Fluorescence microscope

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An upright fluorescence microscope (Olympus BX61) with the fluorescent filter cube turret above the objective lenses, coupled with a digital camera.
Fluorescent and confocal microscopes operating principle




The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e., of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter. Typical components of a fluorescence microscope are a light source (xenon arc lamp or mercury-vapor lamp are common; more advanced forms are high-power LEDs and lasers), the excitation filter, the dichroic mirror (or dichroic beamsplitter), and the emission filter (see figure below). The filters and the dichroic beamsplitter are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen.[1] In this manner, the distribution of a single fluorophore (color) is imaged at a time. Multi-color images of several types of fluorophores must be composed by combining several single-color images.[1]

Most fluorescence microscopes in use are epifluorescence microscopes, where excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e. through the objective). These microscopes are widely used in biology and are the basis for more advanced microscope designs, such as the confocal microscope and the total internal reflection fluorescence microscope (TIRF).

Epifluorescence microscopy[edit]

Schematic of a fluorescence microscope.

The majority of fluorescence microscopes, especially those used in the life sciences, are of the epifluorescence design shown in the diagram. Light of the excitation wavelength is focused on the specimen through the objective lens. The fluorescence emitted by the specimen is focused to the detector by the same objective that is used for the excitation which for greater resolution will need objective lens with higher numerical aperture. Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light and the epifluorescence method therefore gives a high signal-to-noise ratio. The dichroic beamsplitter acts as a wavelength specific filter, transmitting fluoresced light through to the eyepiece or detector, but reflecting any remaining excitation light back towards the source.

Light sources[edit]

Fluorescence microscopy requires intense, near-monochromatic, illumination which some widespread light sources, like halogen lamps cannot provide. Four main types of light source are used, including xenon arc lamps or mercury-vapor lamps with an excitation filter, lasers, supercontinuum sources, and high-power LEDs. Lasers are most widely used for more complex fluorescence microscopy techniques like confocal microscopy and total internal reflection fluorescence microscopy while xenon lamps, and mercury lamps, and LEDs with a dichroic excitation filter are commonly used for widefield epifluorescence microscopes. By placing two microlens arrays into the illumination path of a widefield epifluorescence microscope[5], highly uniform illumination with a coefficient of variation of 1-2% can be achieved.

Sample preparation[edit]

A sample of herring sperm stained with SYBR green in a cuvette illuminated by blue light in an epifluorescence microscope. The SYBR green in the sample binds to the herring sperm DNA and, once bound, fluoresces giving off green light when illuminated by blue light.


Biological fluorescent stains[edit]

Many fluorescent stains have been designed for a range of biological molecules. Some of these are small molecules which are intrinsically fluorescent and bind a biological molecule of interest. Major examples of these are nucleic acid stains like DAPI and Hoechst (excited by UV wavelength light) and DRAQ5 and DRAQ7 (optimally excited by red light) which all bind the minor groove of DNA, thus labeling the nuclei of cells. Others are drugs or toxins which bind specific cellular structures and have been derivatised with a fluorescent reporter. A major example of this class of fluorescent stain is phalloidin which is used to stain actin fibres in mammalian cells.



Immunofluorescence is a technique which uses the highly specific binding of an antibody to its antigen in order to label specific proteins or other molecules within the cell. A sample is treated with a primary antibody specific for the molecule of interest. A fluorophore can be directly conjugated to the primary antibody. Alternatively a secondary antibody, conjugated to a fluorophore, which binds specifically to the first antibody can be used. For example, a primary antibody raised in a mouse which recognises tubulin combined with a secondary anti-mouse antibody derivatised with a fluorophore could be used to label microtubules in a cell.

Fluorescent proteins[edit]

The modern understanding of genetics and the techniques available for modifying DNA allow scientists to genetically modify proteins to also carry a fluorescent protein reporter. In biological samples this allows a scientist to directly make a protein of interest fluorescent. The protein location can then be directly tracked, including in live cells.



Fluorescence microscopy with fluorescent reporter proteins has enabled analysis of live cells by fluorescence microscopy, however cells are susceptible to phototoxicity, particularly with short wavelength light. Furthermore, fluorescent molecules have a tendency to generate reactive chemical species when under illumination which enhances the phototoxic effect.

Unlike transmitted and reflected light microscopy techniques fluorescence microscopy only allows observation of the specific structures which have been labeled for fluorescence. For example, observing a tissue sample prepared with a fluorescent DNA stain by fluorescent microscopy only reveals the organization of the DNA within the cells and reveals nothing else about the cell morphologies.

Sub-diffraction techniques[edit]


Several improvements in microscopy techniques have been invented in the 20th century and have resulted in increased resolution and contrast to some extent. However they did not overcome the diffraction limit. In 1978 first theoretical ideas have been developed to break this barrier by using a 4Pi microscope as a confocal laser scanning fluorescence microscope where the light is focused ideally from all sides to a common focus which is used to scan the object by 'point-by-point' excitation combined with 'point-by-point' detection.[6] However, the first experimental demonstration of the 4pi microscope took place in 1994.[7] 4Pi microscopy maximizes the amount of available focusing directions by using two opposing objective lenses or Two-photon excitation microscopy using redshifted light and multi-photon excitation.

Integrated correlative microscopy combines a fluorescence microscope with an electron microscope. This allows one to visualize ultrastructure and contextual information with the electron microscope while using the data from the fluorescence microscope as a labelling tool.[8]


As well in the 1990s another super resolution microscopy method based on wide field microscopy has been developed. Substantially improved size resolution of cellular nanostructures stained with a fluorescent marker was achieved by development of SPDM localization microscopy and the structured laser illumination (spatially modulated illumination, SMI).[9] Combining the principle of SPDM with SMI resulted in the development of the Vertico SMI microscope.[10][11] Single molecule detection of normal blinking fluorescent dyes like Green fluorescent protein (GFP) can be achieved by using a further development of SPDM the so-called SPDMphymod technology which makes it possible to detect and count two different fluorescent molecule types at the molecular level (this technology is referred to as two-color localization microscopy or 2CLM).[12]


Fluorescence micrograph gallery[edit]

See also[edit]


  1. ^ a b c d Spring KR, Davidson MW. "Introduction to Fluorescence Microscopy". Nikon MicroscopyU. Retrieved 2008-09-28. 
  2. ^ "The Fluorescence Microscope". Microscopes—Help Scientists Explore Hidden Worlds. The Nobel Foundation. Retrieved 2008-09-28. 
  3. ^ Ritter, Karl; Rising, Malin (8 October 2014). "2 Americans, 1 German win chemistry Nobel". Associated Press. Retrieved 8 October 2014. 
  4. ^ Chang, Kenneth (8 October 2014). "2 Americans and a German Are Awarded Nobel Prize in Chemistry". New York Times. Retrieved 8 October 2014. 
  5. ^ F.A.W. Coumans; E. van der Pol; L.W.M.M. Terstappen (2012). "Flat-top illumination profile in an epi-fluorescence microscope by dual micro lens arrays". Cytometry Part A. 81 (4): 324–331. PMID 22392641. doi:10.1002/cyto.a.22029. 
  6. ^ Cremer, C; Cremer, T (1978). "Considerations on a laser-scanning-microscope with high resolution and depth of field" (PDF). Microscopica acta. 81 (1): 31–44. PMID 713859. 
  7. ^ S.W. Hell, E.H.K. Stelzer, S. Lindek, C. Cremer; Stelzer; Lindek; Cremer (1994). "Confocal microscopy with an increased detection aperture: type-B 4Pi confocal microscopy". Optics Letters. 19 (3): 222–224. Bibcode:1994OptL...19..222H. PMID 19829598. doi:10.1364/OL.19.000222. 
  8. ^ Baarle, Kaitlin van. "Correlative microscopy: Opening up worlds of information with fluorescence". Retrieved 2017-02-16. 
  9. ^ Hausmann, Michael; Schneider, Bernhard; Bradl, Joachim; Cremer, Christoph G. (1997), "High-precision distance microscopy of 3D nanostructures by a spatially modulated excitation fluorescence microscope", in Bigio, Irving J; Schneckenburger, Herbert; Slavik, Jan; et al., Optical Biopsies and Microscopic Techniques II (PDF), Optical Biopsies and Microscopic Techniques II, 3197, p. 217, doi:10.1117/12.297969 
  10. ^ Reymann, J; Baddeley, D; Gunkel, M; Lemmer, P; Stadter, W; Jegou, T; Rippe, K; Cremer, C; Birk, U (2008). "High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy" (PDF). Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 16 (3): 367–82. PMID 18461478. doi:10.1007/s10577-008-1238-2. 
  11. ^ Baddeley, D; Batram, C; Weiland, Y; Cremer, C; Birk, UJ (2003). "Nanostructure analysis using spatially modulated illumination microscopy" (PDF). Nature Protocols. 2 (10): 2640–6. PMID 17948007. doi:10.1038/nprot.2007.399. [dead link]
  12. ^ Gunkel, M; Erdel, F; Rippe, K; Lemmer, P; Kaufmann, R; Hörmann, C; Amberger, R; Cremer, C (2009). "Dual color localization microscopy of cellular nanostructures" (PDF). Biotechnology journal. 4 (6): 927–38. PMID 19548231. doi:10.1002/biot.200900005. 

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