Imaging in the far‐red with electronic light microscopy: Requirements and limitations

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Abstract
The acquisition of simultaneous dual confocal images with red and far‐red light has both advantages (e.g. lower autofluorescence) and limitations. An understanding of these requisites is necessary to acquire high‐quality images and to avoid the misinterpretation of experimental data. The poor detection of far‐red light mandates a high optical transfer efficiency for the system, thus the transmittance of the objective lens and its axial and lateral chromatic aberration in the far‐red are important factors for consideration. This technical note is an attempt to ‘demystify’ the process of filter set design for confocal microscopy by discussing the considerations that went into the construction of a filter set for use with the reagents cyanine 3.18 (Cy3) and cyanine 5.18 (Cy5), and thus to encourage users to look beyond the multi‐purpose designs available commercially. The 568‐nm laser line exciting Cy3 is at its emission maximum, which limits the collectable Cy3 fluorescence. High‐transmission optical filters with sharp band pass cutoffs are thus desirable for maximum light throughput. Light path mirror efficiency rapidly degrades above 700 nm, but the loss of this portion of the Cy5 emission spectrum is acceptable since the fluorophore is very bright, and these very long wavelengths are also likely to introduce aberration. While resolution is decreased with far‐red light, there is also greater penetration and less scattering, and it is thus possible to obtain high‐quality images from deeper within the specimen. Although only one make and model of confocal microscope (the Bio‐Rad MRC–600) is considered, similar considerations pertain to the design of filter sets for any confocal microscope that accommodates user‐installed filters.

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