A Thin Layer Imaging with the Total Internal Reflection Fluorescence Microscopy

Subject Areas : Journal of Optoelectronical Nanostructures

Neda Roostaie ¹ , Elham Sheykhi ² , Fariba Japelaghi ³ , Mohammad Amin Bassam ⁴ , Sharareh Tavaddod ⁵ , Batool Sajad ⁶

1 - Department of physics, Alzahra University, Tehran, Iran
2 - Department of physics, Alzahra University, Tehran, Iran
3 - Department of physics, Alzahra University, Tehran, Iran
4 - Department of physics, Malek-Ashtar University of Technology, Tehran, Iran
5 - Department of physics, University of Edinburgh, Scotland
6 - Department of physics, Alzahra University, Tehran, Iran

Received: 2017-05-22 Accepted : 2017-07-23 Published : 2017-08-01

Keywords: Fluorescence, Evanescent wave, microscopy, total internal reflection,

Abstract :

Total internal reflection fluorescence microscopy (TIRFM) is an optical technique that allows imaging of a thin layer of the sample with a thickness of about 100-200 nm. It is used in science of cell biology to study cellular processes, especially near the membranes of living cells. This method is based on the total internal reflection phenomenon, where the evanescent wave is generated in the less dense medium. In fact, the evanescent wave is used to illuminate the sample. Consequently, the possibility of observing a superficial (instead of bulk) part of fluorophore labeled sample is opened up. In this work, a total internal reflection fluorescence microscope based on the light guide has been designed and assembled by means of the inverted microscope to image a thin layer from the surface of the sample. Operated experimental arrangement has been employed for the total internal reflection fluorescence imaging of cadmium selenide (CdSe) quantum dots.

References:

[1]    D. Axelrod, N.L. Thompson, and T.P. Burghardt. Total internal reflection fluorescent microscopy. Journal of microscopy 129 (1) (1983, Jan.) 19-28.

[2]    Fish. K.N. Total internal reflection fluorescence (TIRF) microscopy. Current protocols in cytometry (2009, Oct.) 12-18.

[3]    D. Axelrod, Evanescent excitation and emission in fluorescence microscopy. Biophysical journal 104 (7) (2013, Apr.) 1401-1409.

[4]    D. Axelrod, T.P. Burghardt. and N.L. Thompson. Total internal reflection fluorescence. Annual review of biophysics and bioengineering 13 (1) (1984, Jun.) 247-268.

[5]    Yildiz, Ahmet, and Ronald D. Vale. Tracking movements of the microtubule motors kinesin and dynein using total internal reflection fluorescence microscopy. Cold Spring Harbor Protocols 2015 (9) (2015, Sep.) pdb-prot086355.

[6]    Török, Peter, and Fu-Jen Kao, Total internal reflection fluorescence microscopy, Optical Imaging and Microscopy: Techniques and Advanced Systems, vol. 87, 2007, 195-236.

[7]    Mattheyses, Alexa L., Sanford M. Simon, and Joshua Z. Rappoport. Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci 123 (21) (2010, Nov.) 3621-3628.

[8]    Dos Santos, Marcelina Cardoso, et al. Topography of Cells Revealed by Variable-Angle Total Internal Reflection Fluorescence Microscopy. Biophysical Journal 111 (6) (2016, Sep.) 1316-1327.

[9]    D. Axelrod, Cell-substrate contacts illuminated by totalinternal reflection fluorescence.The Journal of cell biology 89 (1) (1981, Apr.) 141-145.

[10] Truskey, George A., et al. Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances. Journal of cell science 103 (2) (1992, Oct.) 491-499.

[11] Murray, Christopher B., C. R. Kagan, and M. G. Bawendi. Synthesis and characterization of monodisperse nanocrystals and close-packed nanocrystal assemblies. Annual Review of Materials Science 30 (1) (2000, Aug.) 545-610.