Upgrade Your Lab to TIRF

Microscope and the TIRF condenser (at right), pre-installation. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS">Microscope and the TIRF condenser (at right), pre-installation. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS Total internal reflection fluorescence (TIRF) microscopy is a method that resolves molecular events specifically at or near the cell surface (see How It Works). Major microscope manufacturers, including Zeiss, Nikon, and Olympus, offer "turnkey" TIRF systems costing upwards of $100,000. But you can also upgrade an existing microscope to this capability. Berhard Wehrle-Haller, who runs a bioimaging facility at the University Medical Center in Geneva, did exactly that. He already had an old Zeiss microscope, CCD camera, software, and 488-nm laser on hand; all he needed was the TIRF condenser, which he obtained for about $20,000 from TILL Photonics of Gräfelfing, Germany. Though TILL does the installation about half the time, Wehrle-Haller installed his hardware himself. "The toughest thing was to align the laser," he says, "but the rest was very simple. I was done in half an hour." Since then he's added a second laser, as well as system to heat the stage and exchange gases for live-cell imaging. "We now use it several hours per week." Kimmo Tanhuanpää, leader of the light microscopy unit at the Institute of Biotechnology, University of Helsinki, had TILL Photonics install his TIRF system in September 2005. Between a misinstalled lens in the condenser and software conflicts with a new camera, Tanhuanpää has experienced some growing pains. "We got the initial problems ironed out about two months ago so the jury is still out for the final verdict," he writes in an E-mail. However, with the system now performing as expected - a paper, in which the system was used, was published last month1 - "I feel the upgrade was worth the cost," he says. If you'd also like give TIRF a try you'll need a laser (preferably 100 mW), laser fiber, a special objective with high numerical aperture (1.45 or more), and a TIRF condenser - an investment of between $30,000 and $60,000 overall. Though this hardware can be installed on any inverted microscope, the images shown here highlight the major steps in the process of installing TILL Photonics' TIRF system on a Zeiss Axiovert 200, a process that typically takes about two hours. jperkel@the-scientist.com 1. P. Hotulainen, P. Lappalainen, "Stress fibers are generated by two distinct actin assembly mechanisms in motile cells," J Cell Biol, 173:383-94, May 8, 2006. Remove the field and aperture stop retainer and disconnect all electronic connections. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS">Remove the field and aperture stop retainer and disconnect all electronic connections. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS Plug in the TILL TIRF condenser and fasten the screws again. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS">Plug in the TILL TIRF condenser and fasten the screws again. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS Screw the laser fiber into the condenser's rear port. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS">Screw the laser fiber into the condenser's rear port. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS Screw the light guide for epifluorescence illumination into the condenser's side port. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS">Screw the light guide for epifluorescence illumination into the condenser's side port. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS Install the TIRF objective (100x/1.45 NA shown); you'll also need to add an appropriate filter to the microscope's filter turret (not shown). Treat this objective as you would any other oil immersion optical device. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS">Install the TIRF objective (100x/1.45 NA shown); you'll also need to add an appropriate filter to the microscope's filter turret (not shown). Treat this objective as you would any other oil immersion optical device. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS Turn on the laser and use the thumbscrew on the condenser to align the laser. The laser is aligned when you can see laser light coming through the objective. Do not look down the barrel of the objective at this point! Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS">Turn on the laser and use the thumbscrew on the condenser to align the laser. The laser is aligned when you can see laser light coming through the objective. Do not look down the barrel of the objective at this point! Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS Deflect the laser into total internal reflection using the micrometer screw. At this point, you would be able to see a TIRF image. You can also switch between TIRF and epifluorescence modes by opening and closing the appropriate shutters on the condenser. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS">Deflect the laser into total internal reflection using the micrometer screw. At this point, you would be able to see a TIRF image. You can also switch between TIRF and epifluorescence modes by opening and closing the appropriate shutters on the condenser. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS