Introduction to Fluorescence Microscopy
The absorption and subsequent re-radiation of light by organic and inorganic specimens is typically the result of well-established physical phenomena described as being either fluorescence orphosphorescence. The emission of light through the fluorescence process is nearly simultaneous with the absorption of the excitation light due to a relatively short time delay between photon absorption and emission, ranging usually less than a microsecond in duration. When emission persists longer after the excitation light has been extinguished, the phenomenon is referred to as phosphorescence.
British scientist Sir George G. Stokes first described fluorescence in 1852 and was responsible for coining the term when he observed that the mineral fluorspar emitted red light when it was illuminated by ultraviolet excitation. Stokes noted that fluorescence emission always occurred at a longer wavelength than that of the excitation light. Early investigations in the 19th century showed that many specimens (including minerals, crystals, resins, crude drugs, butter, chlorophyll, vitamins, and inorganic compounds) fluoresce when irradiated with ultraviolet light. However, it was not until the 1930s that the use of fluorochromes was initiated in biological investigations to stain tissue components, bacteria, and other pathogens. Several of these stains were highly specific and stimulated the development of the fluorescence microscope.
The technique of fluorescence microscopy has become an essential tool in biology and the biomedical sciences, as well as in materials science due to attributes that are not readily available in other contrast modes with traditional optical microscopy. The application of an array of fluorochromes has made it possible to identify cells and sub-microscopic cellular components with a high degree of specificity amid non-fluorescing material. In fact, the fluorescence microscope is capable of revealing the presence of a single molecule. Through the use of multiple fluorescence labeling, different probes can simultaneously identify several target molecules simultaneously. Although the fluorescence microscope cannot provide spatial resolution below the diffraction limit of specific specimen features, the detection of fluorescing molecules below such limits is readily achieved.
Fundamentals of Excitation and Emission
The basic function of a fluorescence microscope is to irradiate the specimen with a desired and specific band of wavelengths, and then to separate the much weaker emitted fluorescence from the excitation light. In a properly configured microscope, only the emission light should reach the eye or detector so that the resulting fluorescent structures are superimposed with high contrast against a very dark (or black) background. The limits of detection are generally governed by the darkness of the background, and the excitation light is typically several hundred thousand to a million times brighter than the emitted fluorescence.
Illustrated in Figure 1 is a cutaway diagram of a modern epi-fluorescence microscope equipped for both transmitted and reflected fluorescence microscopy. The vertical illuminator in the center of the diagram has the light source positioned at one end (labeled the episcopic lamphouse) and the filter cube turret at the other. The design consists of a basic reflected light microscope in which the wavelength of the reflected light is longer than that of the excitation. Johan S. Ploem is credited with the development of the vertical illuminator for reflected light fluorescence microscopy. In a fluorescence vertical illuminator, light of a specific wavelength (or defined band of wavelengths), often in the ultraviolet, blue or green regions of the visible spectrum, is produced by passing multispectral light from an arc-discharge lamp or other source through a wavelength selective excitation filter. Wavelengths passed by the excitation filter reflect from the surface of a dichromatic (also termed a dichroic) mirror or beamsplitter, through the microscope objective to bath the specimen with intense light. If the specimen fluoresces, the emission light gathered by the objective passes back through the dichromatic mirror and is subsequently filtered by abarrier (or emission) filter, which blocks the unwanted excitation wavelengths. It is important to note that fluorescence is the only mode in optical microscopy where the specimen, subsequent to excitation, produces its own light. The emitted light re-radiates spherically in all directions, regardless of the excitation light source direction.
As presented in Figure 1, the reflected light vertical illuminator comprises an arc-discharge lamphouse at the rear end (usually a mercury or xenon burner). Excitation light travels along the illuminator perpendicular to the optical axis of the microscope, passes through collector lenses and a variable, centerable aperture diaphragm, and then through a variable, centerable field diaphragm (see Figure 1). The light then impinges upon the excitation filter where selection of the desired band and blockage of unwanted wavelength occurs. The selected wavelengths, after passing through the excitation filter, reach the dichromatic beamsplitting mirror, which is a specialized interference filter that efficiently reflects shorter wavelength light and efficiently passes longer wavelength light. The dichromatic beamsplitter is tilted at a 45-degree angle with respect to the incoming excitation light and reflects this illumination at a 90-degree angle directly through the objective optical system and onto the specimen. Fluorescence emission produced by the illuminated specimen is gathered by the objective, now serving in its usual image-forming function. Because the emitted light consists of longer wavelengths than the excitation illumination, it is able to pass through the dichromatic mirror and upward to the observation tubes or electronic detector.
Vibrational energy is lost when electrons relax from the excited state back to the ground state. As a result of the energy loss, the emission spectrum of an excited fluorophore is usually shifted to longer wavelengths when compared to the absorption or excitation spectrum (note that wavelength varies inversely to radiation energy). This well-documented phenomenon is known as Stokes’ Law or Stokes’ shift. As Stokes’ shift values increase, it becomes easier to separate excitation from emission light through the use of fluorescence filter combinations.
Fading, Quenching, and Photobleaching
A wide spectrum of conditions often come into play that ultimately affect the re-radiation of fluorescence emission and thus reduce the intensity. The general term for a reduction of fluorescence emission intensity is fading, a catch-all category that is usually further subdivided into quenching and photobleaching phenomena for more precise descriptions. Photobleaching is the irreversible decomposition of the fluorescent molecules in the excited state because of their interaction with molecular oxygen before emission. The occurrence of photobleaching is exploited in a technique known as fluorescence recovery after photobleaching (FRAP), a very useful mechanism for investigating the diffusion and motion of biological macromolecules. The method is based upon photobleaching a sharply defined region of the specimen by an intense burst of laser light, accompanied by the subsequent observation of the rates and pattern of fluorescence recovery in the photobleached area.
Presented in Figure 4 is a typical example of photobleaching (fading) observed in a series of digital images captured at different time points for a multiply-stained culture of Indian Muntjac deer epidermis fibroblast cells.
Fluorescence Light Sources
The mercury burners do not provide even intensity across the spectrum from ultraviolet to infrared, and much of the intensity of the lamp is expended in the near ultraviolet. Prominent peaks of intensity occur at 313, 334, 365, 406, 435, 546, and 578 nanometers. At other wavelengths in the visible light region, the intensity is steady although not nearly so bright .
In the past few years, optical microscopy has experienced an increase in the application of laser light sources, particularly the argon-ion and argon-krypton (ion) lasers. These lasers have the virtues of small source size, low divergence, near-monochromicity, and high mean luminance.
Luminous Density of Selected Light Sources
The efficiency of detection is a function of the optical collection efficiency and the detector quantum efficiency. A 1.4-numerical aperture objective with 100-percent transmission (an unrealistic condition) has a maximum collection efficiency, limited by the acceptance angle of about 30 percent. The transmission efficiency of the dichromatic mirror is 85 percent and that of the barrier filter is 80 percent. The overall collection efficiency is then about 20 percent or 140 billion photons per second. If the detector is a conventional charge-coupled device (CCD), the quantum efficiency is about 50 percent for the green fluorescein emission (at 525 nanometers), so the detected signal would be 70 billion photons per second or about 10 percent of the emitted fluorescence. Even with a perfect detector (100 percent quantum efficiency), only about 20 percent of the fluorescence emission photons can be detected.
Detecting Single Molecules
Under ideal conditions, it is often possible to detect the fluorescence emission from a single molecule, provided that the optical background and detector noise are sufficiently low. As discussed above, a single fluorescein molecule could emit as many as 300,000 photons before it is destroyed by photobleaching. Assuming a 20-percent collection and detection efficiency, about 60,000 photons would be detected. Using avalanche photodiode or electron multiplying CCD detectors for these experiments, investigators have been able to monitor the behavior of single molecules for many seconds and even minutes. The major problem is adequate suppression of the optical background noise. Because many of the materials utilized in construction of microscope lenses and filters display some level of autofluorescence, efforts were initially directed toward the manufacture of very low fluorescence components. However, it soon became evident that fluorescence microscopy techniques utilizing total internal reflection (TIR) provided the desired combination of low background and high excitation light flux.
Total internal reflection fluorescence microscopy can also be conducted through a modification of the epi-illumination approached utilized in widefield techniques (as illustrated in Figure 7(b)). This method requires a very high numerical aperture objective (at least 1.4, but preferably 1.45 to 1.6) and partial illumination of the microscope field from one side by a small sport or more uniform illumination by a thin annulus. High refractive index lens immersion medium and microscope cover glass are required to achieve the illumination angle resulting in total internal reflection. As presented in Figure 7(b), light rays exiting the objective front lens element at an angle less than the critical angle (denoted asA(1)) in figure 7(b)) are transmitted away from the microscope. When the angle is increased to or beyond the critical angle (indicated a angle A(2) in Figure 7(b)), total internal reflection results.
- The era when optical microscopy was purely a descriptive instrument or an intellectual toy is past. At present, optical image formation is only the first step toward data analysis.
- The microscope accomplishes this first step in conjunction with electronic detectors, image processors, and display devices that can be viewed as extensions of the imaging system. Computerized control of focus, stage position, optical components, shutters, filters, and detectors is in widespread use and enables experimental manipulations that were not humanly possible with mechanical microscopes.
- The increasing application of electro-optics in fluorescence microscopy has led to the development of optical tweezers capable of manipulating sub-cellular structures or particles, the imaging of single molecules, and a wide range of sophisticated spectroscopic applications.