Laser Illumination in Live Cell Microscopy: Scattering and Structured Illumination

Herbert Schneckenburger (Login required)
Aalen University, Institute of Applied Research, Germany

Verena Richter
Aalen University, Institute of Applied Research, Germany

Mathis Piper
Aalen University, Institute of Applied Research, Germany

Michael Wagner
Aalen University, Institute of Applied Research, Germany

Paper #3167 received 2 Mar 2017; revised manuscript received 4 Apr 2017; accepted for publication 7 Apr 2017; published online 12 Apr 2017.

DOI: 10.18287/JBPE17.03.010304


Two types of laser illumination in live cell microscopy with a focus on the sample or in the aperture plane of the microscope objective lens are distinguished. For the second case two examples are described, namely light scattering microscopy with angular resolution and Structured Illumination Microscopy (SIM) with two interfering laser beams. Appropriate applications include morphological studies of cells undergoing apoptosis and mitochondrial imaging with increased resolution.


Living cells; light scattering microscopy; super-resolution microscopy; structured illumination; SIM; apoptosis; mitochondrial imaging

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1. J. Pawley, Handbook of Biological Confocal Microscopy, Plenum Press, New York (1996).

2. R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys. 59, 427-471 (1996).

3. A. Ashkin, “Acceleration and Trapping of Particles by Radiation Pressure,” Phys. Rev. Lett. 24(4), 156-159 (1970).

4. J. Huisken, J. Swoger, F. del Bene, J. Wittbrodt, and E. H. K. Stelzer, “Optical sectioning deep inside live embryos by SPIM,” Science 305(5686), 1007-1009 (2004). Crossref

5. C. Dunsby, “Optically sectioned imaging by oblique plane microscopy”, Opt. Express 16(25), 2306-2316 (2008). Crossref

6. H. Schneckenburger, “Total internal reflection fluorescence microscopy: technical innovations and novel applications,” Curr. Opin. Biotechnol. 16(1), 13-18 (2005).

7. V. Richter, F. Voit, A. Kienle, and H. Schneckenburger, “Light scattering microscopy with angular resolution and its possible application to apoptosis,” J Microsc. 257(1), 1-7 (2015). Crossref

8. B. Angres, H. Steuer, P. Weber. M. Wagner, and H. Schneckenburger, “A membrane-bound FRET-based caspase sensor for detection of apoptosis using fluorescence lifetime and total internal reflection microscopy,” Cytometry 75A(5), 420-427 (2009).

9. M. G. L. Gustafsson, L. Shao, P. M. Carlton, C. J. R. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957-4970 (2008).

10. R. Heintzmann, and C. Cremer, “Lateral modulated excitation microscopy: Improvement of resolution by using a diffraction grating,” Proc. SPIE 3568, 185-196 (1999).

11. R. Förster, H.-W. Lu-Walther, A. Jost. M. Kielhorn, K. Wicker, and R. Heintzmann, “Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator,” Opt. Express 22(17), 20663-20677 (2014). Crossref

12. M. Müller, V. Mönkemöller, S. Hennig, W. Hübner, and T. Huser, “Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ,” Nature Communications, 10980 (2016).

13. C. F. Bohren, and D. R. Huffman, Absorption and Scattering of Light by Small Particles, Wiley-Interscience Publications, New York (1998). Crossref

14. V. Richter, S. Bruns, T. Bruns, P. Weber, M. Wagner, C. Cremer, and H. Schneckenburger, “Axial tomography in live cell laser microscopy,” J. Biomed. Opt. 22(9), 091505 (2017).

15. S. W. Hell, and J. Wichmann, “Breaking the resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 1-3 (1994).

16. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793),1642-1645 (2006).

17. M. J. Rust, M Bates, X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793-796 (2006). Crossref

18. C. Cremer, and B. R. Masters, “Resolution enhancement techniques in microscopy,” Eur. Phys. J. H 38(3), 281-344 (2013). Crossref

19. H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W. S. L. Strauss, and R. Wittig, “Light exposure and cell viability in fluorescence microscopy,” J. Microsc. 245(3), 311-318 (2012). Crossref

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