We have designed and constructed using commercially available components a high speed confocal and widefield microscopy facility for intravital microscopy of the microcirculation in a living mouse. This instrument is a combination of a fluorescence microscope, a fluorescence spectrophotometer and a video camera. This is a digital instrument that allows simultaneous collection of images in up to four separate channels, each with a different fluorescence color or brightfield image. We use an Olympus AX-70 fluorescence microscope with a special stage for physiologic experiments, a long distance condenser and triple ports for the binocular objectives, the confocal scanner, and the widefield port. We have acquired a full set of oil immersion objectives and water immersion objectives. One objective holder is equipped with a piezoelectric driver and its controller to allow computer-controlled changes in the focal plane as rapidly as every 20 msec and movements as small as 0.5 microns; this is used to collect a series of images in the z-plane that can be stacked to generate 3D image reconstructions.
Confocal fluorescence microscopy employs the Yokogawa CSU-10 confocal scanner, a system that is based upon Nipkow disk technology and uses microlenses in the multiple pinholes on the disk. Unlike a point scanning confocal microscope that takes 1-2 sec to collect a full image, the Yokogawa confocal scanner can collect up to 360 images per sec. We are limited by the amount of light and the readout rate of the digital cameras. In practice, this system can run at 10-60 images per second, depending upon the experimental system. This system is equipped with an argon-krypton three-line laser for excitation at 8ex 488 nm, 8ex 568 nm, 8ex 647 nm and a Sutter Lambda L-10 filter wheel on the excitation source.
Widefield fluorescence microscopy uses a Sutter Lambda DG-4 high speed wavelength changer (changing time=1.2 msec) equipped with a high intensity 175 watt Xenon light source. We employ four excitation filters (8ex 360 nm, 8ex 480 nm, 8ex 575 nm, 8ex 655 nm) in this system. The light tube is introduced into the epi-illumination port of the fluorescence microscope. To obtain a brightfield channel in real time while also collecting image data in fluorescence channels, a UniBlitz shutter that is computer-controlled provides transmitted light for a 50 msec pulse.
For both confocal and widefield imaging, light is amplified up to 1000-fold using a Gen III Videoscope image intensifier. A Roper CoolSnap HQ CCD camera captures high resolution (1390 x 1024) images; it has the highest readout speed available for a digital camera (20 MHz) and allows unbinned full images to be captured at about 20 images per second in a single channel. A Cooke SensiCam high speed camera (640 x 480) allows us, with subarrays, to capture up to 60 frames per second-thus enabling us to obtain 20 frames per second for each of three fluorochrome channels. Both high speed digital cameras are cooled to limit dark noise and are computer-controlled. A Uniblitz shutter on the transmission light source is computer controlled and can be opened and closed in 50 msec. This allows us to collect brightfield images while fluorescence images are also being collected.
A Dell workstation is used to control the components of the imaging system and to collect images. This workstation has dual 1 GHz processors, 1.5 GB of RAM, two SCSI hard drives (18 GB and 73 GB), a high end video card, and an 50/90 AID drive for tape storage of data. SlideBook, an imaging software package from Intelligent Imaging Innovations, has been acquired for data capture and control are all of the systems: computer, CCD cameras, excitation shutters, Sutter high speed filter wheels or wavelength changer, Uniblitz shutter, piezoelectric driver. Data analysis is also performed with SlideBook, using a series of modules that allow image reconstruction, deconvolution, statistics, and volume analysis.
A second Dell workstation is offline from the microscope and is configured with SlideBook exclusively for data analysis. The hardware configuration is similar to the online Dell workstation. The advantages of a digital imaging system include: (1) Each data set is permanent; images and subimages retain original resolution; (2) Quantitative analysis of pixel magnitude is enabled; (3) Subarrays, with fewer pixels, can be readout from the CCD camera at higher frame rates without increased noise; (4) Deconvolution technology, including Fourier transform, can be applied to increase signal to noise ratio; (5) Background subtraction, image reconstruction, and color analysis are enabled. Image analysis uses fluorescence data collected in two dimensions; stacking of two dimensional arrays leads to 3D image reconstruction.