![]() ![]() Finally, autofluorescence and/or high background levels should also be defined spectrally (if possible) and treated as an additional fluorophore in order to achieve optimum results. Ironically, including spectra in the calculations that do not match any of the fluorophores in the lambda stack will not affect linear unmixing results (a zero contribution will be assigned to the missing fluorophore). Another critical requirement for linear unmixing is that all fluorophores present in the specimen must be considered in the calculations or the results may be skewed towards the dominant (most concentrated) fluorophore at the expense of less concentrated species. Failure to meet this specification can result in multiple solutions to the spectral separation calculation and a unique result may not be possible. One of the most important considerations is to ensure that the number of spectral detection channels is at least equal to the number of fluorophores present in the specimen. In order to ensure the best chances to obtain successful results when applying linear unmixing algorithms, several experimental criteria must be met. For a particular number of fluorophores ( n), this relationship can be represented as: Thus, each reference spectrum of a pure fluorophore is described as R i(λ) where i = 1,2,3.n represents the index of the fluorophore ( C i). Each pixel in the spectral image is categorized as representing a mixture of fluorophore signals (intensities) when the measured spectrum ( I(λ)) can be deconvolved into the proportion, weight, or concentration ( C) of each individual fluorophore reference spectrum ( R(λ)) when the values are summed. The fundamental concept underlying linear unmixing calculations is relatively simple. Click on the red Auto button adjacent to a selected fluorophore to relocate the cursor to a region of the specimen containing 100 percent of that fluorophore. The white curve is the added spectra, whereas the red curve represents EGFP, the green curve represents Alexa Fluor 488, and the blue curve represents Alexa Fluor 514. As the cursor is translated across the image, the spectral profiles of the various probes are altered to reflect their concentrations at that particular region (denoted by the progress bars in the lower portion of the tutorial window. In order to operate the tutorial, click in the specimen image window and drag the bulls-eye cursor to a point of interest. ![]() The tutorial initializes with a complex spectral data set appearing in the window graph and an image of the triple-stained specimen placed in the upper right-hand corner. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |