Fluorescence, confocal and related techniques

About this technique

---

Fluorescence microscopy

Fluorescence microscopy illuminates the sample through the objective lens, with short wavelength light, which will excite a fluorescent dye, or fluorochrome. The fluorochrome will emit light at a longer wavelength and a wavelength-selective dichroic mirror in the optical path will let this light travel to the eyepieces or camera. Typically excitation is with near-ultraviolet, blue or green light, and emission is respectively in the blue, green or red. Some samples are naturally fluorescent but most will need to be stained. There are three approaches to fluorescent staining:

• Fluorescent dyes
  Some dyes are designed to target specific cellular structures and chemical entities. The best known is DAPI, which stains DNA, but stains are available for most cell structures and organelles. Some (vital stains) will work on living cells, others require the cells to be fixed. Calcium movement within and between cells is commonly studied using calcium-specific fluorescent dyes in living cells.
• Immunolabeling
  This targets specific proteins in the cell with antibodies raised in some other animal (rat, mouse, goat or rabbit). The antibody is coupled to a fluorescent dye. The cells will normally be fixed to allow penetration of the antibody. After staining the cells are usually mounted in a medium (often glycerol-based) containing an anti-fade agent, since fluorescent dyes do fade under continued exposure to light.
• Fluorescent proteins
  The original fluorescent protein, green fluorescent protein (GFP) was isolated from a jellyfish. Now a range of other colours is available, some genetically modified from GFP, others derived from related organisms (corals and sea anemones). These are genetically encoded proteins, which are expressed within the cell and therefore allow us to observe living material. Typically a construct is made which will code for the FP attached to a target molecule, for example tubulin. This is either incorporated into the genome (permanent transfection) or introduced into the cell (temporary transfection). Then the targeted structure (microtubules in this example) will be fluorescent, and can be observed dynamically in a living cell. The sample will be in an aqueous medium, so the same caveats about correct objectives apply as for other living cells. Since this technique involves genetically modified organisms.

Confocal microscopy

A confocal microscope scans the sample with a spot of light in a regular ‘raster’ pattern and so acquires an image point by point. The end result, on this basis, is no different from the image given by a regular microscope. But because only one point is acquired at a time, we can place a pinhole where the spot is imaged, and so eliminate out of focus light. Light that is in focus will be a small spot and will pass through the pinhole, but out of focus light will be a fuzzy blob and will mostly be blocked. This enables true three-dimensional imaging. To get enough light in one point, a laser is usually the illumination source. 

Confocal microscopes can be operated in reflection mode – typically for surface profiling, but in the life sciences they are more often used in fluorescence mode, where out of focus glare can be a major problem. Typically, one uses a confocal microscope either to collect a three-dimensional set of slice images, or to image a single layer in cases where strong surrounding fluorescence would spoil the image in a conventional fluorescence microscope. 

In confocal microscopy one needs to bear in mind that there are only a few, discrete, laser lines for illumination and the stain must be chosen to suit. Excitation (except in some special cases) is only in the visible range, with 405 nm (deep violet) the shortest wavelength available. Also, correct matching of mounting medium, coverslip and objective becomes critically important for 3-D imaging. Specific techniques such as FRET, FRAP, FLIM, FCS/RICS and TIRF are available to address more defined questions and are dealt with separeately

Multiphoton microscopy

Multiphoton microscopy is essentially an extension of confocal imaging. Very short, intense pulses of light in the near infrared are used for illumination and fluorescence is excited when two photons hit the fluorochrome molecule simultaneously, and behave as one photon of half the wavelength. The intensity of a single pulse is very high, but the spaces are much longer than the pulses, so that averaged over time the irradiation is comparable to confocal microscopy. Since two-photon events will only take place at the focus of the excitation spot, optical sectioning is automatic without any confocal pinhole. This means that detection can be made more efficient, particularly in a scattering sample, and it also means that that there will be no bleaching above and below the focal plane.

The longer wavelengths used in multiphoton imaging penetrate better into thick samples, and are also less damaging to living cells, than the wavelengths used for conventional fluorescence. Other key benefits are the ability to excite fluorochromes such as DAPI and calcium ratio dyes which would normally require near UV, and a wide wavelength selection since the lasers used can be tuned through the range 700–1000 nm. 

Second harmonic generation

Certain substances have the ability, when excited by strong pulses of light, to generate the second harmonic – that is, light of twice the frequency (hence half the wavelength). Typically these are molecules with no centre of symmetry, and the more highly crystalline they are the stronger the effect. In biological samples collagen, starch and myosin are all candidates. This means that they can be imaged without any staining or labelling, and since it is a purely physical process there is no fading. The microscope setup is just as for multiphoton imaging, and both can be done at once.