Fluorescence filter combinations designed to image fluorescent proteins must be carefully chosen to maximize the level of emission intensity presented to the detector while simultaneously reducing the number of unwanted photons from autofluorescence or bleed-through by other fluorophores. The broad absorption and emission spectral profiles exhibited by most fluorescent proteins offer a wide range of choice in filters, which are usually optimized for use with a specific detection system (human eye, digital camera, or photomultiplier). This interactive tutorial is designed to enable the identification of critical filter parameters, including the center wavelength, bandwidth region, and dichromatic mirror cut-on wavelength, which are necessary for imaging fluorescent proteins.
The tutorial initializes with the normalized absorption and fluorescence emission spectral profiles of enhanced green fluorescent protein (EGFP) appearing in the Spectral Profileswindow superimposed over the emission spectrum of a mercury arc-discharge lamp. Also included in the window upon initialization is the wavelength cut-on profile of the dichromatic mirror (beamsplitter) appropriate for the fluorophore, as well as suggested starting points for the Excitation Filter Bandwidth and Emission Filter Bandwidth profiles. The mouse cursor can be used to drag the grayscale bandwidth boundaries along the wavelength axis of the Spectral Profiles window to determine the optimum position. The twin slider combination or arrow buttons on the right-hand side of the control for each filter can be used to adjust the bandwidth, and the center wavelength can be altered using the left-hand arrow buttons. Dragging the left slider bar also translates the bandwidth profile across the graph, while dragging the right slider bar increases or decreases the bandwidth size. The current center wavelength and bandwidth for the virtual excitation and emission filters is continuously updated and displayed in red numerals above the sliders.
The Dichromatic Mirror slider controls the cut-on wavelength curve position for this element, and can be toggled on or off using the check box adjacent to the slider. Excitation and emission filter bandwidth displays can also be disabled by removing the checkmarks. Likewise, the mercury lamp spectral profile can be turned off by deactivating the accompanying check box. Absorption (Ab) and emission (Em) spectral curves can be disabled by removing the checkmark from the boxes above the Illumination Sources yellow display window (default: Mercury Arc Lamp). When a laser source is chosen from the illumination pull-down menu, the excitation efficiency percentage based on the molar extinction coefficient at the laser wavelength is displayed in the green box above the illumination display window. Laser lines, which are drawn with colors approximating the wavelength in the Spectral Profiles window, can be deactivated by disabling the Laser Lines check box. In order to retain the current tutorial parameters (excitation and emission filter profiles displayed, etc.) while toggling through the list of probes or illumination sources, the Maintain State check box can be enabled. New fluorescent protein classes can be selected using the set of radio buttons to the right of the Spectral Profiles window along with the Choose A Probe pull-down menu.
Table 1 - Fluorescent Protein Filter Combination Properties
CWL / BW (nm)
CWL / BW (nm)
(% of EGFP)
|GFP (wt)||Argon (488)||450/50||480LP||510/50||48|
|Green Fluorescent Proteins|
|Azami Green||Argon (488)||470/40||495LP||520/30||121|
|Blue Fluorescent Proteins|
|Cyan Fluorescent Proteins|
|CoralHue Cyan||Argon (488)||450/50||480LP||505/35||73|
|Yellow Fluorescent Proteins|
|Orange and Red Fluorescent Proteins|
|CoralHue Orange||He-Ne (543)||525/40||550LP||580/40||92|
|Optical Highlighter Fluorescent Proteins; (N) = Native (P) = Photoconverted|
|PA-GFP (N)||Diode (405)||400/60||465LP||530/50||8|
|PA-GFP (P)||Argon (488)||480/40||505LP||535/40||41|
|PS-CFP (N)||Diode (405)||395/50||430LP||470/60||16|
|PS-CFP (P)||Argon (488)||470/50||500LP||530/40||15|
|PS-CFP2 (N)||Diode (405)||395/50||430LP||470/60||26|
|PS-CFP2 (P)||Argon (488)||470/50||500LP||530/40||32|
|Kaede (N)||Argon (488)||485/40||510LP||535/30||259|
|Kaede (P)||Kr-Ar (568)||540/50||570LP||590/30||59|
|mEosFP (N)||Argon (488)||490/30||510LP||535/30||128|
|mEosFP (P)||Kr-Ar (568)||540/50||570LP||600/40||68|
|Kindling (N/P)||Kr-Ar (568)||560/50||590LP||625/50||12|
|Dronpa (P)||Argon (488)||485/30||505LP||530/30||240|
Presented in Table 1 is a compilation of properties displayed by several of the most popular and potentially useful fluorescent protein variants. Along with the common name and/or acronym for each fluorescent protein, the optimum laser wavelength line, as well as suggested starting points for the excitation and emission filter bandwidths and center wavelengths are listed. Also included in the table is the relative brightness and recommended dichromatic mirror parameters. The computed brightness values were derived from the product of the molar extinction coefficient and quantum yield, divided by the value for EGFP. This listing was created from scientific and commercial literature resources and is not intended to be comprehensive, but instead represents fluorescent protein derivatives that have received considerable attention in the literature and may prove valuable in research efforts.
The absorption and fluorescence emission spectra used in calculating the values for this tutorial were recorded under controlled conditions and are normalized for comparison and display purposes only. In actual fluorescence microscopy investigations, spectral profiles may vary due to environmental effects, such as pH, ionic concentration, and solvent polarity, as well as fluctuations in localized probe concentration, and therefore, may differ from those actually observed under experimental conditions.
Matthew J. Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.