Light Dose is a Limiting Factor to Maintain Cell Viability in Fluorescence Microscopy and Single Molecule Detection

A test system for cell viability based on colony formation has been established and applied to high resolution fluorescence microscopy and single molecule detection. Living cells were irradiated either by epi-illumination or by total internal reflection (TIR) of a laser beam, and light doses where at least 90% of irradiated cells survived were determined. These light doses were in the range of a few J/cm2 up to about 200 J/cm2 depending on the wavelength of illumination as well as on the presence or absence of a fluorescent dye (e.g., the membrane marker laurdan). In general, cells were less sensitive to TIR than to epi-illumination. However, comparably high light doses needed for repetitive excitation of single molecules limit the application of super-resolution microscopy to living cells.

[1]  M. Wagner,et al.  Dose limited fluorescence microscopy of 5-aminolevulinic acid induced protoporphyrin IX in living cells , 2009, BiOS.

[2]  K. König,et al.  Multiphoton microscopy in life sciences , 2000, Journal of microscopy.

[3]  F. Del Bene,et al.  Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy , 2004, Science.

[4]  A. Hermetter,et al.  Conformational effects on the fluorescence of pyrene-labeled alkyldiacyl glycerols in different model membranes , 1994, Journal of Fluorescence.

[5]  H. Schneckenburger,et al.  Nanotomography of Cell Surfaces with Evanescent Fields , 2008 .

[6]  F. Ricchelli.  Photophysical properties of porphyrins in biological membranes. , 1995, Journal of photochemistry and photobiology. B, Biology.

[7]  S. Hell,et al.  STED microscopy with continuous wave beams , 2007, Nature Methods.

[8]  Timothy A. Skimina,et al.  Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Michael D. Mason,et al.  Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. , 2006, Biophysical journal.

[10]  Colin G. Coates,et al.  Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution. , 2004, Journal of biomedical optics.

[11]  J. Swedlow,et al.  Evaluating performance in three-dimensional fluorescence microscopy , 2007, Journal of microscopy.

[12]  S. Vincent.  Oxidative effects of heme and porphyrins on proteins and lipids. , 1989, Seminars in hematology.

[13]  ウェッブ、ウォット・ダブリュー,et al.  Two-photon laser scanning microscope , 1990 .

[14]  Enrico Gratton,et al.  Laurdan and Prodan as Polarity-Sensitive Fluorescent Membrane Probes , 1998, Journal of Fluorescence.

[15]  E. Manders,et al.  Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging , 2007, Nature Biotechnology.

[16]  Michael J Rust,et al.  Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM) , 2006, Nature Methods.

[17]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[18]  Yasushi Sako,et al.  Total internal reflection fluorescence microscopy for single-molecule imaging in living cells. , 2002, Cell structure and function.

[19]  H. Schneckenburger.  Total internal reflection fluorescence microscopy: technical innovations and novel applications. , 2005, Current opinion in biotechnology.

[20]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[21]  T. Wilson,et al.  Method of obtaining optical sectioning by using structured light in a conventional microscope. , 1997, Optics letters.

[22]  T. Dougherty.  Photodynamic therapy. , 1993, Photochemistry and photobiology.

[23]  R. Steiner,et al.  Variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM): realization and application of a compact illumination device , 2003, Journal of microscopy.

[24]  J. Pawley,et al.  Handbook of Biological Confocal Microscopy , 1990, Springer US.