Developing expertise in microscopy, like for any technical field, requires mastering fundamental concepts and skills while also developing fluency in the specific jargon. Although jargon can be convenient for facilitating communication, it often creates barriers for those new to the field. There are numerous resources available for microscopy education, yet it can be hard to know where to start. As part of an effort to make optical microscopy more accessible, we present a brief guide to fluorescent filters and highlight the specialized terms used to describe them.
Fluorescent filters are optical components in the microscope that help separate the light in the microscope by colour (based on wavelength). It is important to familiarize yourself with optical filters because you need to match the filters to the probes when designing and carrying out experiments. You also need to know how to choose the appropriate filters when using multiple fluorophores (also known as fluorescent labels or probes) within a sample. Using fluorophores that are not optimal for your microscope filters can yield a weak signal or signal that is below the level of detection of your system. Alternatively, incorrect filter and fluorophore choice can create false positives during multi-colour imaging, threatening the integrity of data sets. Although we focus on optical filters here, the specifications of other components within your microscope, such as light sources, objectives, and detectors must be considered along with the filters. We will share some practical tips on how their specifications can affect your experiments in future posts.
Filters range from individual glass components to more complex devices. The most common filters used in fluorescence microscopes are individual glass components that work by passively transmitting specific wavelengths while reflecting others, much like placing a colour filter on a camera lens. Figure 1 illustrates a graph or “trace” of filter % transmission as a function of wavelength. As shown, the filter will transmit light from about 465 and 495 nm, while reflecting light outside this range (where the transmission is close to 0). This type of filter is known as a “bandpass” filter because it “passes” or transmits a specific range of wavelengths while rejecting others.
Filter traces are courtesy of Chroma Technology
The bandpass filter shown in Figure 1 is referred to by specific optical specifications, including “FWHM” and the “centre wavelength.” The full width half maximum (FWHM) is defined as the spectral range of the curve when measured at 50% peak transmission (blue dashed line). The centre wavelength is the midpoint of the transmission band, typically also the mid-point of the FWHM (filter trace shown has approximately a 480 nm centre wavelength and is indicated by the green solid line). The FWHM is often listed after the centre wavelength with the number following a slash, as in 480/30, where 480 is the centre wavelength and 30 nm is the FWHM. Other types of filters seen in fluorescence microscopy include long-pass and short-pass filters. Short pass filters transmit shorter wavelengths while reflecting longer ones and long-pass filters transmit longer wavelengths while reflecting shorter ones. We illustrate a long-pass filter in Figure 2. Here rather than referring to the centre wavelength, we refer to the “cut-on wavelength,” the wavelength that corresponds to 50% peak transmission (about 505 nm and indicated by the green solid line). Short-pass filters do the reverse, and we refer to the “cut-off wavelength.”
Filter traces are courtesy of Chroma Technology
An important note of caution; filter traces (provided by manufacturers) are useful guides to how a filter should perform. However, the performance of filters also depends on their optical position and alignment in the system as well as their integrity, as they can show wear over time. For example, if a filter is designed to work at 45 or 90 degrees with respect to the light propagating in the system, the transmission curve can shift if the filter is not precisely placed or slips out of place over time. Also, you may need to replace filters over time. Wear of filters can occur when they are used to select high-intensity ultraviolet wavelengths (such as DAPI) in combination with older light sources such as mercury lamps.
When you design a fluorescence experiment, as a starting point it is important to choose fluorophores that are compatible with your filter sets. Filter information should be available in the acquisition software. If it is not, check the system installation records to track down the manufacturer and/or product codes for the individual filters in your system. If you have lost track of this information, check with the vendor who provided the system. If you want to examine your filters directly, take care because they can be easily damaged if improperly handled. Before inspecting your filters, it is advisable to research appropriate protocols or ask someone experienced for help.
Depending on your microscope, you will use different combinations of filters to excite and detect fluorescent probes from the visible to near-infrared spectrum. Each combination of filters is known as a “filter set” and is designed to work with specific fluorophores. As we will describe in the next post, these combinations help us separate the excitation from the emission and also help us distinguish among different fluorophores. We often categorize the sets by the name of the fluorophore they are designed to work with, as in the “EGFP” filter set. A standard filter set includes filters for the excitation light and a filter for the emission light (often bandpass filters), along with a dichroic filter (often a long-pass filter for standard fluorescence).
In the next post, we delve more into filters, emphasizing how to check for compatibility of fluorophores and filters. We will also provide tips for best practices for carrying out multi-colour fluorescence imaging experiments.