ISO 19056-3:2022 pdf – Microscopes — Definition and measurement of illumination properties — Part 3: Incident light fluorescence microscopy with incoherent light sources.
1 Scope This document specifies procedures for the measurement of illumination brightness, temporal stability and uniformity for incident light fluorescence microscopy. The measurement for uniformity is defined in image planes or intermediate image planes only, when these planes are suitable for detection by electronic imaging devices. This document defines how illumination brightness, temporal stability and uniformity are measured, and how this information is provided to the user. NOTE The scope is intentionally limited to electronic imaging devices and (intermediate) image planes. The visual observation by means of eyepieces would require a different measurement procedure and hence result in ambiguities in the description of measurement procedures. Nevertheless, this document will give useful estimates for the uniformity with visual observation as in this case an eyepiece is used to observe an intermediate image plane (which is under the scope of this document). 2 Normative references There are no normative references in this document. 3 Terms and definitions No terms and definitions are listed in this document. ISO and IEC maintain terminological databases for use in standardization at the following addresses: — ISO Online browsing platform: available at https://www.iso .org/obp — IEC Electropedia: available at https://www.electropedia .org/ 4 Measurands 4.1 General For obtaining the desired image quality, when using a light microscope, the brightness, temporal stability and uniformity of the image play an important role. This holds true for different applications and various types of instruments. The measurement of illumination brightness and uniformity is mandatory. The temporal stability is an optional measurement because it is only suitable for certain types of light sources (e.g. arc lamps).
5 Measurement procedure 5.1 General In addition to defining the measurement geometry and procedure it is necessary to describe essential settings of the microscope in order to eliminate their influence on the measurement of illumination brightness, temporal stability and uniformity. 5.2 Microscope settings The microscope shall be set up in the desired configuration regarding excitation wavelength (band), dichroic filters and/or emission filters. If the microscope offers an adjustable field stop, it shall be set to the smallest setting for which the field of view of the imaging sensor in 5.5 is fully illuminated. If the microscope offers an adjustable aperture stop, it shall be set to the smallest setting for which the pupil of the objective lens used is overfilled. For the measurement of illumination brightness and temporal stability the illumination shall be warmed-up according to the manufacturer ’s specifications. If the manufacturer does not specify a warm-up time, the illumination shall be warmed-up for at least one hour prior to measurement. The light output of the illumination shall be set to its maximum value and, if a filter is used for extinction during measurement, this filter should be specified.
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.