break period – move to lab setting up / adjusting the microscopes for brightfield

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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 - PowerPoint PPT Presentation

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  • 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 IlluminationProf. August Khler: 1866 - 1948Provides for most homogenous IlluminationHighest obtainable ResolutionAllows adjustment of optimal ContrastDefines desired Depth of FieldMinimizes Straylight and unnecessary IradiationHelps in focusing difficult-to-find structuresEstablishes proper position for condenser elements, for all contrasting techniques

  • Necessary components to perform Koehler Illumination:Adjustable Field DiaphragmFocusable and Centerable CondenserAdjustable Condenser Aperture Diaphragm

  • Open Field and Condenser DiaphragmsFocus specimenCorrect for proper Color TemperatureClose Field DiaphragmFocus Field Diaphragm move condenser up and downCenter Field DiaphragmOpen to fill view Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objectives ApertureEnjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)Koehler Illumination Steps:

  • Open Field and Condenser DiaphragmsFocus specimenCorrect for proper Color TemperatureClose Field DiaphragmFocus Field Diaphragm move condenser up and downCenter Field DiaphragmOpen to fill view Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objectives ApertureEnjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

  • Open Field and Condenser DiaphragmsFocus specimenCorrect for proper Color TemperatureClose Field DiaphragmFocus Field Diaphragm move condenser up and downCenter Field DiaphragmOpen to fill view Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objectives ApertureEnjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

  • Open Field and Condenser DiaphragmsFocus specimenCorrect for proper Color TemperatureClose Field DiaphragmFocus Field Diaphragm by moving condenser up or downCenter Field DiaphragmOpen to fill view Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objectives ApertureEnjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

  • Open Field and Condenser DiaphragmsFocus specimenCorrect for proper Color TemperatureClose Field DiaphragmFocus Field Stop by moving condenser up or downCenter Field DiaphragmOpen to fill view Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objectives ApertureEnjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

  • Open Field and Condenser DiaphragmsFocus specimenCorrect for proper Color TemperatureClose Field DiaphragmFocus Field Diaphragm move condenser up and downCenter Field DiaphragmOpen to fill view of observerObserve Objectives Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objectives ApertureEnjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

  • Open Field and Condenser DiaphragmsFocus specimenCorrect for proper Color TemperatureClose Field DiaphragmFocus Field Diaphragm move condenser up and downCenter Field DiaphragmOpen to fill view Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objectives ApertureBFPBetter: Depending on specimens inherent contrast, close condenser aperture to: ~ 0.3 - 0.9 x NAobjective

  • Koehler Steps: Open Field and Condenser DiaphragmsFocus specimenCorrect for proper Color TemperatureClose Field DiaphragmFocus Field Diaphragm move condenser up and downCenter Field DiaphragmOpen to fill view Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular Close Condenser Diaphragm to fill approx. 2/3 of Objectives ApertureObserve Image !Done !

  • Conjugate Planes (Koehler)Illumination PathImaging PathEyepieceTubeLensObjectiveCondenserCollectorEyeField DiaphragmSpecimenIntermediate ImageRetinaLight SourceCondenser Aperture DiaphragmObjective Back Focal PlaneEyepoint

  • Conjugate Planes - Upright MicroscopeImage PlanesAperture Planes

  • 1 Intermediate image plane (photo tube)2 Eyepiece/ Intermediate image/ Eyepoint3 Intermediate image plane (front port)4 Intermediate image plane (base port)5+6 Imaging Beam Path switchers7 Tube lens 8 Analyzer9 Reflector10 Field stop (Reflected light = RL)11 Aperture diaphragm (RL)12 Filter slider (RL)13 HBO Illumination - Source14 HAL Illumination - Source15 Field stop (Transmitted light = TL)16 Polarizer17 Aperture diaphragm (TL)18 Condenser19 Objective BFP (Back Focal Plane)

    Conjugate Planes Inverted Microscope

  • The ObjectiveThe most important Microscope Component

  • The second most important optical componentThe Condenser

  • Why do we need a condenser?

  • Without Condenser (NA condenser = 0),

    only of the resolution could be obtained ! Minimum resolvable distance