ISO 19056-3:2022 pdf download – Microscopes — Definition and measurement of illumination properties — Part 3: Incident light fluorescence microscopy with incoherent light sources.
5.5 Uniformity The measurement for uniformity shall be performed by using an electronic image sensor that can capture the specified image field. This electronic image sensor shall consist of at least 50 rows and columns of individual elements (pixels). Furthermore, the image sensor shall be designed for an entrance pupil at infinity. If the mechanical design of the microscope does not allow the image sensor to be placed in the plane of the image field, no auxiliary optics that would be part of the measurement setup shall be used. In this case an uniformity value cannot be measured according to this document. This image sensor does not need to be calibrated in radiometric units as its output can be related to the brightness measurement described in 4.2 . However, gamma correction shall not be applied to the signal from the image sensor in order to retain a linear relationship between irradiance and sensor signal (hence γ = 1). The specimen shall be a homogeneous fluorescent specimen, e.g. a solution of a fluorescent marker or a broadband fluorescent polymer slide. Solutions of markers are generally preferable over solid samples, since bleached fluorophores in the object field are replaced by unbleached fluorophores due to diffusion. While performing the measurement, it shall be ensured that the complete data recorded originates from the homogeneous fluorescent region of the specimen to avoid misjudgement, which might be caused, e.g. by field of curvature of the optics or a tilted specimen. The signal-to-noise ratio shall be sufficient to clearly determine the uniformity. The signal-to-noise ratio is considered sufficient if the uniformity varies less than 5 percentage points when the illumination brightness is increased by a factor of at least 1,5. Table 1 shows examples for measurements, where signal-to-noise ratio is sufficient for determination of uniformity.
5.6 Spectral information The illumination brightness and uniformity shall be referred to a specific spectral range. The spectral range shall be given as the wavelength of maximum brightness together with the two wavelengths at which the brightness is 50 % of the maximum value. If the brightness value of 50 % is achieved for more than two wavelengths, all spectral ranges shall be indicated. This spectral information can be measured in the focus plane of the objective or it can be calculated from theoretical values of the individual components (light source, filters, transmission of optical components). Examples are given in Annex A . 6 Information provided to the user If information on illumination properties is provided to the user, information on illumination brightness and uniformity are mandatory; information on temporal stability is optional.

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