1. IIIComponents, Microscope SetupDecember 2008 Rudi Rottenfusser – Carl Zeiss MicroImaging

2. Categories 25 Nosepieces, Rings, Adapters 28 Compensators, 6x20mm-type Analyzers 30 Fluorescence Reflectors and Filter Sets 31 Other Sliders & Reflectors f/Infinity Space 32 Components f/ Analyzer slider receptacle 33 Intermediate tubes & modules 34 Randomizers, Tube Carriers, Tube Mounts 35 Tubes, Tube panels, Tube heads, Access. 38 Eyepieces & Projectives - ICS & Stereo 40 Eyepiece reticles, Micrometers 42 Adapters for Still and Video Cameras 43 Attachment Camera Systems 44 Digital High Resolution Camera Systems 45 Zeiss Video Camera Systems 47 Cases, Dust Covers, Cover Plates, Cables 49 Miscellaneous Micro Items 1 Stands, Base Plates 2 Stereo stands & Accessories 3 Fiber Optics Light Sources 4 Power Supplies 5 Lamp Housings, Coll., Sockets, Adapters 6 Bulbs, Arc Lamps, Burners 7 Inserts for Stands, Sliders 8 Filters, 42mm & 32mm diameter 9 Filters, 25mm & 18mm diameter 10 Filters, others 12 Condenser- and Illuminator Carriers 13 Condensers, Lenses, BF, DF, Ph, Pol, DIC 14 Stage Carriers, Stages, Specimen holders 15 Scanning stages, Spec.hldrs, Controllers 16 Cooling / Heating Stages & Accessories 17 Objectives 160mm 18 Objectives ICS (covered specimens) 19 Objectives ICS (for non-covered samples) 23 Objectives for SteMi's, Luminars 24 Objectives (Spc. e.g. Hoffmann; McCrone)

3. Topics • The major Optical Components of the Microscope • Light Sources • “Natural” • Tungsten, Halogen • Arc Lamp • LED • Condenser • Resolution, Numerical Aperture • Objective (more details in part 4) • Eyepiece • Useful Magnification • Markings • Parfocal Setting

4. Topics • Setting up the Microscope for optimal Performance • “Contrast” • Basic Setup for Brightfield • Koehler Illumination • Conjugate Image Planes on Microscopes

5. Cross-section through an ∞ corrected Microscope

6. Light Source for a typical Laboratory Microscope (late 1800’s to mid 1900’s) Light Source • Perfect even illumination • Perfect Color Temperature (“Daylight”) • Evenings? Nights? • Intensity?

7. Artificial Light Sources (incoherent) • Tungsten • Tungsten Halogen

8. Tungsten – Halogen Principle

9. Inexpensive Easy to replace Temporally Stable Spacially Stable No change of Spectral Output during Life Low UV output High IR output Tungsten-Halogen Lamp Visible Light

10. Arc Lamps • Tungsten • Tungsten Halogen • Mercury Arc • Xenon Arc

11. Arc Lamps

12. Arc Lamps HBO 100 XBO 75W Metal Halide Courtesy – Michael Davidson

13. Light Sources • Tungsten • Tungsten Halogen • Mercury Arc • Xenon Arc • LED’s

14. LED Light Sources

15. Long life (10,000+h?) Stable Output over time Clean spectrum Cool No lamp alignment No need for shutter – no vibration Quick switching LED Light Sources

16. Colibri Fast Switching FL Source

17. Light Sources • Tungsten • Tungsten Halogen • Mercury Arc • Xenon Arc • LED’s • Laser (coherent)

18. Lamp Housings and its optical components • Lamp Housing • 100W, 35W Halogen • 100W HBO • 75W Xenon • Colibri (4 LED + HBO) • Collector • Fixed • Focusable, 3-lens or aspheric • Heat Filter • Heat absorbing or reflecting

19. Cross-section through an ∞ corrected Microscope

20. Internal Light Path incorporates: Components between Light Source and Specimen Transmitted Light: • Light Shutter • Filter Turret or Filter Slider with Neutral Density or Color Filters • Luminous Field Diaphragm Reflected (Incident) Light: • Light Shutter • Filter Turret or Filter Slider with Neutral Density, Color Filters, Attenuator • Aperture Diaphragm • Luminous Field Diaphragm

21. Cross-section through an ∞ corrected Microscope

22. The Condenser

23. Resolution (minimum resolved distance between two points): Without Condenser: Objective Specimen Specimen b With Condenser: Condenser When Condenser NA matches Objective NA Highest Resolution !

24. The Objective More details later… Why do we need a condenser?

25. Numerical Aperture (NA) NA = sin a· n b 90° a c  Refractive Index nair = 1 nwater = 1.33 nglycerin = 1.47 noil = 1.518 n How is a sine function defined ?

26. With immersion oil(3)n=1.518 No stray light, no total reflection ! Max. Objective aperture 1.46 (oil) Captured Aperture of specimen below cover slip: 1.46/1.52 = 0.96 (2 = 74°) a1 a2 1 3 2 Why immersion medium affects NA Plan-Apochromat 100x/1.46 Oil   420792-8000 Plan-Apochromat 40x/0.95 corr. 440654-9902 No Oil “Dry” Oil Immersion • Objective • Cover Slip + slide • Immersion Oil • No immersion (dry) • Max. Objective aperture 0.95 ( = 72°) • Captured Aperture of specimen below cover slip: 0.95/1.52 = 0.62 (1 = 39°)

27. Cross-section through an ∞ corrected Microscope

28. Infinity Space • Components in Infinity Space: • DIC sliders • Compensator Sliders • Fluorescence Filters • Analyzer . Requirement for co-localized Images: Components need to be plane-parallel ! Infinity System Specimen off-center

29. Cross-section through an ∞ corrected Microscope

30. Intermediate Tubes and Tubes Tube Lens Turret with up to 3 tube lenses in addition to standard 1x, such as 1.25x 1.6x 2.5x 4.0x

31. Tube Mounts – Upright Microscopes • Primostar • Axiostar / Standard Line • Axiostar tubes fit old (160) Zeiss microscopes, converting them to Infinity Optics ! • Standard, GFL, RA, WL, ACM can get “upgraded”! • Old (finite) condensers work with new objectives! • No upgrade to infinity possible for Universal, Photomicroscope, Ultraphot or UEM • Axioskop 1, Axioskop 40, Axioskop 2FS, Axioplan, Axiophot • Forward and Backward Compatibility between c) and d) via tube adapters! • Axioplan 2, 2i, 2ie • AxioImager A1, D1, M1, Z1 • Stereo Microscopes SV6, SV11, SR, SV8 • SteReo Discovery, Lumar

32. Binocular Tubes (example - Axio Imager) Note: • Tube Lens always included • All Zeiss tubes can be folded • up or down • Optimum angle for most • comfortable viewing: 15-20º

33. Dual Video Adapter • 2 camera ports with 60mm interface – one is adjustable (x, y, z) • interchangeable beam splitting cube for neutral or spectral image or signal splitting • attaches to all camera ports with 60mm interface

34. Camera Adapters for 60mm Interface • C-mount Adapters 1x, 0.63x, 0.5x, 0.4x, Zoom • T2-mount Adapters 1x, 1.6x, 2.5x, 4x • ENG-mount Adapters 1x, 0.8x • Eyepiece tube (for digital cameras) • Adapter for Digital Cameras with built-in objective (37 and 52mm thread diameters)

35. Cross-section through an ∞ corrected Microscope

36. Eyepieces (Oculars) Who needs eye-pieces any more? • Field of View • “Presence” • Detect fine nuances in • color shades • Stereo • Dynamic Range of the Eye

37. Magnification In order to see small objects with the eye the small objects must be magnified to an appropriate size

38. Useful Magnification Limitation #1 – The eye • You will miss fine nuances in the image if the objective projects details onto the intermediate image, which are smaller than the resolving power of your eye (typical at low magnification / high NA) Limitation #2 – The microscope • You will reach “empty magnification” if you enlarge an image beyond the physical resolving power of the optics. Rule of Thumb: Total Magnification of an image to the eyes should be between 500 and 1000 times the objective’s Numerical Aperture

39. 1939

40. Eyepiece Characteristics Example: W PL 10x/23 Foc. W Wide Angle PL Flat Field (“Plan” > old style: “KPL” or “CPL”) 10x Magnification 23 Field of View diameter in mm Foc Focusable

41. Eyepiece Reticles Useful for: • Centering Stage (Pol) • Counting • Measuring distances, circles • Discussions (movable pointers) • Setting of Parfocality

42. Setting your microscope to be “parfocal” Required: Two focusing eyepieces and/or focusing camera adapter

43. Questions? Short break?

44. 0 Units 50 Units 100 Units C ONTRAST 50 50 50 Units 50 – 100 / 50 + 100 = -0.33 50 – 0 / 50 + 0 = 1 50 – 50 / 50 + 50 = 0

45. Contrasting Techniques Examples Brightfield Phase Fluorescence DIC

46. objective specimen condenser Brightfield • For stained or naturally absorbing samples • True Color Representation • Proper Technique for Measurements • Spectral • Dimensional • Koehler Illumination !

47. “Koehler” Illumination (since 1893) Prof. August Köhler: 1866 - 1948 • Provides for most homogenous Illumination • Highest obtainable Resolution • Minimizes Straylight and unnecessary Iradiation • Allows adjustment of optimal Contrast • Defines desired Depth of Field • Helps in focusing difficult-to-find structures • Establishes proper position for condenser elements, for all contrasting techniques

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

49. 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