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.

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A fluorescence microscope (colloquially synonymous with epifluorescence microscope) is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption.^[1][2]

An inverted fluorescence microscope (Nikon TE2000) with the fluorescent filter cube turret below the stage. Note the orange plate that allows the user to look at the sample while protecting his eyes from the excitation UV light.

[edit] Technique

In most cases, a component of interest in the specimen is specifically labeled with a fluorescent molecule called a fluorophore (such as green fluorescent protein (GFP), fluorescein or DyLight 488).^[1] The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit longer wavelengths of light (of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of an emission filter. Typical components of a fluorescence microscope are the light source (xenon arc lamp or mercury-vapor lamp), the excitation filter, the dichroic mirror (or dichromatic beamsplitter), and the emission filter (see figure below). The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen.^[1] In this manner, a single fluorophore (color) is imaged at a time. Multi-color images of several fluorophores must be composed by combining several single-color images.^[1]

Most fluorescence microscopes in use are epifluorescence microscopes (i.e. excitation and observation of the fluorescence are from above (epi–) the specimen). These microscopes have become an important part in the field of biology, opening the doors for more advanced microscope designs, such as the confocal microscope and the total internal reflection fluorescence microscope (TIRF).

Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Special care must be taken to prevent photobleaching through the use of more robust fluorophores, by minimizing illumination, or by introducing a scavenger system to reduce the rate of photobleaching.

[edit] Epifluorescence microscopy

Schematic of a fluorescence microscope.

Epifluorescence microscopy is a method of fluorescence microscopy that is widely used in life sciences. The excitatory light is passed from above (or, for inverted microscopes, from below), through the objective and then onto the specimen instead of passing it first through the specimen. (In the latter case the transmitted excitatory light reaches the objective together with light emitted from the specimen). The fluorescence in the specimen gives rise to emitted light which is focused to the detector by the same objective that is used for the excitation. A filter between the objective and the detector filters out the excitation light from fluorescent light. Since most of the excitatory light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light and this method therefore gives an improved signal to noise ratio. A common use in biology is to apply fluorescent or fluorochrome stains to the specimen in order to image a protein or other molecule of interest.

[edit] Sub-diffraction techniques

It is well known that there is a spatial limit to which light can focus: approximately half of the wavelength of the light you are using. But this is not a true barrier, because this diffraction limit is only true in the far-field and localization precision can be increased with many photons and careful analysis; and like the sound barrier, the diffraction barrier is breakable.

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 ^[3].

[edit] Vertico SMI - SPDMphymod superresolution microscopy

[edit] Localization microscopy/spatially structured illumination

Around 1995, Christoph Cremer commenced with the development of a light microscopic process, which achieved a substantially improved size resolution of cellular nanostructures stained with a fluorescent marker. This time he employed the principle of wide field microscopy combined with structured laser illumination (spatially modulated illumination, SMI^[4]. In addition, this technology is no longer subjected to the speed limitations of the focusing microscopy so that it becomes possible to undertake 3D analyses of whole cells within short observation times (at the moment around a few seconds).

Also since around 1995, Christoph Cremer developed and realized new fluorescence based wide field microscopy approaches which had as their goal the improvement of the effective optical resolution (in terms of the smallest detectable distance between two localized objects) down to a fraction of the conventional resolution (spectral precision distance/position determination microscopy, SPDM).

Combining SPDM and SMI, known as Vertico-SMI microscopy^[5] Christoph Cremer can currently achieve a resolution of approx. 10 nm in 2D and 40 nm in 3D in wide field images of whole living cells^[6]. The Vertico SMI is currently the fastest optical 3D nanoscope for the three dimensional structural analysis of whole cells worldwide.

The Vertico SMI works with high recording speed and processes a complete 3D stack in 40 seconds (2000 frames), the very fast image processing based on specific proprietary algorithms makes the image available after 2min/3min (1-/2-color). The specific wide-field technique captures large areas up to 5000 µm².

SPDMphymod: Superresolution images of standard GFP, RFP, YFP fluorescent dyes

Single YFP molecule detection in a human cancer cell. Typical distance measurements in the 15 nm range (5 nm standard deviation)

Co-localzation microscopy (2CLM) with GFP and RFP fusion proteins (nucleus of a bone cancer cell) 120.000 localized molecules in a wide-field area(470 µm2)

[edit] Use of standard dyes like normal GFP

In 2008 Cremer´s lab discovered that superresolution microscopy was also possible for many standard fluorescent dyes like GFP, Alexa dyes and fluorescein molecules, provided certain photo-physical conditions are present. Using his specific localization microscopy called SPDMPhymod it is possible to detect and count two different fluorescent molecule types (this technology is referred to as 2CLM, 2 Color Localization Microscopy)^[7]

[edit] Gallery

Epifluorescent imaging of the three components in a dividing human cancer cell. DNA is stained blue, a protein called INCENP is green, and the microtubules are red. Each fluorophore is imaged separately using a different combination of excitation and emission filters, and the images are captured sequentially using a digital CCD camera, then overlaid to give a complete image.

Endothelial cells under the microscope. Nuclei are stained blue with DAPI, microtubules are marked green by an antibody bound to FITC and actin filaments are labeled red with phalloidin bound to TRITC. Bovine pulmonary artery endothelial (BPAE) cells

human lymphocyte nucleus stained with DAPI with chromosome 13 (green) and 21 (red) centromere probes hybridized (Fluorescent in situ hybridization (FISH))

Yeast cell membrane visualized by some membrane proteins fused with RFP and GFP fluorescent markers. Imposition of light from both of markers results in yellow color.

[edit] See also

• Microscope
• Mercury-vapor lamp
• Xenon arc lamp
• Stokes shift

[edit] References

[edit] Further reading

• Bradbury, S. and Evennett, P., Fluorescence microscopy., Contrast Techniques in Light Microscopy., BIOS Scientific Publishers, Ltd., Oxford, United Kingdom (1996).
• Rost, F., Quantitative fluorescence microscopy. Cambridge University Press, Cambridge, United Kingdom (1991).
• Rost, F., Fluorescence microscopy. Vol. I. Cambridge University Press, Cambridge, United Kingdom (1992). Reprinted with update, 1996.
• Rost, F., Fluorescence microscopy. Vol. II. Cambridge University Press, Cambridge, United Kingdom (1995).
• Rost, F. and Oldfield, R., Fluorescence microscopy., Photography with a Microscope, Cambridge University Press, Cambridge, United Kingdom (2000).

[edit] External links

• WikiScope
• Fluorophores.org - Database of fluorescent dyes.

v • d • e Optical microscopy Microscope · Optical microscopy Illumination and contrast methods Bright field microscopy · Köhler illumination · Dark field microscopy · Phase contrast · Differential interference contrast (DIC) · Dispersion staining · Second harmonic imaging (SHIM) Fluorescence methods Fluorescence microscopy · Confocal microscopy · Two-photon excitation microscopy · Multiphoton microscopy · Deconvolution Sub-diffraction limit techniques Diffraction limit · Vertico SMI (structured illumination) combining localization microscopy SPDMphymod · Stimulated emission depletion (STED) · Near-field (NSOM/SNOM) · Structured illumination · 4Pi Microscope