Understanding Filters and Dichroic Mirror Configurations in Fluorescence Microscopes

Fluorescence microscopy relies on precise optical control of excitation and emission light. Excitation filters, dichroic mirrors and emission filters work together to ensure that fluorophores are efficiently excited and that only the desired fluorescence signals reach the detector.

This article provides an overview of filter and dichroic mirror configurations commonly available in benchtop fluorescence microscopes, highlighting the advantages and limitations of different approaches.

Fig.1 Schematic illustration of the light path in fluorescence microscopy. The excitation filter, dichroic mirror and emission filter work together to direct selected excitation wavelengths to the sample and selectively transmit fluorescence emission wavelengths to the detector.

Which Filter and Dichroic Configuration Should I Consider when Choosing a Fluorescence Microscope?

Most benchtop fluorescence microscopes are delivered with preconfigured illumination, dichroic mirrors, and filter sets optimised for common fluorophores. However, it is essential to verify that the provided configuration is compatible with your specific fluorophores and experimental requirements.

Excitation Filters

Excitation filters are optical components used in fluorescence microscopes to select the specific band of wavelengths required to excite fluorophores in a sample. Their role is to ensure that only the desired excitation light reaches the specimen, while unwanted wavelengths are blocked.

Excitation filters are essential for shaping the relatively broad spectra of light produced in LED-illuminated widefield fluorescence microscopes. In contrast, laser-based illumination as used in confocal systems, does not require excitation filters, since lasers provide single-wavelength excitation by default.

Dichroic Mirrors

Dichroic mirrors are optical components placed in the detector beam path that spectrally separate excitation light from fluorescence emission.

Used in combination with emission filters, dichroic mirrors ensure that only the desired fluorescence wavelengths reach the detector while reflected excitation light is efficiently rejected. The choice of dichroic configuration has a substantial impact on which fluorophores can be imaged. Choosing the right dichroic configuration is therefore essential for accurate and efficient single colour or multi-colour fluorescence imaging.

Fig.2 - Multi-colour fluorescence image of a pancreatic organoid. Organoid is mounted in hydrogel and imaged with BC43. Cyan: DNA; yellow: laminin A/C; magenta: tubulin. Image credits: Dr. Sebastian Amos and Dr. Yu-Suk Choi, University of Western Australia.

Emission Filters

Emission filters determine which wavelengths of light ultimately reach the detector and therefore play a central role in defining fluorescence channels and ensuring signal specificity. In practice, these filters transmit the fluorescence emitted by the fluorophore of interest while blocking residual excitation light and unwanted emission wavelengths.

The choice of emission filters directly affects image quality and the ability to separate signals in single- and multi-colour imaging experiments.

Fig.3 - BC43 multicolour imaging of a mouse embryonic stem cell. Derived dopaminergic neurons cultured on top of a collagen hydrogel, expressing tyrosine hydroxylase (in magenta),GAP 43 (in yellow), and DNA (in cyan). Image credit: Ana Marote from ICVS, University of Minho and Leonor Ribeiro from INL

Exploring Benchtop Fluorescence Microscopes?

Learn more about the key technologies, components and practical considerations involved in selecting a benchtop fluorescence microscope.

BC43 – Exceptional Performance, Certified Quality, High Productivity

BC43 widefield and confocal imaging systems both use laser illumination and therefore do not require interchangeable excitation filter sets for wavelength selection.

BC43 incorporates a quad-band dichroic mirror that allows clear band-pass separation of all four integrated laser lines. This configuration supports rapid multi-colour imaging without the need for mechanical dichroic switching.

BC43 includes single-band emission filters matching the emission wavelengths of fluorophores excited by the four standard wavelengths used in microscopy (UV, green, red, and far red). Optionally, the system can be equipped with a quad-band emission filter designed to accelerate live-cell imaging and large multidimensional acquisitions.

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