Fluorescence Microscopy


Fluorescence microscopy is an essential technique that allows scientists to visualise molecules (proteins, nucleic acids, ions, metabolites, carbohydrates and lipids), large structures and whole cells in fixed and living specimens as well as single molecules, assemblies and enzymes in vitro. Using multiple different imaging modalities, scientists have adapted fluorescence microscopy to advance our knowledge in all areas of biology and across length scales that range from tens of millimetres to a few nanometres.

Key Concepts

  • Fluorescence is an intrinsic property of some molecules and proteins that causes them to absorb and then emit light at given wavelengths.
  • Fluorescence imaging systems are built to excite molecules and then collect the emitted photons.
  • There are many different imaging techniques that can be used for interrogating different types of specimens and obtaining different kinds of data.
  • Localisation of molecules is only one of many readouts that scientists can obtain from fluorescence microscopy.
  • Imaging resolution has been limited to a diffraction limit based on the nature of our ability to collect fluorescence light through the objective, but new techniques have allowed for imaging to almost arbitrary precision.

Keywords: fluorescence microscopy; CCD camera; confocal laser scanning; spectral analysis; optical filters; living cells

Figure 1. (a) Excitation and emission spectra for FITC are shown. The difference between the excitation and emission spectra is the Stokes shift (image courtesy of Chroma Technology Inc.). (b) A Jablonski diagram shows that an electron interacting with an incoming photon of light of the appropriate wavelength ( 0) and being excited into a higher energy state (S1Vib). Some of the energy is lost by vibrational radiation to reach S1. The electron then returns to a near ground state by emitting a photon of light of a longer wavelength ( 1). (c) The light path of an epifluorescence microscope – white light is directed into an excitation filter that allows one or more selected bands of light to pass; the excitation light is then reflected off a dichroic mirror and focused by the objective into the sample; emitted photons and reflected photons are collected by the objective, hit the dichroic and the emitted photons are passed through to the detector while the excitation photons are blocked. (d) An example of a triple bandpass excitation filter, dichroic beamsplitter and emission filter set that can be used in a filter cube, coupled with separate emission and excitation filters to perform three‐colour fluorescence images. Image courtesy of Chroma Technology Inc.
Figure 2. (a) In a primary immunofluorescence assay, an antibody that is directly conjugated to a fluorophore recognises its antigen, and its position is directly detected by fluorescence microscopy. In indirect immunofluorescence, the fluorophore is attached to an antibody that detects the primary antibody. (b) An example of a fluorescence image that combines indirect immunofluorescence (a mouse antibody that detects tubulin and a rhodamine‐labelled anti‐mouse secondary that detects the primary antibody), a GFP‐tagged protein and DAPI staining of DNA to label chromosomes (scale bar, 5 µm). (c) An example of a confocal light path – light from lasers is fed into an acoustooptic tunable filter (AOTF), which selects the excitation wavelength, and then focused through a lens to a pinhole which blocks nonfocused photons; the lasers are then reflected off a dichroic and focused through an objective lens; emitted light from the focal plane is then refocused back through the objective, passes through the dichroic and a pinhole to reach the detector. Light from above and below the focal plane are blocked by the pinhole. (d) Two‐photon excitation of a fluorophore produces the same excitation as single‐photon excitation.
Figure 3. (a) A voxel contains many fluorescent molecules that are diffusing in and out of the observable volume. The rate of fluorescence counts for rhodamine B over 100 s (upper) and the autocorrelation (G(τ)) of counts over time (τ(s)) (lower) are shown. (b) In a FRAP experiment, a region is photobleached and the fluorescence recovery is measured to generate curves like what is shown in (c). (c) Before photobleaching, the fluorescence is 100%, and the immediate recovery of fluorescence over the first 20 s shows the reassociation of proteins with the bleached structure. (d) An example of a FRET biosensor that detects phosphorylation by a particular kinase. When the substrate is not phosphorylated, FRET does not happen. Upon phosphorylation, the phospho‐tyrosine binding motif will fold back and lead to high‐efficiency FRET.
Figure 4. (a) A structured illumination image of a Drosophila S2 cell expressing myosin regulatory light chain‐GFP before and after processing. After processing, the two ends of the bipolar thick filaments (BTFs) and tightly packed BTFs can be resolved. (b) An example of a PALM experiment. The wide‐field image is shown and example frames showing the stochastic activation of diffuse fluorophores, each of which is fit to a very precise spot (shown by the cross). At the end, the reconstructed PALM image is built of the thousands of frames and all of the localised spots to build structures with much higher resolution. (c) An example of a stimulated emission depletion (STED) setup. In the standard confocal modality, the laser spot is swept through the field, but in STED, a donut‐shaped depletion beam surrounds the diffraction spot and effectively reduces the size of the spot and breaks the Abbe resolution limit. (d) A Jablonski diagram shows the principle of STED. In standard fluorescence, the excited electron moves from S1 to S0Vib and emits a photon of a wavelength ( 1) that can pass through an emission filter. In STED, the longer wavelength ( 2) depletion beam drives the electron to a higher ground state to stimulate the emission of a photon of the same wavelength ( 2) that can be blocked by the emission filter.


Allen JR , Ross ST and Davidson MW (2013) Sample preparation for single molecule localization microscopy. Physical Chemistry Chemical Physics 15: 18771–18783.

Bacia K , Kim SA and Schwille P (2006) Fluorescence cross‐correlation spectroscopy in living cells. Nature Methods 3: 83–89.

de Boer P , Hoogenboom JP and Giepmans BN (2015) Correlated light and electron microscopy: ultrastructure lights up!. Nature Methods 12: 503–513.

Carlsson K , Danielsson PE , Lenz R , et al. (1985) Three‐dimensional microscopy using a confocal laser scanning microscope. Optics Letters 10: 53–55.

Cassidy A and Jones J (2014) Developments in in situ hybridisation. Methods 70: 39–45.

Churchman LS , Okten Z , Rock RS , Dawson JF and Spudich JA (2005) Single molecule high‐resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proceedings of the National Academy of Sciences of the United States of America 102: 1419–1423.

Coons AH , Creech HJ and Jones RN (1941) Immunological properties of an antibody containing a fluorescent group. Experimental Biology and Medicine 47: 200–202.

Coons AH , Creech HJ , Jones RN and Berliner E (1942) The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. The Journal of Immunology 45: 159–170.

Cull MG and Schatz PJ (2000) Biotinylation of proteins in vivo and in vitro using small peptide tags. Methods in Enzymology 326: 430–440.

Dickinson ME , Simbuerger E , Zimmermann B , Waters CW and Fraser SE (2003) Multiphoton excitation spectra in biological samples. Journal of Biomedical Optics 8: 329–338.

Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. Journal of Microscopy 198: 82–87.

Han M , Gao X , Su JZ and Nie S (2001) Quantum‐dot‐tagged microbeads for multiplexed optical coding of biomolecules. Nature Biotechnology 19: 631–635.

Hinner MJ and Johnsson K (2010) How to obtain labeled proteins and what to do with them. Current Opinion in Biotechnology 21: 766–776.

Huang B , Babcock H and Zhuang X (2010) Breaking the diffraction barrier: super‐resolution imaging of cells. Cell 143: 1047–1058.

Johnson I (2010) The Molecular Probes Handbook, a Guide to Fluorescent Probes and Labeling Technologies, 11th edn. Carlsbad: Life Technologies Corporation.

Kovalenko EI , Ranjbar S , Jasenosky LD , et al. (2011) The use of HaloTag‐based technology in flow and laser scanning cytometry analysis of live and fixed cells. BMC Research Notes 4: 340.

Kremers GJ , Gilbert SG , Cranfill PJ , Davidson MW and Piston DW (2011) Fluorescent proteins at a glance. Journal of Cell Science 124: 157–160.

Kumar A , Wu Y , Christensen R , et al. (2014) Dual‐view plane illumination microscopy for rapid and spatially isotropic imaging. Nature Protocols 9: 2555–2573.

Liu Z , Lavis LD and Betzig E (2015) Imaging live‐cell dynamics and structure at the single‐molecule level. Molecular Cell 58: 644–659.

Loren N , Hagman J , Jonasson JK , et al. (2015) Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice. Quarterly Reviews of Biophysics 48: 323–387.

Mahen R , Koch B , Wachsmuth M , et al. (2014) Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells. Molecular Biology of the Cell 25: 3610–3618.

Masters BR (2001) History of the electron microscope in cell biology. In: Encyclopedia of Life Sciences. Chichester: John Wiley & Sons, Ltd. DOI: 10.1002/9780470015902.a0021539.

Nienhaus K and Nienhaus GU (2014) Fluorescent proteins for live‐cell imaging with super‐resolution. Chemical Society Reviews 43: 1088–1106.

Pitrone PG , Schindelin J , Stuyvenberg L , et al. (2013) OpenSPIM: an open‐access light‐sheet microscopy platform. Nature Methods 10: 598–599.

Ploem JS (1967) The use of a vertical illuminator with interchangeable dichroic mirrors for fluorescence microscopy with incidental light. Z Wiss Mikrosk 68: 129–142.

Poulter NS , Pitkeathly WT , Smith PJ and Rappoport JZ (2015) The physical basis of total internal reflection fluorescence (TIRF) microscopy and its cellular applications. Methods in Molecular Biology 1251: 1–23.

Preibisch S , Saalfeld S , Schindelin J and Tomancak P (2010) Software for bead‐based registration of selective plane illumination microscopy data. Nature Methods 7: 418–419.

Quan T , Zeng S and Huang ZL (2010) Localization capability and limitation of electron‐multiplying charge‐coupled, scientific complementary metal‐oxide semiconductor, and charge‐coupled devices for superresolution imaging. Journal of Biomedical Optics 15: 066005.

Rasnik I , French T , Jacobson K and Berland K (2013) Electronic cameras for low‐light microscopy. Methods in Cell Biology 114: 211–241.

Reck‐Peterson SL , Yildiz A , Carter AP , et al. (2006) Single‐molecule analysis of dynein processivity and stepping behavior. Cell 126: 335–348.

Reymond L , Lukinavicius G , Umezawa K , et al. (2011) Visualizing biochemical activities in living cells through chemistry. Chimia (Aarau) 65: 868–871.

Ross ST , Allen JR and Davidson MW (2014) Practical considerations of objective lenses for application in cell biology. Methods in Cell Biology 123: 19–34.

Scalettar BA , Swedlow JR , Sedat JW and Agard DA (1996) Dispersion, aberration and deconvolution in multi‐wavelength fluorescence images. Journal of Microscopy 182: 50–60.

Shaner NC , Patterson GH and Davidson MW (2007) Advances in fluorescent protein technology. Journal of Cell Science 120: 4247–4260.

Sun Y , Rombola C , Jyothikumar V and Periasamy A (2013) Forster resonance energy transfer microscopy and spectroscopy for localizing protein‐protein interactions in living cells. Cytometry. Part A 83: 780–793.

Tanaami T , Otsuki S , Tomosada N , et al. (2002) High‐speed 1‐frame/ms scanning confocal microscope with a microlens and Nipkow disks. Applied Optics 41: 4704–4708.

Trepte O , Rokahr I , Andersson‐Engels S and Carlsson K (1994) Studies of porphyrin‐containing specimens using an optical spectrometer connected to a confocal scanning laser microscope. Journal of Microscopy 176: 238–244.

VanEngelenburg SB and Palmer AE (2008) Fluorescent biosensors of protein function. Current Opinion in Chemical Biology 12: 60–65.

Verveer PJ , Swoger J , Pampaloni F , et al. (2007) High‐resolution three‐dimensional imaging of large specimens with light sheet‐based microscopy. Nature Methods 4: 311–313.

Wang Y , Cai E , Sheung J , et al. (2014) Fluorescence imaging with one‐nanometer accuracy (FIONA). Journal of Visualized Experiments 91: e51774.

Weigert R , Porat‐Shliom N and Amornphimoltham P (2013) Imaging cell biology in live animals: ready for prime time. The Journal of Cell Biology 201: 969–979.

Willig KI , Kellner RR , Medda R , et al. (2006) Nanoscale resolution in GFP‐based microscopy. Nature Methods 3: 721–723.

Wolf DE (2013) Fundamentals of fluorescence and fluorescence microscopy. Methods in Cell Biology 114: 69–97.

Xu C , Zipfel W , Shear JB , Williams RM and Webb WW (1996) Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. Proceedings of the National Academy of Sciences of the United States of America 93: 10763–10768.

Zimmermann T , Rietdorf J and Pepperkok R (2003) Spectral imaging and its applications in live cell microscopy. FEBS Letters 546: 87–92.

Further Reading

Enterina JR , Wu L and Campbell RE (2015) Emerging fluorescent protein technologies. Current Opinion in Chemical Biology 27: 10–17.

Ettinger A and Wittmann T (2014) Fluorescence live cell imaging. Methods in Cell Biology 123: 77–94.

Huang B , Babcock H and Zhuang X (2010) Breaking the diffraction barrier: super‐resolution imaging of cells. Cell 143: 1047–1058.

Matsuda T and Nagai T (2014) Quantitative measurement of intracellular protein dynamics using photobleaching or photoactivation of fluorescent proteins. Microscopy (Oxford) 63: 403–408.

Miyawaki A and Niino Y (2015) Molecular spies for bioimaging – fluorescent protein‐based probes. Molecular Cell 58: 632–643.

Nienhaus K and Nienhaus GU (2015) Where do we stand with super‐resolution optical microscopy? Journal of Molecular Biology, pii: S0022‐2836(15)00711‐1.

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Griffis, Eric R(Mar 2016) Fluorescence Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005780.pub2]