Fluorescence Is Having a Moment
From AI-powered imaging to multiplexed microscopy, researchers are pushing fluorescence further than ever, capturing more signals, more precisely, and all at once.
What is one trend lighting up labs? Multiplexing. The ability to detect multiple fluorophores in a single experiment is transforming how we study complex biological systems, and it all hinges on the right optical filters.
We think of optical filters as the “conductors” of fluorescence — selecting, separating, and balancing light so every signal is heard clearly.
As fluorescence techniques evolve, so does the need for filters with higher precision, better transmission, and smarter design.
Because when the signal matters, clarity is everything.
Overview of Typical Fluorescence Filter Sets
The primary filtering element in an epifluorescence microscope is the set of three filters housed in a fluorescence filter cube (or filter block): the excitation filter, the emission filter, and the dichroic beamsplitter.
The excitation filter (also called the exciter) transmits only those wavelengths of the illumination light that efficiently excite a specific dye. Although shortpass filter designs were used in the past, bandpass filter designs are now used almost exclusively. The emission filter (aka barrier filter or emitter) attenuates all of the light transmitted by the excitation filter and very efficiently transmits any fluorescence emitted by the specimen. This light is always of longer wavelength (more to the red) than the excitation color. The emitter filter can be either a bandpass or longpass filter. The dichroic beamsplitter (aka dichroic mirror or dichromatic beamsplitter) is a thin piece of coated glass set at a 45-degree angle to the optical path of the microscope. This coating has the unique ability to reflect one color, the excitation light, but transmit another color, the emitted fluorescence. Current dichroic beamsplitters achieve this with great efficiency, that is with greater than 90% reflectivity of the excitation along with approximately 90% transmission of the emission. This is a great improvement over the traditional gray, half-silvered mirror, which reflects only 50% and transmits only 50%, giving only about 25% efficiency.
The spectrum characteristics of a particular fluorochrome can be overlayed with the spectral profile of a fluorescence filter set to aid in selecting the most appropriate filter set for any given application. The figure above illustrates what the spectral profile of an “ideal” filter set might look like for the given flourochrome excitation and emission spectrum. As you can see, the excitation filter transmits light spanning the absorption spectrum, and the emission filter transmits a band of wavelengths spanning the peak emission of the fluorochrome. The dichroic attenuates the excitation light and transmits only the light from the emission.
Once an individual exciter, emitter, and dichroic is chosen for the application, they are mounted in a filter cube and are ready for use in a microscope. Most microscopes have a slider or turret that can hold from two to four individual filter cubes. It must be noted that the filters in each cube are a matched set, and one should avoid mixing filters and beamsplitters unless the complete spectral characteristics of each filter component are known.
Common filter blocks are named after the type of excitation filter: UV or U (Ultraviolet excitation for dyes such as DAPI and Hoechst 33342), B (Blue excitation for FITC and related dyes), and G (Green excitation for TRITC, Texas Red®, etc.). Common barrier filter colors are blue or pale yellow in the U-block, green or deep yellow in the B-block, and orange or red in the G-block.
Check out the Fluorescence Filter Types available from Chroma Technology.
About Chroma Technology
Chroma Technology is a leading manufacturer and OEM supplier of high performance optical filters used in a wide range of applications, such as communications, inspection and metrology, astronomy, life sciences and health care, microscopy, calorimetry, machine vision, and Raman spectroscopy. Chroma offers an extensive selection of optical filter sets and filter accessories, as well as custom solutions tailored to customer-specific needs
For more information or a quotation for this or other Chroma products, contact IL Photonics.