A major problem in live cell imaging arises from the use of different fluorochromes with overlapping spectra within one sample, impairing a number of applications.
Even with the use of high quality optical filters it is not possible to separate the spectral information satisfactorily. The consequence is that for example yellow fluorescent protein (YFP)-labelled structures are visible with a green fluorescence protein (GFP) filter set and vice versa which has a severe effect on resulting images. The considerable overlap of excitation and emission spectra of these fluorochromes is exemplified in (Fig. 1). This phenomenon, known as 'bleed-through', strongly reduces colour resolution and constrains scientific conclusions.
Spectral unmixing allows the use of these uncommon fluorescent protein combinations. It's a technique for chromatic resolution enhancement and results in sharply contrasted images. In FRET studies the occurrence of FRET becomes immediately obvious in the images after spectral unmixing even before quantitative analysis is performed. In co-localisation studies bleed-through artefacts are avoided.
cell^R is the first imaging system to implement "Spectral Imaging and Linear Unmixing", a technique originally develloped for satellite imaging and adapted to wide-field fluorescence microscopy. With this powerful method it becomes possible to ascertain the contribution of different fluorochromes to the total signal and, by redistribution of the intensity, to restore a clear signal for each colour channel undisturbed by emission from the other fluorochrome. It is important to note that neither original data are lost during Linear Unmixing nor additional data are added to the image: it is the original image information that is used in the procedure; the overall intensity of pixels will be maintained. Thus, the technique does not create artificially embellished images. After unmixing, quantification analysis is not only still possible but also more precise.
Spectral unmixing in cell^R is comprised of two steps: Calibration and Unmixing. Currently, it is possible to separate two or three different colour channels.
Calibration
To unmix the spectral information of the fluorochromes with strongly overlapping emission spectra, it is necessary to determine the spectral properties of the individual fluorochromes under the same imaging conditions used for the multi-labelled samples (for example, GFP/YFP): The system has to be calibrated for each of the two fluorochromes by taking reference images of single-labelled samples. Here reference images are taken from a GFP-only labelled sample with two corresponding single-band excitation filters (for GFP and for YFP) using the same dichroic mirrors and emission filters. The latter two can be either two single-band filter sets or a dual-band filter set. Then the same procedure is applied to a YFP-only labelled sample. The reference images have only to be taken once for each set of fluorochromes at a given experimental set-up (e.g., filter set, labelling method, and cell type).
The ratio of the average intensities of single fluorochrome-labelled structures (ROIs – regions of interest) measured at these two excitation wavelengths after background correction is a constant which is specific for each fluorochrome under given experimental conditions.
Unmixing
The two reference ratios are the prerequisite for the unmixing of the images of double-labelled samples (GFP/YFP). These images are taken at both excitation wavelengths and combined into a dual-colour image. After background correction, the unmixing algorithm redistributes the intensities of the two colour channels using the calibration ratios.
Figure 2 shows the multi-colour image of a GFP/YFP double-labelled sample, before (left) and after spectral unmixing (right). In this sample GFP was fused with H2B histone protein and YFP with tubulin. The visible result is a pronounced chromatic resolution of green and yellow structures. It is now even possible to detect yellow tubulin on top of bright green nuclei.
