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Handbook of Optical Filters for Fluorescence Microscopy

How to choose the optical filter for your epifluorescence or confocal application

Fluorescence microscopy and other light-based applications require optical filters that have demanding spectral and physical characteristics. Often, these characteristics are application-specific and an optic that might be appropriate and optimal for one is both inappropriate and sub-optimal for another. The Handbook of Optical Filters for Fluorescence Microscopy is a compilation of the principles that the engineers and scientists at Chroma Technology Corp. use to design filters for a variety of fluorescence applications. Although the principles outlined here are geared towards microscopy in general, they are equally valid and important in other, non-microscopy applications.

A basic overview of general fluorescence principles is given, as well as a brief overview of both the typical configuration of an epifluorescence microscope and its history. The three types of optical filters used in a typical epifluorescence microscope (excitation/illumination filter, emission/barrier filter and dichroic mirror) are also detailed.


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Table of Content

Optical filters and their spectral and physical characteristics

The Handbook continues with a thorough description of Chroma terminology and concepts used in the description of an optic and its spectral and physical characteristics. The difference between longpass/shortpass and bandpass filters is explained. The blocking/attenuation properties of filters are described and defined in optical density (OD). A brief overview is given regarding the importance of angle-of-incidence (AOI) and proper functioning of a given optic, followed by a consideration of cone-angle on these properties. The final portion of this section is a description of polarization effects on optics.

Traditional filter materials are illustrated, as well as more recent innovations to filter design and manufacture. Simple filters using colored glass, more advanced thin-film filter design and acousto-optical and liquid crystal tunable filters are all discussed, as are their relative advantages and disadvantages.

Fluorochromes, excitation and illumination sources

The next section contains a consideration of the factors that contribute to achieving excellent imaging results in a microscopy application. Choice of fluorochrome (either to be used alone or in concert with other fluorochromes), choice of excitation/illumination source and considerations of the quality of light reaching the detector are all discussed. Surface qualities (flatness, transmitted and reflected wavefront distortion and wedge, etc.) are reviewed and their effects on optimal beampath generation are described. Typical surface specifications are listed for Chroma-manufactured optics.

Image registration

Image-registration and visual overlap of multiple fluorescences within the microscope are considered. The ability to achieve sub-pixel registration of multiple images within the instrument is considered with a description of the effects of the various filters in this process. More information on this topic can be found here:

Filters for confocal microscopes

A brief description of the filters used in confocal microscopy follows. For more information and a detailed explanation of optics and filters designed specifically for laser-based applications, please refer to our Laser Application Note found here:

Also included is a glossary of terms that are used by Chroma to describe the optical properties of its filters, as well as numerous illustrations of the principles discussed.


Download the complete “Handbook of Optical Filters for Fluorescence Microscopy”:

Leica FluoScout™

The online tool Leica FluoScout™ helps to determine the optimum filter cube and filter cube set to achieve excellent imaging results. Based on the choice of light source and fluorophores, the user gets a recommendation of filter cubes.