Total Internal Reflection Fluorescence Microscopy (TIRFM)
Generally it is not difficult to realize the excitation of fluorescence probe molecules on the sample surface by using the evanescent field based on TIR, and there are many ways to choose. At the beginning of the studies of the, there has been widespread using prism or hemisphere combined with parabolic reflector to realize the TIR. This makes the instrument structure relatively complex, and prism quality and calibration requirement is high. Improved designs paid attention to the objective lens of the microscope in the process of seeking a new implementation method. Through constant improvement in design, the objective lens of total internal reflection microscopy that can be installed directly on the general fluorescence microscope appeared in the year 2000, making it extremely convenient to obtain total internal reflection fluorescence images. Its working principle is shown in Fig.2.6. In addition to the difference of the light on the transmission between the other objective lens, its numerical aperture is greater than 1.38. The experimental results show that the greater the numerical aperture, the higher the image quality.
The Application of TIRFM
TIRFM only excites the fluorescent probe molecules near the interface, and the molecular away from the interface cannot obtain enough energy to generate fluorescence radiation. Figure2.7 compares the different images of the same sample observed by TIRFM and ordinary fluorescence microscope. The fluorescence image obtained by TIRFM has a higher image contrast, suitable for the analysis of emitting mechanism.
Fig. 2.6 Schematic of total internal reflection fluorescence microscope
Fig. 2.7 Comparison of images by TIRFM and ordinary fluorescence microscope