Sensitive and versatile fluorescence detection techniques are of ever-increasing importance and popularity in biological research microscopy. In the now-standard epi-illuminated microscope configuration, the optical filter set performs a critical function in separating the fluorescence emission photons that will form the final image from the more-intense excitation light field. For practical purposes, it is necessary to reduce the excitation light intensity in the detection path by a factor of 106–107. This design objective has to be achieved in parallel with capturing as many of the available fluorescence photons as possible. High capture efficiency allows compensating reductions in overall excitation light levels, with accompanying reductions in dye photobleaching and cellular phototoxicity.
This technical note provides some basic fluorescence microscopy information, including discussions of:
The Fluorescence SpectraViewer (www.invitrogen.com/handbook/spectraviewer) provides an online tool that allows researchers to assess the spectral compatibility of dyes, probes and optical filters in the course of designing experiments (Using the Fluorescence SpectraViewer—Note 23.1).
A set of optical filters for selective excitation and detection of fluorescence typically consists of a minimum of three components: an excitation filter, a dichroic beamsplitter ("dichroic mirror") and an emission filter ("barrier filter") (Figure 23.52). The excitation filter selectively transmits a portion of the spectral output from the light source (Fluorescence excitation sources—Table 23.2). The dichroic beamsplitter then reflects the selected light, directing it to the sample. Fluorescence emission photons traveling from the sample towards the detector are transmitted by the dichroic beamsplitter, while excitation light reflected back from the sample is diverted out of the detection light path. The emission filter blocks unwanted spectral components of the emitted fluorescence (e.g., sample autofluorescence) as well as any residual excitation light. An interactive Java tutorial demonstrating these functions is available online at the Molecular Expressions web site of Florida State University (http://micro.magnet.fsu.edu/primer/java/fluorescence/filtersetprofiles/index.html).
Figure 23.52 Functions of fluorescence microscope filter set components. The desired excitation wavelength (λ2) is selected from the spectral output of the lamp by the excitation filter (EX) and directed to the sample via the dichroic beamsplitter (DB). The beamsplitter separates emitted fluorescence (- - -) from scattered excitation light (—). The emission filter (EM) selectively transmits a portion of the sample's fluorescence emission (λ4) for detection and blocks other emission components (λ5).
For optimal fluorescence detection when using a single dye, the excitation and emission filters should be centered on the dye's absorption and emission peaks. To maximize the signal, one can choose excitation and emission filters with wide bandwidths. However, this strategy may result in unacceptable overlap of the emission signal with the excitation signal, resulting in poor resolution. To minimize spectral overlap, one can instead choose excitation and emission filters that are narrow in bandwidth and are spectrally well separated to increase signal isolation. This approach will reduce optical noise but may also reduce the signal strength to unacceptable levels. When overlapping signals from multiple fluorophores in the same sample are being differentiated, both the spectra of the dyes and their expected intensities must be considered before choosing an optical filter. Complete spectral data for Molecular Probes fluorophores can be found using our interactive Fluorescence SpectraViewer utility (Using the Fluorescence SpectraViewer—Note 23.1). An interactive Java tutorial illustrating the trade-off among these parameters is available online at the Molecular Expressions web site of Florida State University (http://micro.magnet.fsu.edu/primer/java/fluorescence/fluorocubes/index.html).
Filter set selection may involve a straightforward recommendation or a complex analysis of the spectral relationships of dyes and optical filters. Emission filters are available with either longpass or bandpass wavelength transmission profiles. A typical longpass emission filter might transmit all wavelengths ≥530 nm, whereas a typical bandpass filter might transmit only wavelengths between 515 and 545 nm. Longpass filters should be used when the application requires maximum emission collection and when spectral discrimination is not desirable or necessary, which is generally the case for probes that generate a single emitting species in specimens with relatively low levels of background autofluorescence. Longpass filters are also useful for simultaneous detection of spectrally distinct dual emissions such as the monomer and J-aggregate forms of JC-1 (T3168, Probes for Mitochondria—Section 12.2, ) and the monomer and excimer forms of BODIPY FL C5-ceramide (D3521, B22650; Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4; ).
Bandpass filters are designed to maximize the signal-to-noise ratio for applications where discrimination of signal components is more important than overall image brightness. The spectral sensitivity of the detection system should also be considered in order to achieve optimum detector signal-to-noise or accurate color rendition. Some applications, such as confocal laser-scanning microscopy, may require the use of sensitive photomultiplier (PMT) detectors. Alternatively, an electron-multiplying charge-coupled device (EMCCD) may be employed for quantitative imaging or microspectrofluorometry. Dual-, triple- and quadruple-band filter sets enable microscopists to excite and detect two, three or four fluorophores simultaneously instead of performing sequential image acquisitions with intervening filter changes (Figure 23.53).
Selecting optimal filter sets for fluorescence microscopy applications requires matching optical filter specifications to the spectral characteristics of dyes. Comparisons should be made with care because some dyes have significantly different spectral properties in a particular application than those reported for the dye in solution. For example, the spectral characteristics of many nucleic acid stains depend on whether the dyes are in aqueous solution or bound to DNA or RNA. Similarly, styryl dyes such as FM 1-43 (T3163, T35356; Tracers for Membrane Labeling—Section 14.4, Probes for Following Receptor Binding and Phagocytosis—Section 16.1) and di-4-ANEPPS (D1199, Fast-Response Probes—Section 22.2) have emission maxima that depend on whether they are dissolved in solvent or associated with membranes. To provide selection guidelines, we have compiled excitation and emission spectra for many of the most widely used probes in fluorescence microscopy in an online tool, the Fluorescence SpectraViewer. Spectral characteristics of Molecular Probes dyes—Table 23.1 provides fluorescence excitation and emission maxima for the most common environment in which Molecular Probes dyes would be found in typical experimental specimens.
Figure 23.53 Optical transmission characteristics of a triple-band filter set (XF467, Omega Optical Inc.) designed for simultaneous imaging of DAPI, fluorescein, and Texas Red dyes. Transmission curves for the individual filter set components are shown in blue (excitation filter), green (dichroic beamsplitter) and red (emission filter). Graphic supplied by and used with permission of Omega Optical Inc., Brattleboro, VT.
We invite customers to call our Technical Assistance Department for help in selecting the correct optical filter for a specific application. When calling, please be prepared to describe the dye(s), instrumentation and method of detection being used. A technical support scientist will then offer advice on the most effective filter configuration for the specified purposes. Alternatively, we recommend contacting Chroma Technology Corp., Omega Optical, Inc. or Semrock, Inc. or the microscope manufacturer for this information. Chroma Technology, Omega Optical and Semrock provide complete transmission curves and information on their specialty filters for ratio imaging, uncaging, multiphoton and other applications at their respective web sites (www.semrock.com, www.omegafilters.com, www.chroma.com).
For Research Use Only. Not for use in diagnostic procedures.