# Procedure to measure illumination field **URL:** \ **Category:** Hardware\ **Created:** [August 14, 2023, 2:29pm UTC](https://forum.microlist.org/t/procedure-to-measure-illumination-field/2242 "2023-08-14T14:29:06Z")\ **Posts on this page:** 3\ **Page:** 1
**Author:** ![Yossi\_Steinberger](https://sea2.discourse-cdn.com/flex020/user_avatar/forum.microlist.org/yossi_steinberger/32/1272_2.png) [@Yossi\_Steinberger](https://forum.microlist.org/u/Yossi_Steinberger)\ **Post date:** [August 14, 2023, 2:29pm UTC](https://forum.microlist.org/t/procedure-to-measure-illumination-field/2242/1 "2023-08-14T14:29:06Z")
Hello - What is a good sample for measuring the illumination field? The laser on my commercial system - Dragonfly 505 series - is concentrated via a telescoping lens - and should only be illuminating the center 512x512 pixels of the 2048x2048 sensor. However, when I use a chroma autofluorescent slide I get a high background all over the sensor, besides the somewhat increased signal at the center 512x512 region. Does the light somehow reflect within the slide, broadening the apparent illumination area? What is the recommended procedure and materials for properly measuring the illumination field? Best, Yossi ---
**Author:** ![talley](https://sea2.discourse-cdn.com/flex020/user_avatar/forum.microlist.org/talley/32/3_2.png) [@talley](https://forum.microlist.org/u/talley)\ **Post date:** [August 14, 2023, 2:44pm UTC](https://forum.microlist.org/t/procedure-to-measure-illumination-field/2242/2 "2023-08-14T14:44:17Z")
We like the concentrated dye solutions proposed by Michael Model, he’s written a number of articles on the method: > **[A standard for calibration and shading correction of a fluorescence microscope...](https://pubmed.ncbi.nlm.nih.gov/11500847/)** > > Standardization of fluorescent images can be achieved by normalizing them to the image of a concentrated solution of a fluorophore. Due to its simplicity and efficiency, this method can be used in clinical analysis as well as in routine laboratory... > **[Intensity calibration and shading correction for fluorescence microscopes -...](https://pubmed.ncbi.nlm.nih.gov/18770832/)** > > Standardization in image cytometry involves intensity calibration and shading correction. This unit presents a method using concentrated solutions of fluorophores. A drop of highly concentrated dye solution placed between a slide and a coverslip... > **[Intensity calibration and flat-field correction for fluorescence microscopes...](https://pubmed.ncbi.nlm.nih.gov/24692055/)** > > Standardization in fluorescence microscopy involves calibration of intensity in reproducible units and correction for spatial nonuniformity of illumination (flat-field or shading correction). Both goals can be achieved using concentrated solutions of... ---
**Author:** ![Yossi\_Steinberger](https://sea2.discourse-cdn.com/flex020/user_avatar/forum.microlist.org/yossi_steinberger/32/1272_2.png) [@Yossi\_Steinberger](https://forum.microlist.org/u/Yossi_Steinberger)\ **Post date:** [August 22, 2023, 3:24pm UTC](https://forum.microlist.org/t/procedure-to-measure-illumination-field/2242/4 "2023-08-22T15:24:59Z")
Dear all - The issue remains unresolved. I used the fluoroscene, or my dye solution (Janelia Fluor far-red dye), or the chroma autofluorescent slides, and in all cases - although I should only get signal in a 512x512 pixel area, because I concentrate the light with a telescoping lens, nonetheless I get signal across the image sensor. Moreover the signal is strongest and homogenous in a 256x256 pixel area. Note in the screenshot below, there are two different lookup tables to **(A)** highlight either the weak signal across the entire image sensor, or, to **(B)** show the highest signal in a 256x256 region. A. weak signal across the entire image sensor ![image](https://us1.discourse-cdn.com/flex020/uploads/microlist/original/2X/8/87835ee6fe898d59db14c1eedb08d5a55ea0ef62.jpeg) B. highest signal in a 256x256 region. Note that ZOOMED IN. ![image](https://us1.discourse-cdn.com/flex020/uploads/microlist/original/2X/2/24d966fec3ba5f5a8a705fa64fbe0746c1cb7bd6.png) Is this because of leaky excitation, and I need to assume only 10% of the excitation light reaches the proper 512x512 area? Or - is this because of leaky emission, and the excitation light truly only passes through the 512x512 area? _How does one measure the illumination area?_ Looking forward to hearing from you - Yossi p.s. based on the systems specification, I would expect an illumination area of 512x512 pixels, or 30x30 microns^2, with a power density of around 3 kW/cm^2. These were my acquisition parameters. - Dragonfly 505 - Widefield - Sona back illuminated sCMOS (scientific complementary metal oxide semiconductor camera). 6.5 um pixels. 2048x2048 pixels. - 100x/1.47 objective - 6x illumination zoom, using a telescoping lens (“Pd4” on the dragonfly) - 1% laser intensity - 10ms exposure - 637nm wavelength - 3.25x3.25 illumination aperture. - 16-bit - 100 Digital numbers, DN (also known as A.U., arbitrary units, gray values) is the set offset by the camera manufacturer.