Upgrade Your Lab to TIRF

Why spend six figures on a new total internal reflection fluorescence system when you can upgrade your existing microscope for just $30,000?

Jeffrey M. Perkel
Jun 1, 2006
<figcaption>Microscope and the TIRF condenser (at right), pre-installation. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS</figcaption>
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...

<figcaption>Remove the field and aperture stop retainer and disconnect all electronic connections. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS</figcaption>
Remove the field and aperture stop retainer and disconnect all electronic connections. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS
<figcaption>Plug in the TILL TIRF condenser and fasten the screws again. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS</figcaption>
Plug in the TILL TIRF condenser and fasten the screws again. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS
<figcaption>Screw the laser fiber into the condenser's rear port. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS</figcaption>
Screw the laser fiber into the condenser's rear port. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS
<figcaption>Screw the light guide for epifluorescence illumination into the condenser's side port. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS</figcaption>
Screw the light guide for epifluorescence illumination into the condenser's side port. Credit: IMAGE COURTESY OF MATTHIAS ASCHERL, TILL PHOTONICS
<figcaption>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</figcaption>
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
<figcaption>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</figcaption>
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
<figcaption>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</figcaption>
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