Fluorescence requires a source of excitation energy. There are several main types of light sources that are used to excite fluorescent dyes. This section introduces the types of commonly used excitation sources and presents some of the ways that filters can be used to optimize your experimental result.
There are three families of light sources used for fluorescence. 1) The most popular sources used for exciting fluorescent dyes are broadband sources such as the mercury-arc and tungsten-halogen lamps. These produce white light that has peaks of varying intensity across the spectrum. 2) In contrast, laser excitation sources offer one or a few well-defined peaks, allowing more selective illumination of your sample. 3) More recently, high-output light-emitting diodes, or LEDs, have gained popularity due to their selective wavelengths, low cost and energy consumption, and long lifetime.
Examples of light sources. A) Mercury-arc lamp light profile and photograph. B) Argon laser light profile and photograph. C) Green light emitting diode (LED) light profile and photograph.
The high intensities and selective wavelengths of lasers make them convenient excitation sources for many dyes. The best performance is achieved when the dye’s peak excitation wavelength is close to the wavelength of the laser. Compact violet 405 nm lasers are replacing expensive UV lasers for most biological work. The most commonly used lasers are the 488 nm blue-green argon laser, 543 nm helium-neon green laser and 633 nm helium-neon red laser. Mixed-gas lasers such as the krypton-argon laser can output multiple laser lines and therefore may still require optical filters to achieve selective excitation.
While a given dye’s excitation maximum may not exactly match the laser’s peak wavelength, the high power of the laser can still produce significant fluorescence from the dye when exciting at a suboptimal wavelength.
Laser excitation. A) Light profiles of common lasers. B) Demonstration of efficiency of fluorophore excitation and emission when using suboptimal laser excitation light.
Map of common lasers used with Invitrogen Alexa Fluor dyes.
LEDs are relatively new light sources for fluorescence excitation. Single-color LEDs are ideal for low-cost instrumentation, where they can be combined with simple longpass filters (see below) that block the LED excitation and allow the transmission of the dye signal. However, the range of wavelengths emitted from each LED is still relatively broad. Currently their application may also require the use of a filter to narrow the bandwidth.
Light profiles of common LEDs.
When using broadband white light sources it is necessary to filter the desired wavelengths needed for excitation; this is most often done using optical filters. Optical filters can range from simple colored glass to highly engineered interference filters that selectively allow light of certain wavelengths to pass while blocking out undesirable wavelengths. For selective excitation, a filter that transmits a narrow range of wavelengths is typically used. Such a filter is called a bandpass excitation filter.
Types of excitation filters. A) Basic excitation filter that blocks all light up to a specific wavelength and allows all light above that wavelength to transmit. B) Example of a bandpass filter that allows only a small range of light to be transmitted while blocking all light on either side of that range.
Filters are important for selecting excitation wavelengths. They are also important for isolating the fluorescence emission emanating from the dye of interest. Detecting the fluorescence emission of a sample is complicated by the presence of stray light arising from sources other than the emitting fluorophore—for example, from the excitation source. This stray light must be kept from reaching the light-sensitive detector in order to ensure that what the instrument "sees" is due only to the fluorescence of the sample itself.
Longpass filter—When a single dye is used, a filter that blocks out the excitation light to reduce background noise but transmits everything else is often a good choice to maximize the signal collected. Such a filter is called a longpass emission filter.
Use of simple longpass emission filter for single dye detection. A) Image of single dye-stained cell detected using no emission filter. B) Same stained cell in (A) captured using simple emission filter with all light below 520 nm being blocked and all light above 520 nm being transmitted to the detector.
Bandpass filter—If multiple dyes are used in the sample, a bandpass emission filter can be used to isolate the emission from each dye. Careful filter selection helps to ensure that the detector registers only the light you are interested in—the fluorescence emitted from the sample.
Use of bandpass emission filter for multiple dye detection.
There are many options for light sources for fluorescence. Selecting the appropriate light source, and filters for both excitation and emission, can increase the sensitivity of signal detection to an astounding degree, making fluorescence labeling one of the most sensitive detection technologies available.
The complete tutorial is also available on this video.
Plot and compare spectra and check the spectral compatibility of multiple fluorophores.
For Research Use Only. Not for use in diagnostic procedures.