• Break Period – move to labBreak Period – move to lab
• Setting up / adjusting the Setting up / adjusting the microscopes for Brightfieldmicroscopes for Brightfield
Brightfield The most basic illumination technique
How to set it up for best results
“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
Necessary components to perform “Koehler” Illumination:
• Adjustable Field Diaphragm
• Focusable and Centerable Condenser
• Adjustable Condenser Aperture Diaphragm
1) Open Field and Condenser Diaphragms
2) Focus specimen3) Correct for proper Color
Temperature4) Close Field Diaphragm5) Focus Field Diaphragm – move
condenser up and down6) Center Field Diaphragm7) Open to fill view 8) Observe Objective’s Back Focal
Plane via Ph Telescope or by removing Ocular
9) Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture
10)Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)
Koehler Illumination Steps:
1) Open Field and Condenser Diaphragms
2) Focus specimen3) Correct for proper Color
Temperature4) Close Field Diaphragm5) Focus Field Diaphragm – move
condenser up and down6) Center Field Diaphragm7) Open to fill view 8) Observe Objective’s Back Focal
Plane via Ph Telescope or by removing Ocular
9) Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture
10)Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)
1) Open Field and Condenser Diaphragms
2) Focus specimen3) Correct for proper Color
Temperature4) Close Field Diaphragm5) Focus Field Diaphragm – move
condenser up and down6) Center Field Diaphragm7) Open to fill view 8) Observe Objective’s Back Focal
Plane via Ph Telescope or by removing Ocular
9) Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture
10)Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)
1) Open Field and Condenser Diaphragms
2) Focus specimen3) Correct for proper Color
Temperature4) Close Field Diaphragm5) Focus Field Diaphragm by moving condenser up or down1) Center Field Diaphragm2) Open to fill view 3) Observe Objective’s Back Focal
Plane via Ph Telescope or by removing Ocular
4) Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture
5) Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)
1) Open Field and Condenser Diaphragms
2) Focus specimen3) Correct for proper Color
Temperature4) Close Field Diaphragm5) Focus Field Stop by moving
condenser up or down6) Center Field Diaphragm7) Open to fill view 8) Observe Objective’s Back Focal
Plane via Ph Telescope or by removing Ocular
9) Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture
10)Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)
1) Open Field and Condenser Diaphragms
2) Focus specimen3) Correct for proper Color
Temperature4) Close Field Diaphragm5) Focus Field Diaphragm – move
condenser up and down6) Center Field Diaphragm7) Open to fill view of observer8) Observe Objective’s Back Focal
Plane via Ph Telescope or by removing Ocular
9) Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture
10)Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)
1) Open Field and Condenser Diaphragms
2) Focus specimen3) Correct for proper Color
Temperature4) Close Field Diaphragm5) Focus Field Diaphragm – move
condenser up and down6) Center Field Diaphragm7) Open to fill view 8) Observe Objective’s Back Focal
Plane via Ph Telescope or by removing Ocular
9) 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
Koehler Steps: 1) Open Field and Condenser
Diaphragms2) Focus specimen3) Correct for proper Color
Temperature4) Close Field Diaphragm5) Focus Field Diaphragm – move
condenser up and down6) Center Field Diaphragm7) Open to fill view 8) Observe Objective’s Back Focal
Plane via Ph Telescope or by removing Ocular
9) Close Condenser Diaphragm to fill approx. 2/3 of Objective’s Aperture
10)Observe Image !
Done !
Conjugate Planes (Koehler)
Illumination Path
Imaging Path
Eyepiece
TubeLens
Objective
Condenser
Collector
Eye
Field Diaphragm
Specimen
Intermediate Image
Retina
Light Source
Condenser Aperture Diaphragm
Objective Back Focal Plane
Eyepoint
Conjugate Planes - Upright Microscope
Image Planes
Aperture Planes
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)
Conjugate Planes – Inverted Microscope
The Objective
The most important Microscope The most important Microscope Component…Component…
The second most important The second most important optical component…optical component…
The Condenser
Why do we need a condenser?
dmind
Without Condenser (NA condenser = 0),
only ½ of the resolution could be obtained !
CondenserObjective NANAd
min
Minimum resolvable distance
