RRC-CMF: Laser TIRF
|Zeiss Laser TIRF Microscope / Imaging System|
The Zeiss Laser TIRF imaging system is a microscope capable of visualising molecular level processes at or near cell membranes via the Total Internal Reflection Fluorescence (TIRF) principle. When properly configured, this imaging technique can yield narrow imaging depths of less than 100 nm from a glass-specimen interface. (Note that TIRF requires a microscope objective with a high numerical aperture; such as the 100x/1.45 alpha Plan-Fluar is the only available option on the current system.)
The TIRF microscope is also outfitted with a complete Pecon XL TIRF S incubation system, which allows for the stabilization of temperature, CO2, and humidity with various stage inserts. These environmental variables can be controlled directly and easily through the AxioVision acquisition software to completement any live cell imaging approaches.
Even if the TIRF imaging is not feasible for a given sample, the system and its environmental controls can be used in transmitted light (bright field, phase contrast, differential interference contrast) or fluorescent imaging modes (the latter including reflectors for DAPI, CFP, GFP, YFP, and RFP-like signals). The microscope base features a fully motorized stage that, when initialized through the software, can scan preset areas of a single dish. The result is an ability to capture multiple time course experiments simultaneously from a single dish, which is well-suited for studies of cell migration, proliferation, or dynamic cell morphology.
Also available in the AxioVision software is the MosaiX module, an imaging processing algorithm that makes use of the motorized stage to stitch multiple images into a single seamless, tiled layout. Entire dishes or coverslips can thus be scanned and reconstructed as enormous, zoomable images.
|Laser Unit||Wavelength||Maximum Power||Status|
|Argon||458, 488, 514 nm||25 mW||ok|
|Diode 561-40||561 nm||40 mW||ok|
|Magnification||Model Name||Immersion Type||NA||Working Distance|
|10x||EC Plan-Neofluar||dry||0.30||5.2 mm|
|100x||alpha Plan-Fluar||oil||1.45||0.11 mm|
The Pecon XL TIRF S incubation system features several overlapping units such as the Heating Unit XL S and the TempModule S (see image, first right) that together heat the ambient air surrounding the enclosed microscope stage by convection. The latter is also used to control the temperature of the Heating Insert P S (see image, second right), a stage adapter that heats dishes directly by conduction.
In addition, the CO2-Module S can be used to regulate concentrations of carbon dioxide, as delivered through a transparent insert cover (not shown).
1. Locate the main power strip attached to the right side of the microscope table. Press the power switch on the front face.
2. Turn on the microscope base and touchscreen by pressing the round silver button on the back left of the microscope.
3. If fluorescence will be viewed using the microscope eyepieces, turn on the metal halide lamp by pressing the power switch.
4. If imaging green/yellow signals with laser excitation, power up the Lasos argon laser by pressing the black power switch. Next, turn the key to the right (clockwise) to activate the cooling fan.
5. The Lasos argon laser can function in STANDBY POWER position. However, to set the argon laser intensity to 50% of its total, flip the silver switch on the controller to the RUN position. A green light labeled OPTIMAL should be visible. Next, dial up the current (with the black knob) until the red light marked REDUCED LIFETIME appears. Dial the black knob back again until this warning light cuts out; this is the 50% point.
6. The computer should already be on standby; if not, power it on. Log on the system using the username and password that was created during microscope training.
7. Double-click the AxioVision software icon, position the specimen on the stage, and begin imaging.
1. When finished with imaging, shut down any incubation controls that may have been activated in the AxioVision softare. Save all files, exit the AxioVision program, and log out of Windows XP.
2. If it was in use, turn off the fluorescence illuminator by pressing the power switch.
3. Dial down the current of the argon laser, and flip the silver switch down to the STANDBY POWER position.
4. Turn the key on the Lasos power controller to the left (counterclockwise) and wait 2-3 minutes for the cooling fan to stop. Once quiet, push the black power switch to OFF.
5. Turn off the microscope base and touchscreen by pressing the round silver button on the back left of the microscope.
6. Disconnect all idle power running to the components by simply pressing the switch on the power strip. Following this, everything should be off except the PC - which can be left on standby.