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The Nelson laboratory has a Zeiss Axiophot fluorescence microscope for high resolution immunofluoresence microscopy equipped with CCD camera for digital image capture. We have unlimited access to a confocal microscope with capacity for high resolution DIC microscopy and time-lapse video recording in the laboratory of Dr. Stephen Smith in this Department. However we have recently purchased a new microscope for live cell imaging that has the specifications needed for the proposed experiments, and therefore will described in detail. The MarianasTM with FRAP microscopy system designed for live cell imaging was assembled in collaboration with Intelligent Imaging Innovations, a company specialized in the development of both software and hardware for live-cell imaging applications. This system provides us with the ability to perform advanced microscopy-based methods such as 6D microscopy (dual-color 3-dimensional time lapse imaging), fluorescence recovering after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET). These methods will allow us to image and dissect molecular processes as they happen in unperturbed living cells. The microscope is equipped with a motorized Zeiss Axiovert 200M microscope that includes Plan-Apo 63X 1.4 NA and 100X 1.4 NA objectives with DIC prisms as well as a Coolsnap HQ, an interlined high speed cooled CCD camera. For experiments that require prolonged periods of imaging, an objective heater system (heater collar and controller) prevents the objectives from drifting in the z-axis. The Zeiss 200M is also equipped with a x-y motorized stage that includes 0.1 m linear encoders on each axis and joystick control. This allows us to select and record locations in x,y and z for multiple point visitations. For 3-D imaging of rapid intracellular phenonema (ie. cytoskeleton dynamics and vesicle fusion), the microscope is equipped with a piezoelectric focusing collar (for rapid movement of the objective in the z-axis), a xenon dual-galvanometric fast excitation source (for rapid switch between excitation filters), and a Solamere XR/MEGA-12 XR intensified CCD camera (for minimal exposure time). The combination of these three devices allows us to collect stacks of images in the order of 100s of milliseconds and gives us capability for ratiometric imaging experiments. In addition to these devices, a motorized infocus spherical aberration collar has been purchased so that aberrations arising from the use of oil objectives to image samples in acqueous solutions are eliminated. The result is an image of increased optical clarity and fluorescence intensity. The microscope includes a FRET/FRAP photoablation system that consists of a fiber optically pumped dye laser, a computer controled beam position and intensity, and a diffraction limited spot size. This device allows us to perform fluorescence recovery after photobleaching (FRAP) at intracellular regions of variable size (from diffraction limited spots to areas of a several square microns). In addition, FRET experiments can be performed in fixed samples. The microscope is equipped with a quad filter set for DAPI/FITC/CY3/CY5, a CFP/YFP filter set for FRET and an EGFP/dsRed dual emission filter. In addition, a bright field shutter has been purchased for switching between fluorescence and DIC images. The system is operated by the SlideBook 4.0 software (Image Capture Integration module, 3-D deconvolution module, 3D dimensional analysis, rendering and exploration module, stereology and montage module, FRET module, 4D analysis and particle tracking module, spherical aberration correction module and FRAP module) in Dell Precision workstations (dual 2.0GHz Xeon processors, dual 73GB Hard Drives and 2.0GB RDRAM). |
