BS ISO 19056-3-2022 pdf free download.Microscopes — Definition and measurement of illumination properties Part 3: Incident light fluorescence microscopy with incoherent light sources
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.
5.3  Illumination brightness
The measurement is performed with a 10× objective and a calibrated power meter placed so that the detection area of the power meter is slightly underfilled with light. The detection range of the power meter shall be in the linear range. The detector of the power meter shall not be saturated.
The integration time of the power meter shall be between 0,2 s and 1 s. The focal plane of the objective shall include a circular, centred diaphragm of (0,4 ± 0,1) mm in diameter. Figure 1 shows the position of the power meter during the measurement.
NOTE 1 Because the actual measured value of the area of the diaphragm enters the calculation for illumination brightness in 4.2 the diameter need not have a tight tolerance.
NOTE 2 If no diaphragm is used, the measured value of radiant flux can still be used to compare the same instrument at different points in time.
5.4  Temporal stability
The measurement is performed with a 10× objective and a power meter placed so that the detection area of the power meter is underfilled with light. The detection range of the power meter shall be in the linear range. If a 10× objective is not available, the measurement shall be performed without an objective and the power meter shall be placed at the position of the pupil of the illumination, thus the objective needs to be removed. The power meter shall be mounted in a fixed position with regard to the objective or the objective nosepiece respectively. External influences on the measurement, e.g. changes in room temperature or changes in the ambient lighting conditions, have to be eliminated.
Figure 2 shows an alternative position of the power meter when used without objective. The detector of the power meter shall not be saturated. The integration time of the power meter shall be between 0,2 s and 1 s.
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.BS ISO 19056-3 pdf download.