TIRF - Total Internal Reflection Fluorescence

How is TIRF technically realized in the TILL setup?

Changing the example described above to a more biological one - by using a cell surrounded by media instead of a water droplet and a cover slip instead of simple glass − shows the possibility to investigate biological applications.

As shown in the previous section, one has to control the angle of the incident light. In our setup we use a laser as an excitation source. The laser is focused into the back focal plane of the objective. Assuming the laser is focused into the center of the back focal plane, the light exits the objective along the optical axis of the system. Moving the focus away from the center towards the edge of the back focal plane leads to the fact that the light exits the objective under a certain angle. The further you move the focus away from the center, the bigger the angle of the exiting light gets. To be able to reach the critical angle necessary for total internal reflection, you need a least an objective with an NA of > 1.39 for a glass/water interface. Using a standard 1.4 NA objective makes it nearly impossible to adjust a TIRF system. Therefore objectives with extremely high NA have been developed. The standard TIRF objective has an NA of 1.45. The highest NA objective commercially available is the 1.65 from Olympus. The NA of the objective limits the achievable angle of the excitation light, and therefore also limits the minimal penetration depth!