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Probing molecular interactions by nano-scale microscopy----Microtubule kinetics in living cells using TIRFM Chien-Hua Chen1, Shu-Jung Yu2, Chi-Hung Lin1 and Din-Ping Tsai2 Institute of Biophotonics, Nation Yang Ming University, Taipei 10617, Taiwan, R.O.C1 Department of Physics, Nation Taiwan University, Taipei 10617, Taiwan, R.O.C2

1. Introduction The use of total internal reflection fluorescence microscopy (TIR-FM) in living cells has shed new light on important cellular processes taking place near the plasma membrane. It provides a way to selectively excite fluorephores in an aqueous or cellular environment that is very close to a substrate. TIR-FM is based on the surface evanescent field that occurred when light is internally reflected at the interface between two materials that have different refraction indices. Because the intensity of evanescent field decays with the increase of distance normal to the interface, excitations of the fluorescent molecules happened only in the near-zone of the interface. The background signals originated from the excitation light can therefore be removed, resulting in a high signal to noise ratio (SNR) at the nano-scale depth of interface. Sample 60X, NA=1.45 Oil objective

Diode pump solid state laser λ=532nm Or Argon-ion laser λ=488 n m

Dichroic mirror

CCD

Fiber illuminator 40x, NA = 0.65 Objective Field aperture

Fig. 1 Scheme of the experimental setup.

TIR-FM is a particularly useful technique for studying surface-associated cellular processes at a molecular resolution level of cell membrane. The rapidly decaying evanescent field reduces the optical and thermal damages to the bio-samples. The minimized intensity at the interface of the cell membrane enables us to perform long-term observation of living cell. In this paper, we use this technique to observe the kinetics of a cytoskeletal component, the microtubule. 2. Materials and methods A. Microscopy A schematic view of the objective-based TIRFM system is shown in Fig. 1. Imaging was performed by using an inverted optical microscope (IX70, Olympus Inc.) with an objective-based total internal reflection setup. A 100X (oil Iris, Olympus Inc.) and 60X (BFP1, Olympus Inc.) high numerical aperture (NA) oil objective lens were used in the experiments. An argon-ion laser with the wavelength of 488nm and a diode-pump solid state laser with the wavelength of 532nm were used as excitation light sources. B. Bio-samples CHO (Chinese Hamster Ovary) cells, from a cancerous cell line, were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with nonessential amino acid and 15% fetal

Fig. 2 (a) An Epi-fluorescence image of a living CHO cell, containing fluorescently labeled microtubules(b) The same cell was visualized under TIR-FM

bovine serum at 370C. GFP-fused protein, β-

The closer the distance to the cover glass substrate, the brighter image of the microtubule can be captured. The TIRFM images displayed in time lapse format in Fig. 3, the dynamic kinetics of microtubule is discerned evidently. The extension and shrink of the microtubule may have close relationship with the intracellular trafficking. Fig. 4 shows the zoom in pictures of Fig. 3.

tubulin gene was transfected to CHO cell via a pEYFP vector. 3. Results and Discussions Epi-fluorescence and TIRFM images of CHO living cell containing fluorescently labeled microtubules are shown in Fig. 2a and 2b, respectively. Note the reduction of the fluorescence background signals in the cytosol under TIR-FM greatly improves the contrast of the image qualities. The optical sensitivity of such observation of microtubules is therefore increased remarkably. Figure 3 shows the image captured in a fixed time interval of 6 seconds. Because the microtubule can be excited and seen clearly in near-zone of the evanescent field, the motion of the living microtubules can be observed and studied temporally.

Fig. 4 The dynamic kinetics of microtubule is discerned evidently in the TIRFM image with the time lapse of 6 seconds.

4. Summary TIRFM provides high sensitivity and contrast of the microtubules in a living cell is evidently. Dynamic kinetics of the microtubules in a CHO living cell was successfully imaging with a time lapse of 6 seconds per frame by a high sensitivity CCD. Molecular detection of the GFP on the microtubules using TIRFM creates a great means for the imaging in the nano-scale. 5. References 1.

Danial, A. Methods in cell biology, 30, 245 (1989)

2.

Thompson, N.L. and Lagerholm, B. C.(1997) Total internal reflection fluorescence applications in cellular biophysics.Curr. Opin. Biotechnol.8,58-64

3.

Derek, T. and Diermar J. Manstein(2001) Light up the cell surface with evanescent wave microscopy. Trends in cell biology Vol11. No7.

4.

Steyer, J. A. and Almers, W. (2001) A real-time view of life within 100nm of the plasma membrane. Nature reviews

Fig. 3 TIRFM image of the microtubule of CHO living cell with the time lapse of 24 seconds.