The dramatic increase in multi-color fluorescence microscopy applications witnessed over the past decade is due, in part, to the significant advances in instrument and detector design as well as the introduction of a vast array of new fluorophores, including synthetics and quantum dots. In addition, live-cell imaging has been revolutionized by the introduction of ever increasingly useful genetically-encoded fluorescent proteins spanning the entire visible spectral region. Among the primary advantages of using multiple fluorescent labels in fixed and living cells and tissues is the ability to observe the spatial relationship and temporal dynamics of subcellular constituents, and to monitor potential molecular interactions between differently labeled entities. A number of advanced microscopy techniques have been applied using multi-color fluorescence labeling, including fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), fluorescence (or Förster) resonance energy transfer (FRET), fluorescence in situ hybridization (FISH), and fluorescence lifetime imaging (FLIM). Many of these methods benefit significantly from the ability to use specifically targeted fluorescent proteins in live-cell imaging experiments.

Spectral imaging combined with linear unmixing is a highly useful technique (see Figure 1) that can be used in combination with other advanced imaging modalities to untangle fluorescence spectral overlap artifacts in cells and tissues labeled with synthetic fluorophores that would be otherwise difficult to separate. Illustrated in Figure 1 is an Indian Muntjac fibroblast cell stained with a combination of SYTOX Green (nucleus), Alexa Fluor 488 conjugated to phalloidin (filamentous actin network), and Oregon Green 514 conjugated to goat anti-mouse primary antibodies (targeting mitochondria). These dyes have emission maxima at 523, 518, and 528 nanometers, respectively, and each appears green to the eye when viewed in widefield fluorescence using a standard FITC cube (Figure 1(a); imaged in confocal mode with a 488-nanometer argon-ion laser). Using laser scanning confocal microscopy coupled with a spectral imaging detector, the entire spectrum of fluorophores in the specimen was first gathered (Figure 1(a)), and then linearly unmixed and assigned pseudocolors (Figure 1(b); nucleus, cyan; actin, green; mitochondria, red) to more clearly delineate the relative proximity of the various structures.
Shortly after enhanced green fluorescent protein (EGFP) was introduced as a viable probe for live-cell imaging in the mid-1990s, investigators began to fuse the purified DNA sequence to that of a wide variety of endogenous proteins having highly specific intracellular targets. The resulting genetically-expressed chimeras are able to serve as molecular beacons to enable tracking of virtually any protein of interest in living cells. Fluorescence imaging of EGFP alone is relatively straightforward and can be readily conducted using a single longpass emission filter that covers the entire green-to-red spectral region (approximately 520 to 650 nanometers). For abundant targets, a high level of signal can be captured with virtually any digital camera system using this configuration. However, with the emergence of new fluorescent proteins in the lower wavelength emission regions (blue and cyan), as well as those emitting at longer wavelengths (yellow, orange, red, and far red), the use of bandpass emission filters becomes necessary in order to segregate the emission of closely overlapping spectra in experiments using two or more fluorescent proteins.
Although recent advances in synthetic fluorophore technology and the development of biological quantum dot conjugates promises to ultimately yield tremendous flexibility in labeling of fixed cells and tissues, live-cell imaging techniques currently rely heavily on the use of fluorescent proteins fused to target peptides and proteins that label specific proteins and subcellular organelles. However, even though a wide variety of new fluorescent protein color variants have become available in the past few years, the number of proteins that can be simultaneously imaged in multi-color investigations is limited by the broad, overlapping spectral profiles of these fluorophores. For example, several of the most useful variants, including enhanced cyan, green, and yellow fluorescent protein (ECFP, EGFP, and EYFP), perform superbly in fusions and are very useful when imaged alone. Unfortunately, these probes have strongly overlapping emission spectra that are virtually impossible to separate into specific channels during colocalization experiments using traditional interference filters. In most cases, narrow bandwidth emission filters must be utilized to discriminate between two or more fluorescent proteins, which results in the significant loss of otherwise useful signal. In live-cell imaging experiments, however, it is necessary to gather as much signal as possible to minimize the required expression level of fluorescent proteins and to reduce the intensity of excitation light in order to avoid phototoxicity.

The emission profile of a typical fluorophore is usually non-symmetrical (with the exception of quantum dots) such that the slope of the curve is far steeper for wavelengths shorter than the maximum (peak) and much broader for longer wavelengths past the peak, which contain most of the total fluorescence emission, as illustrated in Figure 2(b). The full width at half maximum (FWHM; the measure of bandwidth size of a spectral profile at 50-percent of the maximum intensity) of most synthetic fluorophores used in fluorescence imaging ranges from 20 nanometers to over 60 nanometers, whereas that of fluorescent proteins averages between 40 and 150 nanometers. Given that the visible light spectrum spans a wavelength range of approximately 300 nanometers (400 to 700 nanometers), it is apparent that only a limited number of synthetic fluorophores or fluorescent proteins can be simultaneously imaged in this region without incurring bleed-through artifacts due to significant spectral overlap. Note that bleed-through is often referred to as crosstalk in the scientific literature; however, the two terms are considered synonymous. As the desire to conduct experiments using specimens having more than two fluorescent labels increases, the ability to quantitatively assess resulting datasets is often compromised by a number of factors, including the inability to limit emission to a single detection channel. Thus, the unambiguous identification of specific fluorescence information is not always possible, particularly when there exists a mixture of strong and weak signals or if the choice of probes is limited, as is often the case for fluorescent proteins.