Procedure to measure illumination field

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?


We like the concentrated dye solutions proposed by Michael Model, he’s written a number of articles on the method:

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

B. highest signal in a 256x256 region. Note that ZOOMED IN.

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 -

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.