Break Period – move to lab

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Presentation transcript:

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

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

“Koehler” Illumination Prof. August Köhler: 1866 - 1948 “Koehler” Illumination 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

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

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)

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)

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)

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)

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)

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)

BFP 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

Done ! 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 !

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

Conjugate Planes - Upright Microscope Image Planes Aperture Planes

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)

The most important Microscope Component… The Objective

The second most important optical component… The Condenser

Why do we need a condenser?

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