INTRODUCTION TO FLUORESCENCE MICROSCOPY

Fluorescence microscopy offers a unique approach to the study of living and fixed cells because of its sensitivity, specificity and versatility. Fluorescence emitted from the biological sample can be simultaneously detected as both an image and as photometric data using the microscope, and has great potential for qualitative and quantitative studies on the function and structure of cells.

Stokes identified the phenomenon of fluorescence in the mid 19th century, and the first fluorescence microscopes were developed at the beginning of the 20th Century. These were used to study autofluorescence in organic and inorganic compounds. Imaging of secondary fluorescence (whereby specific tissues and bacteria which did not autofluoresce were labeled with a fluorescent marker and subsequently imaged) was developed in the 1930’s by Haitigen, and by the 1950’s, Koons and Caplan were using fluorescence microscopy to observe the location of antigens labeled with a fluorophore-tagged antibody (Wang and Lansing Taylor, 1989).

More recently, increasingly elaborate techniques including fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), and fluorescence resonance energy transfer (FRET) have been developed that enable the visualization and analysis of ever more complex events in cells, organelles and sub-organelle components within the biological specimen. Our experience in this central facility, however, is that these techniques are often shrouded in jargon and can baffle the inexperienced microscopist to such an extent that they would rather try and avoid doing it altogether. This short article therefore attempts to dispel some of the mysticism surrounding FLIM, FRET and FRAP by describing the basic principles of these techniques, how they are performed on a variety of fluorescence microscopes, and what practical benefit they might be to the cell biologist.

Before we consider the technicalities and practicalities of FRAP, FLIM and FRET, it is worth reminding ourselves of the principles of fluorescence and fluorescence microscopy. Each of these techniques in some way takes advantage of a particular aspect of the processes by which fluorophores are excited and damaged during excitation, or undergo non-radiative decay prior to photon emission, and a given system will necessarily have to employ different hardware to achieve this.

Fluorescence is a type of luminescence where light is emitted from molecules for only a short period of time following the absorption of light. When the delay between absorption and emission is in the order of nanoseconds or less, the emitted light is called fluorescence. When this delay is in the order of microseconds, it is called delayed fluorescence, and a delay greater than this is called phosphorescence.

In fluorescence microscopy, naturally occurring autofluorescent molecules and introduced fluorophores targeted to cellular structures of interest are irradiated with high intensity light. When these molecules absorb a quantum of light, a valence electron is boosted up into a higher energy orbit, creating an excited state. When this electron returns to its original, lower energy orbit, termed the ground state, a quantum of light may be emitted (see Fig 1).

Absorption occurs only at wavelengths of light whose quantum energy is equivalent to the difference in energy between the ground electronic state and the excited state. Consequently, a given fluorescent molecule will have a discrete wavelength at which it will become excited; this is know as its excitation spectrum (lakowicz, 1999).

Basic model of fluorescence excitation. When a fluorophore is excited, it can relax back to the ground state by releasing energy in the form of light. Other relaxation events, such as vibrational relaxation, also occur. This means that light emitted by the fluorophore has less energy than the light used to excite it.

Fluorescence microscopy - what it means in practice
The optical paths for image formation of the specimen are similar in fluorescence microscopy to those of a standard bright field microscope. The fundamental differences between bright field and fluorescence microscopy lie in the requirements of the fluorescence microscope to maximize the collection of emitted fluorescent light, while minimizing the collection of the incident excitation light. One of the key benefits of fluorescence microscopy, after all, is the increase in resolution through contrast made available by the selectivity of fluorophores to specific regions of the sample. Image contrast is critically dependent on the ability of the microscope to pass fluorescent light to the detector (typically a CCD camera or photomultiplier tube) while substantially blocking the excitation light.

Any light microscope relies on 3 components: an illumination source, a magnifying lens, and an image acquisition device. In the simplest light microscope this might consist of a candle, a convex lens, and the human eye. In a widefield microscope, the entire sample is illuminated simultaneously, and the image can be viewed directly either by eye or a camera. For a widefield fluorescence microscope, the candle is replaced by a high power lamp (typically a mercury or xenon source), which causes the fluorescently labeled sample to emit light, and images are typically acquired using a CCD camera. As we have seen already, fluorophores have characteristic excitation spectra, so an excitation filter (usually a band pass filter) is placed between the lamp and the sample to narrow the bandwidth of light reaching the sample. We have also seen that emitted light will be at a longer wavelength than the excitation light, and therefore an emission filter (either a long pass or band pass filter) is placed between the sample and the camera to block the excitation light from the image.

Successful fluorescence excitation relies on an intense source of light. In the past 20 years or so, lasers have provided an alternative excitation source to mercury and xenon lamps, and are commonly utilised in confocal and mulitphoton laser scanning microscopy to illuminate the sample. Lasers generally produce high intensity, monochromatic light. Because the light source is monochromatic, no excitation filter is required. An emission filter is still necessary to stop the laser reaching the acquisition device. Laser scanning microscopes operate by scanning the laser over the sample and building up an image, pixel by pixel, throughout the duration of the scan by collecting emitted light with a photomultiplier tube. In the case of confocal laser scanning microscopy, the mercury lamp is replaced by the laser, the excitation filter is removed, and the camera is replaced by a photomultiplier tube. The respective merits and application requirements that have led to the development of widefield fluorescence, fluorescence deconvolution, confocal laser scanning, and multiphoton microscopes are beyond the scope of this article (see reviews in Pawley, 1995 for further information).