Most spectrofluorometers can record both excitation and emission spectra. An emission spectrum is the wavelength distribution of an emission measured at a single constant excitation wavelength. In comparison, an excitation spectrum is measured at a single emission wavelength by scanning the excitation wavelength.

Specific light sources are chosen depending on the application.

Arc and incandescent xenon lamps

The high-pressure xenon (Xe) arc is the most versatile light source for steady-state fluorometers now. It can provides a steady light output from 250 - 700 nm (Figure 2), with only some sharp lines near 450 and 800 nm. The reason that xenon arc lamps emit a continuous light is the recombination of electrons with ionized Xe atoms. These ions produced by collision between Xe and electrons. Those sharp lines near 450 nm are due to the excited Xe atoms that are not ionized.

Figure 2: Spectral irradiance of arc-discharge lamps.

During fluorescence experiment, some distortion of the excitation spectra can be observed, especially the absorbance locating in visible and ultraviolet region. Any distortion displayed in the peaks is the result of wavelength-dependent output of Xe lamps. Therefore, we need to apply some mathematic and physical approaches for correction.

Most of the light sources used provide only polychromatic or white light. However, what is needed for experiments are various chromatic light with a wavelength range of 10 nm. Monocharomators help us to achieve this aim. Prisms and diffraction gratings are the two main kinds of monochromators used, although diffraction gratings are most useful, especially in spectrofluorometers.

Dispersion, efficiency, stray light level and resolution are important parameters for monochromators. Dispersion is mainly determined by slit width and expressed in nm/mm. It is prepared to have low stray light level. Stray light is defined as light transmitted by the monochromator at wavelength outside the chosen range. Also, a high efficiency is required to increase the ability to detect low light levels. Resolution depends on the slit width. There are normally two slits, entrance and exit in a fluorometers. Light intensity that passes through the slits is proportional to the square of the slit width. Larger slits have larger signal levels, but lower resolution, and vice verse. Therefore, it is important to balance the signal intensity and resolution with the slit width.

Optical filters are used in addition to monochromators, because the light passing through monochromator is rarely ideal, optical filters are needed for further purifying light source. If the basic excitation and emission properties of a particular system under study, then selectivity by using optical filters is better than by the use of monochromators. Two kinds of optical filter are gradually employed: colored filters and thin-film filters.

The standard detector used in many spectrofluorometers is the InGaAs array, which can provides rapid and robust spectral characterization in the near-IR. And the liquid-nitrogen cooling is applied to decrease the background noise. Normally, detectors are connected to a controller that can transfer a digital signal to and from the computer.

Tryptophan (trp), tyrosine (tyr), and phenylalanine (phe) are three natural amino acid with strong fluorescence (Figure 3). In tryptophan, the indole groups absorbs excitation light as UV region and emit fluorescence.

Figure 3: The structure of (a) tryptophan, (b) tyrosine and (c) phenylalanine.

Green fluorescent proteins (GFP) is another natural fluorophores. GFP is composed of 238 amino acids (Figure 4), and it exhibits a characteristic bright green fluorescence when excited. They are mainly extracted from bioluminescent jellyfish Aequorea vicroria, and are employed as signal reporters in molecular biology.

Figure 4: Green fluorescent proteins (GFP) ribbon diagram.

Most bio-molecules are nonfluorescent, therefore it is necessary to connect different fluorophores to enable labeling or tracking of the biomolecules. For example, DNA is an example of a biomolecule without fluorescence. The Rhodamine (Figure 5) and BODIPY (Figure 6) families are two kinds of well-developed organic fluorophores. They have been extensively employed in design of molecular probes due to their excellent photophysical properties.

Figure 5: The structure of Rhodamine 123.

Figure 6: The structure of selected boron-dipyrromethane (BODIPY) derivatives with their characteristic emission colors.

With the development of fluorophores, red and near-infrared (NIR) dyes attract increasing attention since they can improve the sensitivity of fluorescence detection. In biological system, autofluorescence always increase the ratio of signal-to-noise (S/N) and limit the sensitivity. As the excitation wavelength turns to longer, autopfluorescence decreases accordingly, and therefore signal-to-noise ratio increases. Cyanines are one such group of long-wavelength dyes, e.g., Cy-3, Cy-5 and Cy-7 (Figure 7), which have emission at 555, 655 and 755 nm respectively.

Figure 7: The structure of (a) Cy-3-iodo acetamide, (b) Cy-5-N-hydroxysuccinimide and (c) Cy-7-isothiocyanate.

Almost all of the fluorophores mentioned above are organic fluorophores that have relative short lifetime from 1-10 ns. However, there are also a few long-lifetime organic fluorophore, such as pyrene and coronene with lifetime near 400 ns and 200 ns respectively (Figure 8). Long-lifetime is one of the important properties to fluorophores. With its help, the autofluorescence in biological system can be removed adequately, and hence improve the detectability over background.

Figure 8: Structures of (a) pyrene and (b) coronene.

Although their emission belongs to phosphorescence, transition metal complexes are a significant class of long-lifetime fluorophores. Ruthenium (II), iridium (III), rhenium (I), and osmium (II) are the most popular transition metals that can combine with one to three diimine ligands to form fluorescent metal complexes. For example, iridium forms a cationic complex with two phenyl pyridine and one diimine ligand (Figure 9). This complex has excellent quantum yield and relatively long lifetime.

Figure 9: The structure of the cationic iridium complex, (ppy)₂Ir(phen).