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Near-field imaging of the microtubules of a CHO living cell Shu-Jung Yu1,2, Chien-Hua Chen3, Chi-Hung Lin3,4 , Cheng-Chi Chen1,5 and Din Ping Tsai1,2 1

Center of Nanostorage Research, National Taiwan University, Taipei 10617, Taiwan 2 Department of Physics, National Taiwan University, Taipei 10617, Taiwan 3 Institute of Biophotonics, 4Institute of Microbiology and Immunology, National Yang Ming University, Taipei, 112, Taiwan 5 Graduate Institute of Electro-Optical Engineering, National Taiwan University, Taipei 10617, Taiwan Phone: +886-2-3366-5099 Fax: +886-2-2365-6061 E-mail: [email protected] 1.

Introduction Molecular reactions occurring near or on the plasma membrane are fascinating and important studying objects. We used the characteristic of total internal reflection fluorescence microscopy (TIR-FM) to select fluorescent molecules excited near the plasma membrane to understand the formation and degradation of microtubules. The brightness of microtubules is utilized to distinguish the distance between fluorescent molecules and the interface. In this paper, we used TIRFM to investigate the dynamic kinetics of cytoskeleton called microtubule. Principle TIR-FM is based on the evanescent field when light internally reflects (incident angle exceeds critical angle, θ c ) at the interface between two materials with different refractive indices,

3.

Experimental Setup

Sample

c 60x, 1.45NA Oil objective Field aperture 40x, 0.65NA objective Laser

CCD

d

Dichroic mirror

2.

θ c = sin

−1

( n2

n1 ) ,

where n1 and n2 (n1>n2) are the refractive indices. The intensity of the evanescent field exponentially decays with the increasing distance z from the interface,

I Z = I o e −Z d d=

(

,

λo 2 2 n1 sin 2 θ 1 − n2 4π

)

−1 2

,

where Io is intensity at the interface i.e. at z = 0, d is the penetration depth, and λo is the wavelength of the incident light.

The penetration depth is defined as the distance

where the intensity decreases to Io/e and depends on the incident angle, wavelength and polarization of light, as well as the refractive indices of the substrate and sample.

Fiber illuminator

Figure 1. The schematic of the experimental setup. 3.1 TIRFM A schematic of the objective-based TIRFM (IX70, Olympus) system with a 100X (UPlanApo, 1.35NA oil Iris, ∞/0.17, Olympus) or 60X (PlanApo, 1.45NA oil, BFP1, ∞/0.17, Olympus) high NA oil objective are shown in Fig. 1. We used an Ar-ion laser (λ= 488 nm) or a diode-pump Nd:YVO4 solid state laser (λ= 532 nm) to excite the sample. The setup contains two modes: TIRF (linec) and epi-fluorescence (lined). The reflective laser is filtered by a dichroic mirror, and then CCD captures the images in fixed time interval. 3.2 Cell sample CHO (Chinese Hamster Ovary tumor cell) is a kind of cell which has strong vitality and does not mutate after repeatedly reproductions. 3.3 CCD We used the CCD to capture images in life science for high precision quantitative analysis in low light, brightfield, darkfield, and fluorescence. (32-0021B-197, RETIGA, cooled mono 12-bit)

4. Results and discussions 4.1 Epi-fluorescence and TIRF images The images of CHO living cell for epi-fluorescence and TIRF are shown in Fig. 2a and 2b, respectively. Their microtubules are labeled by the technology of green fluorescent protein (GFP). Evidently, the TIRF image can help to remove the fluorescent background and improves the signal to noise ratio (SNR) at the nano-scale depth of interface, so we can clearly see the microtubules. The optical sensitivity of the observation of microtubules is increased remarkably. In Fig. 2a, because the laser goes through the cell, and then the microtubules in whole cell are excited, the nucleus can be observed.

Fig. 2a Epi- fluorescence image of CHO living cell

within a CHO living cell is clearly shown. (the white arrow indicates a bundle of microtubules). From Fig. 4a to Fig. 4f display the formation of microtubules, and from Fig. 4g to Fig. 4i show the degradation of microtubules. Figure 4i exhibits that the microtubules rapidly vanished.

a

b

c

d

e

f

g

h

i

Fig. 4Time interval = 24sec Fig. 2b TIRF image of CHO living cell

4.2. The changing brightness of microtubules Figure 3a consists of 16 images which are captured in a time sequence of 6 seconds interval. The bright protrusion in the middle of the images is the microtubule tips. The intensity of the signal indicates the distance from the membrane. The aligned images show the movements of the microtubule tips.

Fig. 3a TIR images with time interval of each imaging frame of 6 secs The zoom-in images of the microtubule tips are shown in Fig. 3b with a time interval of 18 seconds. We utilize the brightness of microtubule tips to discern the distance from the membrane.

Fig. 3b TIR images with time interval of each imaging frame of 18 secs 4.3 The formation and degradation of microtubules Figures 4 display a constant time sequence of the captured images. The dynamics of the microtubules

5.

Summary TIRFM is used to study the microtubules near the plasma membrane of a CHO living cell. Evidently, there is a better contrast of the microtubules within the near-field zone. The kinetics and dynamics of the microtubules and microtubule tips can be observed temporally. Molecular detection of the GFP on the microtubules using TIRFM creates a powerful way for imaging in the nano-scale. Acknowledgement We would like to thank Mr. Toshio Haneda, Olympus Optical Co. Ltd. and Yuan Li Instrument Co. Ltd. for their helps and supports on the TIRFM. D. P. Tsai and S. J. Yu also thank the financial supports from National Science Council, Taiwan, R.O.C. under the grand number NSC-92-2120-M-002-008, and the Ministry of Economic Affairs of Taiwan, R.O.C. under the grand number 92-EC-17-A-08-S1-0006. References [1] J.A. Steyer and W. Almers; Nature (2001); A real-time view of life within 100nm of the plasm membrane; pp.268-pp.276; April; Vol. 2 [2] N.L. Thompson and B.C. Lagerholm; Current Opinion in Biotechnology (1997); Total internal reflection fluorescence: application in cellular biophysics; 8:58-64 [3] T. Derek and J.M. Diermar; Trends cell biology (2001); lighting up the cell surface with evanescent wave microscopy; pp.298-pp.303; vol. 11 No.7 July [4] M. Oheim, D. Loerke, W. Stühmer and R.H. Chow; Eur. Biophys. J. (1998); The last few milliseconds in the life of a secretory granule; 27:83-98