instrument were a key to us obtaining two PPG grants (NIDDK and NHLBI), a NIDDK GI Core center grant, and several RO1s, all of which are ongoing. Service of all BioRad multiphoton instruments have been discontinued for the last 3 years and parts for the microscope, scanner, detectors and our old generation of Tsunami laser for multiphoton excitation (Spectral Physics) have been discontinued as well. Replacement of this instrument is a necessary step to assure the successful experimental continuity of these NIH awards. This microscope has 4 major and 3 minor users in three broad research areas: diarrheal diseases, cancer and cardiology. All of the major and minor users have funded NIH projects, the aims of which require use of a multiphoton microscope to accomplish the specific aims. As in the case of the current instrument, the proposed multiphoton system will be a part of the Ross Research Building Fluorescent Microscopy Core which is part of an NIH/NIDDK GI Core Center, of which Dr. Donowitz is the PI and Dr. Kovbasnjuk is the Imaging Core Co-Director and both of whom are major users. This Ross Microscopy Core (://www.hopkinsmedicine.org/confocal/) has provided instruments and training for Hopkins scientists for 15 years and has been used by over 100 laboratories at JHUSOM, including advanced imaging applications which include Forster Resonance Energy Transfer (FRET), Fluorescence Recovery After Photobleaching (FRAP), deconvolution, Second Harmonic Generation (SHG), quantitative imaging protocols for pH-sensitive SNARF-4Fdye, Ca indicator dyes, NAD(P)H, GFP, as well as deep tissue immunofluorescence protocols, etc. User charges in part along with NIH funding support the service contracts for the multiphoton as well as our new Zeiss LSM 510/META confocal system and for a Core Manager who oversees both administrative and scientific Core activities. Institutional support provides back up in case of NIH funding lapses or major equipment malfunction. The proposed instrument will be only the second system at Hopkins available for the general JHUSOM research community.
