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

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

3. “Koehler” Illumination
Prof. August Köhler:
“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

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. 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:
~ x NAobjective

12. 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 !

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