Axiovert 200M microscope, stage incubator, META scanning module, UV laser, visible laser module, image acquisition computer
The laser scanning confocal microscope (LSM) is configured to undertake all confocal microscope techniques with fixed and living cells except two-photon excitation. The LSM includes a Zeiss Axiovert 200M inverted microscope equipped with high-resolution optics for fluorescence microscopy and Normarski differential interference microscopy; a scanning module with two independent fluorescence channels and a third fluorescence channel, whose output is directed to a 32-PMT array; a UV laser with excitation wavelengths of 351 and 364 nm; an Argon laser with excitation wavelengths of 458, 488, and 514 nm; two HeNe lasers with excitation wavelengths of 543 and 633 nm; and a data acquisition computer with two 21” monitors.
Optics, Normarski differential interference contrast, Filter module, Condenser, Lamps, Stage incubator
The Axiovert 200M inverted microscope is equipped with plan-neofluar and plan-apochromat objectives from 10- to 100-fold magnification (plan-neofluar 10X/0.30, planapochromat 20X/0.75, plan-neofluar 40X/0.85 Pol, C-apochromat 63X/1.2 water immer., plan-apochromat 100X/1.4 oil immer.); Normarski differential interference contrast sliders for objectives of 20, 40, and 63X magnification; a filter module with fluorescence filter sets for Dapi/Hoechst, fluoroscein/fluo-3, and rhodamine; an achromatic/aplanatic condenser (0.9 NA for bright field, dark field, phase contrast, and differential interference contrast), a 50-watt HBO lamp for incident illumination and a 100-watt halogen lamp for transmitted light illumination, and a model S stage incubator.
CTI controller, Temperature controller, Stage insert and stage incubator S
A Zeiss stage incubator model S, together with CTI, digital temperature, and humidity controllers, is available for experiments involving living cells. This device is particularly useful for those investigators doing time-course and/or physiologic studies. The stage incubator will accommodate microscope slide cultures (1” x 3”) and maintain these cultures at 37°C or other temperatures at a set CO2 concentration and relative humidity, while monitoring the stage insert temperature, air temperature, CO2 concentration and relative humidity.
Fluorescence channels, META PMT array, pinholes, ATOF, independent fiberoptic inputs for UV and visible lasers
The UV/visible META scanning module with individual pinholes for each detector provides identical optical slice thickness with multi-channel fluorescence analysis. The positioning of the pinholes and their size are controlled by the instrument operator to maximize confocal Z resolution. The scanning module is configured with two independent fluorescence channels with single PMTs and a third fluorescence channel in which the fluorescence emission is separated by wavelength and recorded by a 32-PMT array. The images acquired from the two independent fluorescence channels and the META-PMT array can be recorded and displayed simultaneously. Individual Acousto-Optical Tunable Filters (AOTFs) are used to attenuate and select UV and visible wavelengths. Up to eight images can recorded and displayed using user-selected combinations of excitation wavelength, filters and detectors (Multi-Tracking). Multi-tracking features fast switching of laser excitation and eliminates bleed through. The AOTFs also permit excitation (and bleaching) of a region of interest (ROI) of any size or shape, as selected by the operator. The scanning module is equipped with UV beam splitters at 375, 488, 543, & 633 nm; main beam splitters at 477 & 543 nm; primary color dividers at 458 & 514 nm; secondary color dividers at 490, 514, 545, & 635 nm; bandpass filters of 385-470, 435-485, 475-525, 505-530, 505-550, 530-600, 560-615 nm; and longpass filters at 385, 505, 560, 650 nm. Image size is user-selectable with an image rate up to 2600 lines per second and image depth from 8- to 12- bit digitization (256 to 4096 gray levels). Digital scan control together with other LSM510 features facilitates 1 to 8X zoom, ROI scans, and scan rotation.
UV excitation, nuclear localization, Ca2+ measurements
The Coherent Enterprise 653 II UV laser (Ar) has an output of 81 mWatts with excitation lines at 351 and 364 nm. The UV laser makes it possible to excite Dapi/Hoechst stain and to definitively locate the nucleus of eucaryotic cells. The availability of UV excitation also permits the use of indo-1 to measure intracellular free Ca2+ concentrations ([Ca2+]i).
Visible laser excitation, Argon visible laser, HeNe lasers
The visible laser module consists of an Argon laser (25 mWatt) with three laser lines (458, 488, & 514nm) and two HeNe lasers (1 mWatt, 5 mWatt) with one laser line each (543, 633nm, respectively)
Computer, programs, output devices
The image acquisition computer is configured with a Pentium IV processor at 1.8 GHz, 512 Mb RAM at 400 MHz, 40 Gb ultra-wide SCSI hard drive, Matrox Millunium G450 video card, and two 21” monitors. Output devices include a 24X RW CD-ROM, 3.5” floppy-drive, and Codonics NP-1600 and other printers. The operating system is Windows NT. Software includes Zeiss LSM 510 W.S., v3.0; Zeiss PHYSIOLOGY, v3.0; and Zeiss image VISART, v3.0, and METAMORPH, v6.0.