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4f Koehler Transmitted Illumination Condenser for Teaching and Low-Cost Microscopic Imaging

Jorge Madrid-Wolff and

Manu Forero-Shelton

Article Category: Research Article

Volume/Issue: Volume 1: Issue 2

Online Publication Date: Aug 13, 2020

DOI: 10.35459/tbp.2019.000113

Page Range:

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Fig 2; Collimation of the LED. Move the collector lens until you collimate the LED, so the light does not diverge too much. You should not have an image of the LED. If you do, you are not collimating the beam but focusing it.

Jorge Madrid-Wolff and

Manu Forero-Shelton

Fig 2

Collimation of the LED. Move the collector lens until you collimate the LED, so the light does not diverge too much. You should not have an image of the LED. If you do, you are not collimating the beam but focusing it.

Fig 1; (A) Schematic and (B) photograph of the optical setup. The system is all configured in 4f, conjugating 2 sets of planes. The upper section of the diagram illustrates the conjugation of the sample plane with the field diaphragm and (not shown in the schematic) with the image plane on the camera sensor. In these planes the image of the LED is lost after having been Fourier transformed by the collector lens and spatially homogenized by the diffusing surface.

Jorge Madrid-Wolff and

Manu Forero-Shelton

Fig 1

(A) Schematic and (B) photograph of the optical setup. The system is all configured in 4f, conjugating 2 sets of planes. The upper section of the diagram illustrates the conjugation of the sample plane with the field diaphragm and (not shown in the schematic) with the image plane on the camera sensor. In these planes the image of the LED is lost after having been Fourier transformed by the collector lens and spatially homogenized by the diffusing surface.

Fig 3; Placement of the field diaphragm (iris) at the focal plane of the collector lens. Using a power meter or a light meter on your phone, move the iris until you find the maximum light intensity, which should be the same as the focal length of the condenser lens.

Jorge Madrid-Wolff and

Manu Forero-Shelton

Fig 3

Placement of the field diaphragm (iris) at the focal plane of the collector lens. Using a power meter or a light meter on your phone, move the iris until you find the maximum light intensity, which should be the same as the focal length of the condenser lens.

Fig 4; Once again, you want to collimate the beam with this lens. Because the closed diaphragm is more similar to a point source than the original LED, it should be easier to collimate than in the first step.

Jorge Madrid-Wolff and

Manu Forero-Shelton

Fig 4

Once again, you want to collimate the beam with this lens. Because the closed diaphragm is more similar to a point source than the original LED, it should be easier to collimate than in the first step.

Fig 6; Placement of the condenser lens. Keep the field diaphragm open and the aperture diaphragm closed. Insert the second spherical lens, with its curved face pointing toward the light source, in the cage plate and adjust its position until a collimated beam is achieved. As before, do not expect perfect collimation, but try to achieve the least diverging beam.

Jorge Madrid-Wolff and

Manu Forero-Shelton

Fig 6

Placement of the condenser lens. Keep the field diaphragm open and the aperture diaphragm closed. Insert the second spherical lens, with its curved face pointing toward the light source, in the cage plate and adjust its position until a collimated beam is achieved. As before, do not expect perfect collimation, but try to achieve the least diverging beam.

Fig 5; Placement of the aperture diaphragm at the back focal plane of the condenser lens. Once again, move the iris, which will work as the aperture diaphragm, to about 1 focal length of the spherical lens. Make sure you open the other iris (field diaphragm) and move the second iris until you maximize the intensity on your power meter or phone.

Jorge Madrid-Wolff and

Manu Forero-Shelton

Fig 5

Placement of the aperture diaphragm at the back focal plane of the condenser lens. Once again, move the iris, which will work as the aperture diaphragm, to about 1 focal length of the spherical lens. Make sure you open the other iris (field diaphragm) and move the second iris until you maximize the intensity on your power meter or phone.

Fig 7; Coupling the illumination to the rest of the microscope. Close the field diaphragm and open the aperture diaphragm. Move the illuminator perpendicularly to the optical axis of the microscope until an image of the field diaphragm appears on the camera. Move the illuminator along the optical axis (shown as a horizontal dotted line) until the image of the field diaphragm appears sharp, and center it by translating the illuminator. Having the setup on a micrometric translational stage makes this step easier, but this can also be achieved manually with some patience.

Jorge Madrid-Wolff and

Manu Forero-Shelton

Fig 7

Coupling the illumination to the rest of the microscope. Close the field diaphragm and open the aperture diaphragm. Move the illuminator perpendicularly to the optical axis of the microscope until an image of the field diaphragm appears on the camera. Move the illuminator along the optical axis (shown as a horizontal dotted line) until the image of the field diaphragm appears sharp, and center it by translating the illuminator. Having the setup on a micrometric translational stage makes this step easier, but this can also be achieved manually with some patience.

Fig 8; Field homogeneity. (A) Relative intensity profile for the system presented here over a typical field of view with 10× magnification. (B) Intensity profile along the dotted diagonal in panel A. (C, D) Analogous to panels A and B, but for a single LED flashlight in direct illumination.

Jorge Madrid-Wolff and

Manu Forero-Shelton

Fig 8

Field homogeneity. (A) Relative intensity profile for the system presented here over a typical field of view with 10× magnification. (B) Intensity profile along the dotted diagonal in panel A. (C, D) Analogous to panels A and B, but for a single LED flashlight in direct illumination.

Fig 9; Sectioning capability of the 4f system with respect to a commercial condenser and an LED flashlight in direct illumination. Different optical planes (sections) of a petal from Phalaenopsis amabilis, captured with a 10× 0.3 numerical aperture (NA) air objective. (Top row) 4f system with the condenser aperture fully open (NA = 0.2). (Middle row) Commercial Koehler system with the condenser aperture fully open (NA = 0.52). (Bottom row) Single LED source. Scale bar 50 μm.

Jorge Madrid-Wolff and

Manu Forero-Shelton

Fig 9

Sectioning capability of the 4f system with respect to a commercial condenser and an LED flashlight in direct illumination. Different optical planes (sections) of a petal from Phalaenopsis amabilis, captured with a 10× 0.3 numerical aperture (NA) air objective. (Top row) 4f system with the condenser aperture fully open (NA = 0.2). (Middle row) Commercial Koehler system with the condenser aperture fully open (NA = 0.52). (Bottom row) Single LED source. Scale bar 50 μm.