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Köhler Illumination

Proper illumination for biological samples is of paramount importance for achieving high-resolution and contrast images. Hot spots or uneven illumination can greatly reduce the usefulness of an optical system or create artifacts that obscure important features. In 1893, August Köhler developed an illumination technique that created improved, glare-free illumination for microscopy applications when using non-uniform sources.  This was done by creating parallel ray bundles instead of imaging the light source at the illumination plane. This illumination technique can reduce stray light and artifacts encountered when imaging samples such as the human retina.
In the 3DOptix cloud-based simulation tool, we can simulate illumination applications using the included tools and analysis features. We will overview how to set up and analyze an optical system with Köhler illumination.
Our optical system will consist of a simple optical system with the following components:
  1. Eskma Optics 111-0218E, bi-convex
  2. Eskma Optics 110-0502E, plano-convex
  3. Eskma Optics 110-0504E, bi-convex
  4. 3x Light Sources
    • Point Source
    • Wavelength – Top Hat Distribution
      • 400-700 nm range
      • 10 nm step size
    • Power, 0.010 W
    • Unpolarized
  5. Detector: Illumination
    • Spot: Incoherent Irradiance
    • Analysis Rays: 1 million
    • 500×500 pixels
You can see the image of our optical system below. The 3DOptix simulation file can be downloaded to see additional information about the optical system such as component spacing and analysis detectors. Lens 1 includes a light block to stop light from going directly to lens 2 from the source.

The light source that we are simulating is a filament lamp that emits in all directions, but initially, it needs to be analyzed for a few important source points and where they land at the illumination plane. We need to determine the best lens positions that will generate these parallel ray bundles and these trace rays will allow us to visually align the system. The three POINT SOURCES initially created will be turned off for this part of the analysis. 

To start the analysis, we will create the trace rays.
  • 4x Light Sources
    • Plane Wave
    • Circular 0.0001 mm radius
    • 3d Layout Rays: 1
    • 532 nm Wavelength
    • Power, 0.010 W
    • Unpolarized
To make the visualization of the rays easier we will change the source color to correspond to their source point position. The top trace rays will be red and the center green.
The rays will be placed in line with the center and top of lens 1 at its front focus, 100 mm.  Both center rays are angled to hit the edges of the lens. One top ray is angled to hit the bottom of the lens. These will only be for analysis.
Lens 2 and 3 can now be added and moved along the optical axis to steer the rays to the illumination plane.  With a few tries in lens 2 and 3 position and observing the ray pairs at the illumination plane we have achieved an initial Köhler illumination pattern; parallel ray bundles at the illumination plane.

Since we have an initial setup that seems to be a correct solution, we can now remove the traced rays and reintroduce the POINT SOURCES. Once we do this, we can start to analyze the illumination pattern to determine if better positioning of the lenses is necessary.

As a note, the three POINT SOURCES have TOP HAT spectral profiles emulating a broadband filament source.

The analysis was run for three different positions to show how the uniformity changes as the detector is moved to the nominal position. The images below demonstrate the illumination pattern becoming larger and more even as the detector is moved closer to the ideal position. This position corresponds to the point along the optical axis where all the ray bundles exiting lens 3 converge and overlap.
To go further with the analysis, we want to determine numerically the uniformity. Generally, we would have a target illumination parameter of some percentage to generate the desired image quality. To do this we can use the last analysis where the illumination looked best and limit the pixel values to only the ones that are within some value of nominal.

The target we will use is 20% around the nominal intensity value. We can download the detector data and look at the central portion of the output to determine the nominal pixel value.
Using this number, we can then limit the min and max thresholding of the analysis window to view only the pixels that satisfy this condition. The nominal value used will be 0.0025 counts and the threshold will be 0.0020 to 0.0030 counts. Although not perfect we do get a very good spot of even illumination.

Further analysis and optimization of lens positioning and diaphragms can improve the performance of the system by increasing the illumination effective area, non-uniformity, and overall image quality.
Using Köhler’s technique, we remove artifacts from directly imaging the light source onto the image plane and increase the illumination uniformity.
This can generate better image quality overall for the system.

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Available on January 30th, 2023