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Fluorescence Microscopy

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Definition: a technique for acquiring microscopic images using fluorescence which is excited in the sample

fluorescence microscope

Figure 1: Setup of a confocal fluorescence microscope

Fluorescence microscopy is a technique for acquiring microscopic images of samples (often biological materials) using fluorescence within the sample, which is usually excited with a sharply focused diffraction-limited laser beam. The focus of the laser beam is raster scanned through the sample (e.g. using galvo mirrors), and the fluorescence light excited in the sample is collected with some optics and monitored with a photodetector. Residual laser light can be eliminated with an optical filter before the detector. The fluorescence microscope is usually operated with a computer (a PC or a laptop), which controls the scan, records the fluorescence intensities, and finally generates, processes and stores the obtained images.

It is common to use a confocal geometry (Figure 1), where the light from the focus in the sample is imaged onto a pinhole, which therefore suppresses light coming from other regions (e.g. from before the focus). This allows the use of relatively thick samples, compared with conventional microscopy.

The fluorescence can originate from molecules which occur naturally in the sample, or from dye molecules which are introduced during sample preparation. The image contrast can arise from varying concentrations on the fluorescent molecules, or from influences on these molecules which e.g. modify their fluorescent lifetime (see below).

In the simplest form of fluorescence microscopy, the laser is operated continuously, and its wavelength is chosen to be in an absorbing spectral region of the fluorescent molecules, which can therefore be excited with single-photon absorption.

fluorescence microscopy image of cells

Figure 2: Fluorescence microscopy image of cells, acquired at the Integrated Microscopy Facility, University of California, Santa Barbara and kindly provided by Dr. Kalju Kahn.

Multiphoton Microscopy

An important variant of fluorescence microscopy utilizes multiphoton excitation of the fluorescence. Here, femtosecond pulses from a mode-locked laser are used, where the laser wavelength is not in a region of linear absorption, but multiphoton absorption (typically two-photon or three-photon absorption) occurs due to the high peak intensities. The laser wavelength is then often in the near-infrared region. Multiphoton fluorescence microscopy requires a more expensive laser system, but its advantages are manifold and substantial:

Another variant of the technique is stimulated emission depletion microscopy (STED microscopy) [3]. Here, the fluorescence marker substance is first excited with a Gaussian-shaped pump pulse. Shortly thereafter, a second pulse with a doughnut (donut) shape (having zero intensity at the center) is sent to the same spot. The second pulse has a slightly longer wavelength, and as a consequence it mainly causes stimulated emission, which depletes the excited region preferentially in a ring around the center of the original spot. As a result, a smaller excited region remains, and this can be exploited to achieve a significantly higher spatial resolution of below 100 nm, sometimes even below 20 nm. This resolution is substantially better than is normally possible for optical microscopy (→ breaking Abbe's resolution limit), except for methods of near-field microscopy.

Applications

As mentioned above, fluorescence microscopy is mostly used for biological samples, e.g. for medical diagnosis. It is currently investigated in which form fluorescence microscopy can be used e.g. for the detection of cancer. It is also useful for imaging living cells. Particularly with multiphoton fluorescence microscopy, it is possible, e.g., to watch cell division without severely influencing the cells, whereas cells often cannot tolerate single-photon excitation for extended time intervals.

Bibliography

[1]W. Denk et al., "Two-photon laser scanning fluorescence microscopy", Science 248, 73 (1990)
[2]National High Magnetic Field Laboratory, Optical microscopy primer on fluorescence microscopy, (http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorhome.html)
[3]S. W. Hell and J. Wichmann, "Breaking the diffraction resolution limit by stimulated emission", Opt. Lett. 19 (11), 780 (1994)
[4]S. W. Hell, and M. Kroug, "Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit", Appl. Phys. B 60, 495 (1995)
[5]T. A. Klar et al., "Breaking Abbe's diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes", Phys. Rev. E 64 (6), 066611 (2001)
[6]M. Dyba and S. W. Hell, "Focal spots of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution", Phys. Rev. Lett. 88, 163901 (2002)

See also: fluorescence, laser microscopy

Category: methods

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