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Molecular Probes The Handbook

Optical Filters for Fluorescence Microscopy - Section 23.5

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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 section of the Handbook provides basic information on optical filters and tabulated spectral data that are useful in selecting the optimal filters for many of our dyes (Fluorescence excitation sources - Table 23.10, Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11). In addition, Molecular Probes now carries the Semrock BrightLine series of high-performance optical filters for fluorescence microscopy.

The Optical Filter Set

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 23.10). 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).

The Trade-Off in Optical Filter Set Design

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. The absorption and emission maxima for a wide variety of our dyes and some appropriate optical filters are given in Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11. 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).

Selecting an Optical Filter Set

Filter set selection may involve a straightforward recommendation or a complex analysis of the spectral relationships of dyes and optical filters.ref 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, photo) and the monomer and excimer forms of BODIPY FL C5-ceramide (D3521, B22650; Probes for the Endoplasmic Reticulum and Golgi Apparatus - Section 12.4; photo).

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 (Figure 23.53). 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, a linear photometric charge-coupled device (CCD), diode array or intensified video camera 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 Optical transmission characteristics of a triple-band filter set (XF63, 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), black (dichroic beamsplitter) and red (emission filter). Graphic supplied by and used with permission of Omega Optical Inc., Brattleboro, VT.


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, Endocytosis and Exocytosis - 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 filter set recommendations for some of our most widely used dyes and probes for fluorescence microscopy applications (Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11). This table includes fluorescence excitation and emission maxima for the most common environment in which the dye would be found in typical experimental specimens, as well as links to plots of the full spectra.

Semrock BrightLine Optical Filters

Semrock BrightLine optical filters sets are durable, high-performance excitation and emission filters for fluorescence microscopy. BrightLine optical filters are made using Semrock's patented dense hard-coating technology that, unlike the coating of traditional filters, has no adhesives in the optical path. This unique design, combined with Semrock's advanced ion-beam sputtering deposition systems, delivers excellent optical performance, including:

  • Sharper, brighter fluorescence imaging
  • Greater durability — BrightLine filters can be cleaned and handled like any standard glass optics
  • No noticeable degradation when used with xenon and halogen broadband light sources for prolonged periods
  • Highest possible transmission for less background and a better signal-to-noise ratio
  • "Zero-pixel shift" performance for perfect multicolor images

A wide selection of unmounted and mounted BrightLine optical filter sets are available directly from Molecular Probes (Semrock BrightLine filter sets available through Molecular Probes - Table 23.12).

Technical Support

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 Semrock, Inc., Omega Optical, Inc., Chroma Technology Corp. or the microscope manufacturer for this information. Semrock, Omega Optical and Chroma Technology provide complete transmittance curves and information on their specialty filters for ratio imaging, uncaging, multiphoton and other applications at their respective web sites (http://www.semrock.com/, http://www.omegafilters.com/, http://www.chroma.com/).