User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

Fluorescence Microscopy: A Concise Guide to Current Imaging Methods

Christian A. Combs¹

¹NHLBI Light Microscopy Facility, NIH, Bethesda, Maryland

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 2.1

DOI:

10.1002/0471142301.ns0201s50

Online Posting Date:

January, 2010

GO TO THE FULL TEXT:

PDF or HTML at Wiley Online Library

Are you the author of this protocol? Login or register and return to this page.

Abstract

The field of fluorescence microscopy is rapidly growing, providing ever increasing imaging capabilities for cell and neurobiologists. Over the last decade, many new technologies and techniques have been developed which allow for deeper, faster, or higher resolution imaging. For the non-expert microscopist, it can be difficult to match the best imaging technique to the biological question to be examined. Picking the right technique requires a basic understanding of the underlying imaging physics for each technique, as well as an informed comparison and balancing of competing imaging properties in the context of the sample to be imaged. This unit provides concise descriptions of a range of commercially available imaging techniques and provides a tabular guide to choosing among them. Techniques covered include structured light, confocal, total internal reflection fluorescence (TIRF), two-photon, and stimulated emission depletion (STED) microscopy. Curr. Protoc. Neurosci. 50:2.1.1-2.1.14. © 2010 by John Wiley & Sons, Inc.

Keywords: confocal; two-photon; structured light; STED

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Introduction
Wide-Field Fluorescence Microscopy (WFFM) Techniques
Modern Confocal Microscopy
Total Internal Reflection Fluorescence (TIRF) Microscopy
Two-Photon Fluorescence Microscopy (TPFM)
Stimulated Emission Depletion (STED) Fluorescence Microscopy
Final Considerations
Acknowledgements
Literature Cited
Figures
Tables

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 2.1.1

Diagram of some of the critical opposing factors in an imaging experiment. The best image is one that can balance these factors to obtain the necessary information while avoiding photobleaching or phototoxic effects. Table 2.1.1 outlines how these factors differ among the various commercialized microscopy techniques discussed in this unit.

View Image
Figure 2.1.2

The basic principles of structured light microscopy are shown in panels A, B, and C. If an unknown pattern (such as a biological sample) represented in (A) is multiplied by a known regular illumination pattern (B) then a beat pattern (moiré fringes) will appear (C). The pattern is the difference between the sample and the regular illumination pattern and is coarse enough to be seen through the microscope even if the original pattern in the sample was not resolvable. By moving the grid and the sample in space and computationally processing the resulting data an image can be generated that has resolution at least 2× better than a conventional wide-field image. Confocal (D) and structured light (F) images of the edge of a Hela cell showing the actin cytoskeleton. E and G show enlargements of the images in D and F. The apparent fiber diameters are 110 to 120 nm in the structured light images, compared to 280 to 300 nm in the confocal image. Panels A, B, and C are reproduced from Gustafsson (2005), and panels D and E are reproduced from Gustafsson (2000), with permission of the National Academy of Sciences U.S.A. Panels A to E were originally published in color and have been altered here to black and white.

View Image
Figure 2.1.3

Basic architecture of a modern confocal microscope. Excitation light from the laser is passed through the various collimating optics in a scan-head to either a variable dichroic mirror (Nikon, Zeiss, or Olympus) or an AOBS (Acousto-Optical Beam Splitter; Leica) where it is reflected through the objective and focused to a point on the sample. Moveable mirrors in the scan-head before the objective scan the excitation beam over the sample a point at a time to build the image. Fluorescence emission light passes back through the objective, through the dichroic or AOBS to the light sensing photo-multiplier tubes (PMTs). An aperture (pinhole) placed in the conjugate image plane to the point of focus in the sample allows only light from the focal plane to impinge on the sample, and out-of-focus light is blocked. The pinhole can be made larger to allow for larger optical sectioning capability, allowing more out of focus light to impinge on the PMT(s). In some models a diffraction grating or prism placed in the beam-path of the emission light can act as a variable band-pass filter or as a spectral detector if the polychromatic light is spatially spread on a number of PMTs.

View Image
Figure 2.1.4

Maximum projection reconstruction from confocal images obtained through a 65-µm stack of mouse cerebellum labeled with a combination of fluorescent proteins. In this image one can see the unique colors produced and spectrally detected by the genetic combinations of individual fluorescent proteins (XFPs). These colors were used to trace and map the various synaptic circuits. This panel of the figure was reproduced from Livet et al. (2007), with permission of the Nature Publishing group.

View Image
Figure 2.1.5

This figure shows the basics of how TIRF microscopy excites a shallow region above the coverslip using oblique laser excitation, which is totally internally reflected and produces an evanescent wave for fluorophore excitation. (A) In internal reflection: a light propagating through the periphery of a high numerical aperture objective (>1.38) is totally internally reflected by the coverslip and sent down the opposing side of the objective. (B) An evanescent wave is formed when the critical angle _C is reached and the light is totally reflected. The reflection at the coverslip is due to the oblique angle of illumination and the mismatch of refraction index (n) between the oil and coverslip. Note that the evanescent wave only excites fluorophores where the cell attaches or is touching the coverslip. A wide-field (C) and TIRF (D) image of GFP-tagged myosin V from Drosophila embryo hemocytes. Comparing the two images it is evident where the Myosin 5 is closest to the coverslip. In the top cell much of the cell is near the coverslip. In the bottom cell only areas in the periphery are near the coverslip (highlighted by arrows). Hemocytes courtesy of Amy Hong, NHLBI, NIH. Panel B was reproduced with permission from Mike Davidson (Florida State University and the National High Magnetic Field Laboratory) and the Molecular Expressions Web site.

View Image
Figure 2.1.6

Principles of two-photon fluorescence microscopy (TPFM). (A) Regular one-photon (e.g., confocal) and TPFM energy transitions in a Jablonski diagram. In TPFM two photons are absorbed nearly simultaneously to produce twice the energy. In this example, GFP is excited with 960-nm light for TPFM and 488 nm higher energy light for a confocal experiment. The emission is the same for both cases. It should be noted that TPFM absorption spectra for most fluorophores, including GFP, are very broad (in some cases hundreds of nanometers), and that the maximum is roughly a little less than twice the one-photon absorption maxima. (B) Two-photon fluorescence is generated in only one plane when a laser pulse train propagating through an objective is focused to a spot. Fluorescence is generated only at the point where the maximal photon crowding occurs and falls off from this plane at a rate of the fourth power from the center of the focal spot. (C) In vivo TPFM image of a mouse neocortex genetically labeled with a chloride indicator. This image shows the remarkable depth to which TPFM imaging is possible. Panel C is reproduced from Helmchen and Denk (2005) with permission of the Nature Publishing group.

View Image
Figure 2.1.7

Technical principals of stimulated emission depletion (STED) microscopy. (A) The combination of the normal excitation beam with the phase modulated STED beam produces a sub-diffraction emission spot. The images on the right in (A) show the doughnut pattern produced by the phase modulation of the STED beam. This beam, when overlapped with the diffraction-limited excitation spot, quenches emission where the beams overlap leaving the middle, sub-diffraction sized, spot for spontaneous fluorescence. (B) Comparison of confocal (left) and STED (right) images of a labeled preparation reveals a marked increase in resolution by STED because more labeled particles are visualized. Scale bar, 500 nm. Figure reproduced from Willig et al. (2006b) with permission of the Nature Publishing group.

View Image

Literature Cited

Literature Cited
	Boccacci, P. and Bertero, M. 2002. "Image-restoration methods: Basics and algorithms". In Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances (A. Diaspro, ed.) pp. 253-269. Wiley-Liss, New York.
	Diaspro, A., Bianchini, P., Vicidomini, G., Faretta, M., Ramoino, P., and Usai, C. 2006. Multi-photon excitation microscopy. Biomed. Eng. Online 5:36.
	Eisenstein, M. 2006. Helping cells to tell a colorful tale. Nat. Methods 3:647-655.
	Gustafsson, M.G. 2000. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198:82-87.
	Gustafsson, M.G. 2005. Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. U.S.A. 102:13081-13086.
	Hell, S.W. 2007. Far-field optical nanoscopy. Science 316:1153-1158.
	Helmchen, F. and Denk, W. 2005. Deep tissue two-photon microscopy. Nat. Methods 2:932-940.
	Hibbs, A.R. 2004. Confocal Microscopy for Biologists. Springer, New York.
	Inoue, S. and Spring, K.R. 1997. Video Microscopy: The Fundamentals. Springer, New York.
	Kellner, R.R., Baier, C.J., Willig, K.I., Hell, S.W., and Barrantes, F.J. 2007. Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy. Neuroscience 144:135-43.
	Lichtman, J.W. and Conchello, J.A. 2005. Fluorescence microscopy. Nat. Methods 2:910-919.
	Livet, J., Weissman, T.A., Kang, H., Draft, R.W., Lu, J., Bennis, R.A., Sanes, J.R., and Lichtman, J.W. 2007. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450:56-62.
	Pawley, J.B. (Ed.) 2006. Handbook of Biological Confocal Microscopy. Springer, New York.
	Pearson, H. 2007. The good, the bad and the ugly. Nature 447:138-140.
	Schermelleh, L., Carlton, P.M., Haase, S., Shao, L., Winoto, L., Kner, P., Burke, B., Cardoso, M.C., Agard, D.A., Gustafsson, M.G., Leonhardt, H., and Sedat, J.W. 2008. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320:1332-1336.
	Shaner, N.C., Steinbach, P.A., and Tsien, R.Y. 2005. A guide to choosing fluorescent proteins. Nat. Methods 2:905-909.
	Suzuki, T., Matsuzaki, T., Hagiwara, H., Aoki, T., and Takata, K. 2007. Recent advances in fluorescent labeling techniques for fluorescence microscopy. Acta Histochem. Cytochem. 40:131-137.
	Svoboda, K. and Yasuda, R. 2006. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50:823-839.
	Toomre, D. and Manstein, D.J. 2001. Lighting up the cell surface with evanescent wave microscopy. Trends Cell Biol. 11:298-303.
	Wallace, W., Schaefer, L.H., and Swedlow, J.R. 2001. A workingperson's guide to deconvolution in light microscopy. Biotechniques 31:1076-1078, 1080, 1082 passim.
	Willig, K.I., Kellner, R.R., Medda, R., Hein, B., Jakobs, S., and Hell, S.W. 2006a. Nanoscale resolution in GFP-based microscopy. Nat. Methods 3:721-723.
	Willig, K.I., Rizzoli, S.O., Westphal, V., Jahn, R., and Hell, S.W. 2006b. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440:935-939.
	Willig, K.I., Harke, B., Medda, R., and Hell, S.W. 2007. STED microscopy with continuous wave beams. Nat. Methods 4:915-918.