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Break Period – move to lab Setting up / adjusting the microscopes for Brightfield

Break Period – move to lab Setting up / adjusting the microscopes for Brightfield. Brightfield The most basic illumination technique How to set it up for best results. “Koehler” Illumination. Prof. August K ö hler: 1866 - 1948. Provides for most homogenous Illumination

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Break Period – move to lab Setting up / adjusting the microscopes for Brightfield

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  1. Break Period – move to lab • Setting up / adjusting the microscopes for Brightfield

  2. Brightfield • The most basic illumination technique • How to set it up for best results

  3. “Koehler” Illumination Prof. August Köhler: 1866 - 1948 • Provides for most homogenous Illumination • Highest obtainable Resolution • Allows adjustment of optimal Contrast • Defines desired Depth of Field • Minimizes Straylight and unnecessary Iradiation • Helps in focusing difficult-to-find structures • Establishes proper position for condenser elements, for all contrasting techniques

  4. Necessary components to perform “Koehler” Illumination: • Adjustable Field Diaphragm • Focusable and Centerable Condenser • Adjustable Condenser Aperture Diaphragm

  5. Koehler Illumination Steps: • Open Field and Condenser Diaphragms • Focus specimen • Correct for proper Color Temperature • Close Field Diaphragm • Focus Field Diaphragm – move condenser up and down • Center Field Diaphragm • Open to fill view • Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular • Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture • Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

  6. Open Field and Condenser Diaphragms • Focus specimen • Correct for proper Color Temperature • Close Field Diaphragm • Focus Field Diaphragm – move condenser up and down • Center Field Diaphragm • Open to fill view • Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular • Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture • Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

  7. Open Field and Condenser Diaphragms • Focus specimen • Correct for proper Color Temperature • Close Field Diaphragm • Focus Field Diaphragm – move condenser up and down • Center Field Diaphragm • Open to fill view • Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular • Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture • Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

  8. Open Field and Condenser Diaphragms • Focus specimen • Correct for proper Color Temperature • Close Field Diaphragm • Focus Field Diaphragm by • moving condenser up or down • Center Field Diaphragm • Open to fill view • Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular • Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture • Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

  9. Open Field and Condenser Diaphragms • Focus specimen • Correct for proper Color Temperature • Close Field Diaphragm • Focus Field Stop by moving condenser up or down • Center Field Diaphragm • Open to fill view • Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular • Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture • Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

  10. Open Field and Condenser Diaphragms • Focus specimen • Correct for proper Color Temperature • Close Field Diaphragm • Focus Field Diaphragm – move condenser up and down • Center Field Diaphragm • Open to fill view of observer • Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular • Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture • Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

  11. Open Field and Condenser Diaphragms • Focus specimen • Correct for proper Color Temperature • Close Field Diaphragm • Focus Field Diaphragm – move condenser up and down • Center Field Diaphragm • Open to fill view • Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular • Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture BFP Better: Depending on specimen’s inherent contrast, close condenser aperture to: ~ 0.3 - 0.9 x NAobjective

  12. Koehler Steps: • Open Field and Condenser Diaphragms • Focus specimen • Correct for proper Color Temperature • Close Field Diaphragm • Focus Field Diaphragm – move condenser up and down • Center Field Diaphragm • Open to fill view • Observe Objective’s Back Focal Plane via Ph Telescope or by removing Ocular • Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture • Observe Image ! Done !

  13. Conjugate Planes (Koehler) Retina Eye Eyepoint Eyepiece Intermediate Image TubeLens Imaging Path Objective Back Focal Plane Objective Specimen Condenser Condenser Aperture Diaphragm Field Diaphragm Illumination Path Collector Light Source

  14. Conjugate Planes - Upright Microscope Image Planes Aperture Planes

  15. Conjugate Planes – Inverted Microscope 1 Intermediate image plane (photo tube) 2 Eyepiece/ Intermediate image/ Eyepoint 3 Intermediate image plane (front port) 4 Intermediate image plane (base port) 5+6 Imaging Beam Path switchers 7 Tube lens 8 Analyzer 9 Reflector 10 Field stop (Reflected light = RL) 11 Aperture diaphragm (RL) 12 Filter slider (RL) 13 HBO Illumination - Source 14 HAL Illumination - Source 15 Field stop (Transmitted light = TL) 16 Polarizer 17 Aperture diaphragm (TL) 18 Condenser 19 Objective BFP (Back Focal Plane)

  16. The most important Microscope Component… The Objective

  17. The second most important optical component… The Condenser

  18. Why do we need a condenser?

  19. d dmin Minimum resolvable distance Without Condenser (NA condenser = 0), only ½ of the resolution could be obtained !

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