Modern fluorescence microscope instrumentation employs a combination of interference filters and a dichromatic beam splitter to satisfy the excitation and emission requirements of the fluorescent probe(s) used to label the specimen. When these components are chosen appropriately, the microscope provides an essential mechanism for selective excitation of specimen fluorophores, and the subsequent isolation of much weaker fluorescence emission necessary for image formation. By carefully matching excitation and emission filter properties with the function of the dichromatic beamsplitter, labeled specimen features are imaged on a dark background with maximum sensitivity. This interactive tutorial enables visitors to determine optimum choices among current Nikon fluorescence filter blocks for maximizing the efficiency of excitation and emission with specific fluorescent probes.
To operate the tutorial, first choose a filter combination using the Nikon Filter Set pull-down menu on the right-hand side of the tutorial window. Next, choose a family of fluorescent dyes using the Probe Class pull-down menu. For each class of probes, a selection of fluorophores is provided for access through the Fluorophore pull-down menu (Hint: for faster operation, after selecting the desired probe, toggle rapidly through the filter set selections using the up and down arrow keys).
The tutorial initializes with the spectral profiles of an blue-absorbing fluorescent probe (Alexa Fluor 488) superimposed on the spectra characterizing a suitable Nikon filter set in the graph entitled Spectral Profiles. The normalized intensities of the selected fluorophore excitation and emission spectra are indicated on the right-hand graph ordinate (Relative Intensity), while the left-hand ordinate designates spectral transmission values for components of the selected filter combination (Transmittance). The Spectral Cross-Sections are denoted by blue and light red shading of excitation and emission overlap regions, respectively. The excitation cross-section (blue) corresponds to overlap between the fluorophore absorption and excitation filter transmission regions, while the cross-section for emission (red) represents the overlap of the fluorophore emission and emission filter transmission regions. Each of the cross-sectional overlap colors can be toggled on and off individually using the Excitation and Emission check boxes. The color shading is similarly removed when either of the Fluorophore Spectra check boxes is turned off, removing the fluorophore Absorption and/or Emission spectra. Spectra for individual filter set components can also be deleted by removing the appropriate checkmark from the Filter Spectra check boxes. Transmission curves for the Exciter, Dichroic, and Emitter are color coded blue, green, and red, respectively, and are toggled on and off with the corresponding check boxes. Note that important filter set spectral parameters and the fluorescent probe peak wavelengths for excitation and emission are indicated next to the filter and probe check boxes.
Adam Rainey and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.