Filters can make or break fluorescence microscopy experiments. They are a critical component of every fluorescence microscope setup and even have the potential to make breakthroughs in the high-speed, high-contrast imaging provided by the latest LED microscope illumination. However, the selection and configuration of the best filters can be challenging. In this section, we'll walk you through the answers to your questions and help you choose the best filter for your application.
You'll learn: How to choose the best filter for your experiment
Which Pinkel and Sedat configurations enable high-speed imaging
Five important tips about optical filters when using LED lighting.
When designing fluorescence microscopy experiments, scientists often need to consider the choice of light source, filter, and fluorophore in order to obtain high-quality data. Many of our technical resources focus on the features and benefits of different light sources, and this *** will also provide guidance on "Selecting Optical Filters for Widefield Fluorescence Microscopy".
Learn about optical filters.
A standard fluorescence microscope setup typically consists of three components housed in a fluorescence cube—an excitation filter, a dichroic mirror beam splitter (also known as a dichroic filter), and an emission filter, as shown in Figure 1 below
Excitation filter – the wavelength of the light from the light source is selected to match the absorption spectrum of the fluorophore.
Dichroism – selects and transmits the fluorescence emitted by the sample and transmits the longer wavelengths emitted by the sample.
Emission Light Filters – Select only the wavelengths that correspond to the fluorophore emission curve while increasing contrast by blocking autofluorescence, stray light from the room, or reflected LED light.
Filters and light sources for GFP imaging. Single-wavelength imaging of GFP using a 470 nm LED of CoolLED PE-100 (blue panel) excites GFP along its excitation spectrum (light green). The excitation light path passes through the excitation filter (violet) and wavelengths below 495 nm are reflected onto the sample by a dichroic mirror (red). This energy is absorbed and released in the form of fluorescence, resulting in a GFP emission spectrum (dark green) and a dichroism mirror (red) transmitting light above 495 nm to the detector. Background noise is further removed by the emission light filter (yellow) in the emission light path.
There are thousands of filters and dichroscopes to choose from, and this choice depends on the individual fluorophore and light source. The suitability of a filter depends primarily on the wavelength it blocks or transmits, as shown in Figure 2. It shows the spectral pattern of GFP, excited by an LED at 470 nm and an optimized single-band filter set.
First, we can see that the blue LED spectrum overlaps with the absorption spectrum of the light green GFP, so the light source is compatible with the fluorophore. The red dichroism reflects light with a wavelength of less than 495 nm and directs the excitation path to the sample. In the emission path, it transmits wavelengths above 495 nm to the detector. This is ideal for GFP because there is a gap between peak absorption and emission**, i.e., the relevant excitation wavelength of GFP is reflected onto the sample and the associated emission wavelength reaches the detector.
The violet excitation filter is the relevant wavelength of the LED specifically designed to transmit the GFP absorption spectrum. Note the LED "tail" that extends up to 525 nm: Since dichroic mirrors do not provide perfect beam splitting, certain wavelengths beyond 495 nm are reflected onto the sample or reflected into the emission path and form a background. However, it is first blocked by the excitation filter, and then the energy absorbed by GFP is released in the form of dark green fluorescence emission. In this case, the yellow emission light filter further blocks all light from outside the GFP emission peak range.
Multiplex Fluorescence: How to Configure Filters Effectively?
Imaging a single fluorophore is relatively straightforward and requires only a fluorescence block to be mounted in the microscope. However, capturing multiple fluorophores in a single experiment requires additional considerations for balancing speed, contrast, and budget.
Single-band filters—Multiple single-band fluorescence excitation blocks, as shown in Figure 1, can be mounted in a motorised microscope filter cube turret to achieve high-contrast imaging, as each excitation block contains a filter set specific to each fluorophore. However, switching between excitation blocks is slow, limiting the speed of dynamic observation of multi-labeled samples to the second level.