The tutorial initializes with a graph of three fluorescent protein spectra (ECFP, EGFP, and EYFP) in the upper left-hand corner along with a pseudocolored image of a triple-labeled specimen expressing the probes targeted to the nucleus, mitochondria, and actin network, respectively. A stack of lambda sections appears in the lower left-hand corner. In order to operate the tutorial, use the Lambda Stack Depth slider to examine individual images from the lambda stack, which are displayed in a window directly to the right of the stack. Alternatively, use the AUTO button to automatically play through the stack. As different sections are presented, the unmixed and pseudocolored versions are simultaneously displayed in the specimen image window and an arrow above the spectra indicates the position of the bandpass region.

In order to better understand the lambda stack concept (also commonly referred to in the literature as an image cube or spectral cube), a single pixel location in the lateral image dimension (having coordinates xi,yi) can be examined along the wavelength (zλ) axis. As illustrated in Figure 1(a), the intensity and/or color of the pixel i changes as a function of fluorescence emission signal strength and wavelength, respectively, when monitored from one end of the lambda stack to the other. By plotting pixel intensity versus wavelength on a linear graph (see Figure 1(b)), the emission spectral profile of the particular fluorophore spatially located at pixel i can readily be determined. It should be noted that the accuracy and resolution of an emission spectrum obtained using this technique is a function of the number of lambda stack images gathered at distinct wavelength bands, the spectral width in nanometers of each wavelength band (shorter bandwidths produce higher resolution), the physical quality of the specimen under investigation, and the photon sensitivity (quantum efficiency) of the detector.

A real-world example of a lambda stack acquired on a Nikon A1 laser scanning confocal microscope in living cells using three fluorescent proteins having overlapping spectra is presented in Figure 2. The fluorescent protein markers used in this experiment are enhanced green fluorescent protein (EGFP from jellyfish; emission maximum at 507 nanometers), enhanced yellow fluorescent protein (EYFP from jellyfish; emission maximum at 527 nanometers), and the monomeric version of Kusabira Orange (mKO, emission maximum at 561 nanometers), a high-performance probe developed from a naturally-occurring coral protein. In this case, the individual lambda stack images were scanned in 10-nanometer wavebands ranging from 480 to 640 nanometers (Figure 2(a)) to generate a total of 16 spectral sections for the fluorescent protein mixture.

The first image of the lambda stack reveals the spectral signature of the specimen in the emission range of 480 to 490 nanometers, while the second image contains emission data from 490 to 500 nanometers (see Figure 2(b)). Note that virtually all of the fluorescence emission in the first two lambda sections arises from the short-wavelength tail of EGFP alone with only a very minor contribution from EYFP in the longer wavelength section (490 to 500 nanometers). In the next two lambda sections (500 to 510 nanometers and 510 to 520 nanometers), the contribution from EYFP steadily increases as the emission from EGFP reaches a plateau. In the three lambda sections between 520 and 550 nanometers, The EGFP signal begins to decrease as the contribution from EYFP emission reaches a maximum at approximately 530 nanometers. Likewise, the emission contribution from mKO becomes more significant in the band between 540 and 550 nanometers. Thus, in the 550 to 560 nanometer band, the relative contributions from the fluorescent proteins are approximately 10, 25, and 65 percent respectively for EGFP, EYFP, and mKO.

Emission contributions from EGFP and EYFP become diminished in the final wavelength bands (560 to 640 nanometers) as the emission from mKO dominates. In review, the wavelength bands at the extremes of the lambda stack (between 480 to 500 nanometers and between 590 to 640 nanometers) feature emission contributions that are dominated by the shortest and longest wavelength-emitting proteins, EGFP and mKO, respectively. Those wavelength bands in the center of the lambda stack (500 to 590 nanometers) contain fluorescence emission that represents some contribution from all three fluorescent proteins. As will be discussed below, the distribution of the mixed emission signal across the wavelength bands of the lambda stack can be linearly unmixed using reference emission spectral profiles from each probe to clearly separate the contribution of the individual fluorescent proteins.