Although spectral imaging and linear unmixing is a very powerful tool to reduce or eliminate signal bleed-through artifacts in fluorescence microscopy when imaging multiply labeled specimens, one should also be aware of potential artifacts and errors that can be introduced when linear unmixing is applied without adhering to the proper experimental conditions. Among the most important considerations is to note that the success of this technique depends upon a number of factors that are within the control of the microscopist. However, perhaps the two most critical aspects for obtaining reasonable results are attention to detail during specimen preparation and the application of reference spectra that faithfully represent the true profiles of fluorophores present in the specimen. Thus, control specimens containing individual fluorophores to be used as "fingerprint" references should be imaged under the same conditions as the specimen containing a mixture of probes to be distinguished. In addition, the best results will be obtained when the signal-to-noise ratio of all fluorophores under investigation has been optimized. In all cases, saturation of the detector should be avoided and the background noise level should be kept to a minimum. Furthermore, the optical components of every microscope system are highly variable and will impose some degree of bias, rendering it virtually impossible to reliably use spectra collected from another microscope as references. As a rule of thumb, the optimum reference spectra are obtained using identical conditions to those used for acquiring the lambda stack. These conditions include using the same objective, offset, amplifier gain, wavelength range, and dichromatic mirrors. If possible, reference spectra can be measured in regions of the specimen that are labeled with only a single probe, or they can be chosen from non-overlapping regions within the lambda stack itself.

Another potential source of artifacts in spectral imaging is high background signal arising from the mounting media, the specimen itself (autofluorescence), or from lipophilic reagents often used to introduce plasmids via transfection. Reflections from laser excitation sources may also contribute to high background levels. In general, these background sources should be treated as a spectral component whenever possible (in effect, by gathering reference spectra) during linear unmixing in order to successfully separate background from specific fluorophore signals. During analysis, spectral signals that do not match those in the reference library are considered residual and are not assigned. In cases where a residual image is part of the output, however, it is possible to examine how well spectra from the reference library fit the acquired spectra. In many cases, residual signals arise from saturated pixels or from high background levels.
When preparing specimens for spectral imaging and linear unmixing, the best policy is to ensure that expression of two or more genetically-encoded fluorescent proteins is balanced and that synthetic dyes are applied in concentrations that yield similar intensities. Dramatic mismatches in intensity will saturate the detector for the most concentrated probes while those of lower intensity can easily be lost in the noise floor (in the worst case scenario). Achieving a high signal-to-noise ratio for each fluorophore in the specimen will also help to produce the best results with linear unmixing, as will carefully planning the staining regime to avoid using probes having spectra that overlap too closely. In addition, lambda stacks should be composed of wavelength bands that are representative of the spectral differences between the fluorophores. The best choice in deciding acquisition parameters for lambda stacks is to choose enough wavelength bands to record data from throughout the emission spectrum of all the fluorophores present in the specimen.
Illustrated in Figure 1 are examples of spectral imaging and linear unmixing in multiply stained adherent cell cultures having fluorophore intensities that are either unbalanced (Figures 1(a) and 1(b)) or balanced (Figures 1(c) and 1(d)). Figure 1(a) shows a bovine pulmonary artery endothelial cell (BPAE line) labeled with three green emitting fluorophores: Alexa Fluor 488 conjugated to phalloidin (filamentous actin), Alexa Fluor 514 conjugated to a cytochrome C targeting antibody (mitochondria), and SYTOX Green (nucleus). Note that the intensity of SYTOX Green is so disproportionate with respect to the other labels that signal from the two Alexa Fluor dyes is seriously compromised in the unmixed image (Figure 1(b)). In contrast, the same cell line stained with three orange fluorophores: Alexa Fluor 555 (filamentous actin), SYTOX Orange (nucleus), and MitoTracker Red CMXRos (mitochondria) produces far superior results when the fluorophore concentrations are adjusted to produce similar intensities (Figure 1(c)). After reassignment of the labeled structures with pseudocolors in the unmixed image (Figure 1(d)), The blue nucleus is visualized on a bed of red mitochondria surrounded by green actin stress fibers.
Although linear unmixing is capable of separating fluorescence spectra that are highly overlapping, there are limits in the ability of most algorithms to distinguish between fluorophores with emission spectra that are virtually superimposed. A comprehensive analysis of common green fluorophores (having emission maxima ranging from 506 to 530 nanometers; see Figure 2) demonstrated that, when mixed together in a single specimen, many of the dye combinations could be separated using linear unmixing despite their similarity. In cases where the fluorescence signal level is balanced with respect to intensity, the spectral profiles of fluorophores having wavelength maxima variations of only 4 to 5 nanometers (for example, fluorescein and Oregon Green 488; maxima at 519 and 523 nanometers, respectively) can be successfully unmixed. For probes having emission maxima separated by more than 7 nanometers (such as EGFP and SYTOX Green; maxima at 509 and 524 nanometers, respectively), linear unmixing can reliably separate the spectra, even under conditions where different signal levels occur. It should be noted, however, that fluorescent probes having spectral profiles with an excessively high degree of overlap (in effect, almost completely), such as fluorescein and Alexa Fluor 488 or SYTOX Green and Oregon Green 488, cannot be distinguished with spectral imaging and linear unmixing.

In summary, there are several practical considerations that will assist in ensuring the greatest potential for success in real-world investigations using spectral imaging and linear unmixing. Perhaps the most important aspect is to carefully gather reference spectra for all fluorophores that faithfully represent the spectra likely to be obtained from the test specimen. In this regard, all controls must (without deviation) be imaged under identical conditions. Pixel saturation and high background noise should be avoided, and autofluorescence should be taken into consideration as a component spectrum in cases where it cannot be eliminated. In addition, unwanted signal from laser lines and mounting media discontinuities can affect linear unmixing results. Finally, low signal levels arising from dispersing the emission signal over a broad spectral range can adversely affect the results of quantitative experiments. Whenever possible, fluorophore concentrations (or at least signal levels) should be as closely matched as possible to ensure optimum results.