Research in the So Lab

Standing-wave Total Internal Reflection Fluorescence Microscopy (SW-TIRM)

Microscopy for high resolution beyond the diffraction limit

Investigators: Euiheon Chung & Daekeun Kim

Light microscopy is widely used in biomedical research to study living biological systems. A major limitation of optical imaging is its inability to resolve objects with separation below several hundred nanometers. Even though there are several scanning-probe methods with higher resolution such as atomic force microscopy, there is significant resolution degradation in soft biological specimens and this method is inherently slow due to point-by-point scanning. Thus there is a need to develop a wide-field optical imaging method with below 100nm resolution.

To obtain lateral resolution beyond the diffraction limit in optical measurements, standing-wave total internal reflection fluorescence (SW-TIRF) microscopy has been developed. SW-TIRF uses the modulation of super-diffraction-limited evanescent excitation field to extract high spatial frequency content through the diffraction-limited optical imaging system. This system can obtain an effective point-spread function (PSF) with a full width at half maximum (FWHM) that is better than one sixth of the emission wavelength.

Fig.1 Schematic of objective-launch standing-wave total internal reflection fluorescence microscopy (SW-TIRF)

The use of standing evanescent wave imaging in a total internal reflection geometry have shown that lateral resolution better than 1/6 of emission wavelength can be achieved. The enhanced image results from the high-spatial frequency modulation to the conventional point-spread function.

A high NA objective lens-based standing wave total internal reflection fluorescence microscopy has been developed. One dimensional lateral resolution improvement using SW-TIRF is demonstrated with FWHM of about 100 nm that is approximately one third of the diffraction limit [1].
If this development is successful, the technology should have interesting applications in cell biology, single molecular level detection, protein studies and the examination of pathological specimens.

Fig. 2 Comparison of imaging 44 nm fluorescent beads

(a) conventional TIRF and (b) SW-TIRF. (a), (b) Planar images using TIRF and SW-TIRF and (c), (d) corresponding intensity profiles.

(The images were taken in the same setup while TIRF imaging was taken by blocking one excitation beam, A, B: regions of interest , NA=1.65, index of refraction of cover glass 1.788.)

Fig. 3. (a1), (a3) and (b1), (b3) are the enlarged images and their intensity profiles in y-direction of region A under TIRF.

(a2), (a4) and (b2), (b4), are the enlarged images and their intensity profiles in y-direction of region B under SW-TIRF.

1. E. Chung, D. Kim, and P.T.C. So, "Ultra-High Resolution Optical Imaging beyond the Diffraction Limit," The 2nd International Symposium on Nanomanufacturing, KAIST, Daejeon, Korea, Nov. 2004, pp. 158-163