The tutorial initializes with the individual spectral profiles contained within a lambda stack captured from a nucleus stained with EGFP, Alexa Fluor 488, and SYTOX Green appearing in the spectral window. Beneath the spectra are pseudocolored images of a single nucleus as it appears in each 10-nanometer wavelength band, with the emission intensity being directly proportional to the amount of fluorophore present. An image of the mixed spectra is presented in the lower right-hand corner of the tutorial. In order to operate the tutorial, use the Lambda Stack Section slider to translate through the various wavebands and observe the mixed image as well as the intensity proportions displayed in the spectral graph. A new fluorophore (cyan, green, or orange) can be selected with the Spectral Profile pull-down menu.

In order to measure the spectrum of an absorbing dye, fluorophore, or complete specimen with multiple labels, the transmitted or emitted light is first dispersed into its component wavelengths and the intensity at each wavelength or a very narrow band of wavelengths is measured. The spectral resolution is dependent upon the bandwidth of each measurement and increases as the bandwidth of the sampled channels decreses. A variety of different techniques can be used to disperse light, and most of them have been applied (at least in prototype instruments) to microscopy scenarios. Among the most important characteristics to consider when measuring spectra are resolution, wavelength range, and dynamic range. Spectral resolution is determined by the closest wavelengths that can be distinguished from one another and is a critical parameter for highly accurate spectral imaging measurements. Spectral range refers to the total number of wavelengths (in effect, the bandwidth) in a particular measurement. Finally, the detection limit and dynamic range define the minimum level of signal necessary for conducting measurements and number of distinguishable levels in a particular measurement, respectively. All of these values can vary for each fluorophore or absorbing species as a function of the spectral profile.

Illustrated in Figure 1 is a typical set of spectral images acquired in sequential bandwidths of six nanometers spanning the wavelength range of 500 to 692 nanometers to generate a lambda stack (discussed in detail below) containing 32 images. The specimen is a culture of adherent human cervical carcinoma (HeLa line) cells in which DNA and RNA was stained using Acridine Orange and imaged with a Nikon A1 spectral confocal microscope system. Images (512 x 512 pixels) were recorded using the 32-channel multianode photomultiplier at 24 frames per second using 488-nanometer laser excitation. Such a high acquisition speed, which is of significant benefit in live-cell imaging, is made possible by advanced signal processing technology coupled with fast analog-to-digital conversion circuitry that operates in tandem with the photomultiplier.