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.
