5.3 Objective-Based TIRFM

Figure 5.11: Schematic of a TIRFM

TIRFM is short for total internal reflection fluorescence microscopy.

5.3.1 Total Internal Reflection

Figure 5.12: Law of Refraction in TIRFM

The law of refraction states the following in this case:

$$\begin{equation} 1.52 \times \sin(\theta_1) = 1.33 \times \sin(\theta_2) \end{equation}$$

When a colliminated light beam goes from glass into water, the refractive angle $\theta_2$ is larger than the incident angle $\theta_1$. As $\theta_1$ increases, so does $\theta_2$ - the refracted light is dimmer and the reflected light stronger.

When $\theta_1 \ge 60^\circ$ (i.e., the critical angle $\theta_c$), the incident ray is reflected back into the glass. Otherwise, when $\theta_2 = 90^\circ$, the direction of refracted light is parallel to the interface.

5.3.2 Evanescent Field

Figure 5.13: An Evanescent Field

This is a result of the total internal reflection at the glass and aqueous interface. The field is essentially a small portion of light (at about 100 to 200 nm) that illuminates a cell at the glass and aqueous solution interface.

Within this field, light intensity drops as distance from the interface increases. Only adherent cells’ plasma membranes can be observed by TIRFM.

5.3.3 Basic Approaches to TIRFM

Figure 5.14: Components of Objective-Based TIRFM

A high NA objective is needed.