Presentation is loading. Please wait.

Presentation is loading. Please wait.

III Components, Microscope Setup December 2008 Rudi Rottenfusser – Carl Zeiss MicroImaging.

Similar presentations


Presentation on theme: "III Components, Microscope Setup December 2008 Rudi Rottenfusser – Carl Zeiss MicroImaging."— Presentation transcript:

1 III Components, Microscope Setup December 2008 Rudi Rottenfusser – Carl Zeiss MicroImaging

2 Categories 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) 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

3 Topics I.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 II.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 1800s to mid 1900s) Perfect even illumination Perfect Color Temperature (Daylight) Evenings? Nights? Intensity? Light Source

7 Artificial Light Sources (incoherent) Tungsten Tungsten Halogen

8 Tungsten – Halogen Principle

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

10 Tungsten Tungsten Halogen Mercury Arc Xenon Arc Arc Lamps

11

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

13 Tungsten Tungsten Halogen Mercury Arc Xenon Arc LEDs Light Sources

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

16 Colibri Fast Switching FL Source

17 Tungsten Tungsten Halogen Mercury Arc Xenon Arc LEDs Laser (coherent) Light Sources

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 Components between Light Source and Specimen Internal Light Path incorporates: 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 Specimen Resolution (minimum resolved distance between two points): Without Condenser: With Condenser: When Condenser NA matches Objective NA Highest Resolution ! Objective Condenser Specimen

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

25 Numerical Aperture (NA) c b a 90° Refractive Index n air = 1 n water = 1.33 n glycerin = 1.47 n oil = n NA = sin · n How is a sine function defined ?

26 Why immersion medium affects NA 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°) No immersion (dry) Max. Objective aperture 0.95 ( = 72°) Captured Aperture of specimen below cover slip: 0.95/1.52 = 0.62 ( 1 = 39°) 1)Objective 2)Cover Slip + slide 3)Immersion Oil No Oil Dry Oil Immersion Plan-Apochromat 40x/0.95 corr Plan-Apochromat 100x/1.46 Oil

27 Cross-section through an corrected Microscope

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

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 a)Primostar b)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 c)Axioskop 1, Axioskop 40, Axioskop 2FS, Axioplan, Axiophot Forward and Backward Compatibility between c) and d) via tube adapters! d)Axioplan 2, 2i, 2ie e)AxioImager A1, D1, M1, Z1 f)Stereo Microscopes SV6, SV11, SR, SV8 g)SteReo Discovery, Lumar

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

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 Who needs eye- pieces any more? Eyepieces (Oculars) 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. Total Magnification of an image to the eyes should be between 500 and 1000 times the objectives Numerical Aperture Rule of Thumb:

39 1939

40 Eyepiece Characteristics Example: W PL 10x/23 Foc. W Wide Angle PL Flat Field (Plan > old style: KPL or CPL) 10xMagnification 23Field 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 1 Go to highest magnification possible with your system 2Focus carefully via focusing knobs 3 Go to the lowest magnification possible; leave focus alone 4 Refocus system with your two focusing eyepieces (or camera focusing adjustment)

43 Questions? Short break?

44 C ONTRAST 50 – 0 / = 1 50 – 100 / = – 50 / = 0 50 Units0 Units100 Units 50 Units 50

45 Examples Brightfield DIC Fluorescence Phase Contrasting Techniques

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

47 Koehler Illumination (since 1893) Prof. August Köhler: 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) 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

50

51 Image Planes Aperture Planes Conjugate Planes in the Upright Microscope

52 1 Intermediate image plane (photo tube) 2 Eyepiece (Intermediate Image inside) 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 (Arc) 14 HAL Illumination (Filament) 15 Field stop (Transmitted light = TL) 16 Polarizer 17 Aperture diaphragm (TL) 18 Condenser 19 Objective (Back Focal Plane inside) Conjugate Planes in the Inverted Microscope

53 1)Open Field and Condenser Diaphragms 2)Focus specimen 3)Correct for proper Color Temperature 4)Close Field Diaphragm 5)Focus Field Diaphragm – move condenser up and down 6)Center Field Diaphragm 7)Open to fill view 8)Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular 9)Close Condenser Diaphragm to fill approx. 2/3 of Objectives Aperture 10)Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution) Koehler Illumination Steps:

54 1)Open Field and Condenser Diaphragms 2)Focus specimen 3)Correct for proper Color Temperature 4)Close Field Diaphragm 5)Focus Field Diaphragm – move condenser up and down 6)Center Field Diaphragm 7)Open to fill view 8)Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular 9)Close Condenser Diaphragm to fill approx. 2/3 of Objectives Aperture 10)Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

55 1)Open Field and Condenser Diaphragms 2)Focus specimen 3)Correct for proper Color Temperature 4)Close Field Diaphragm 5)Focus Field Diaphragm – move condenser up and down 6)Center Field Diaphragm 7)Open to fill view 8)Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular 9)Close Condenser Diaphragm to fill approx. 2/3 of Objectives Aperture 10)Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

56 1)Open Field and Condenser Diaphragms 2)Focus specimen 3)Correct for proper Color Temperature 4)Close Field Diaphragm 5)Focus Field Diaphragm by moving condenser up or down 1)Center Field Diaphragm 2)Open to fill view 3)Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular 4)Close Condenser Diaphragm to fill approx. 2/3 of Objectives Aperture 5)Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

57 1)Open Field and Condenser Diaphragms 2)Focus specimen 3)Correct for proper Color Temperature 4)Close Field Diaphragm 5)Focus Field Stop by moving condenser up or down 6)Center Field Diaphragm 7)Open to fill view 8)Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular 9)Close Condenser Diaphragm to fill approx. 2/3 of Objectives Aperture 10)Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

58 1)Open Field and Condenser Diaphragms 2)Focus specimen 3)Correct for proper Color Temperature 4)Close Field Diaphragm 5)Focus Field Diaphragm – move condenser up and down 6)Center Field Diaphragm 7)Open to fill view of observer 8)Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular 9)Close Condenser Diaphragm to fill approx. 2/3 of Objectives Aperture 10)Enjoy Image (changing Condenser Diaphragm alters Contrast / Resolution)

59 1)Open Field and Condenser Diaphragms 2)Focus specimen 3)Correct for proper Color Temperature 4)Close Field Diaphragm 5)Focus Field Diaphragm – move condenser up and down 6)Center Field Diaphragm 7)Open to fill view 8)Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular 9)Close Condenser Diaphragm to fill approx. 2/3 of Objectives Aperture BFP Better: Depending on specimens inherent contrast, close condenser aperture to: ~ x NA objective

60 Koehler Steps: 1)Open Field and Condenser Diaphragms 2)Focus specimen 3)Correct for proper Color Temperature 4)Close Field Diaphragm 5)Focus Field Diaphragm – move condenser up and down 6)Center Field Diaphragm 7)Open to fill view 8)Observe Objectives Back Focal Plane via Ph Telescope or by removing Ocular 9)Close Condenser Diaphragm to fill approx. 2/3 of Objectives Aperture 10)Observe Image ! Done !

61 Lets Review the steps to achieve Koehler Illumination… This is how to optimize contrast by Koehler Illumination

62 Koehler Illumination Steps: 1.Turn light on; open field and condenser diaphragms 2.Focus specimen 3.Consider neutral background (set rheostat to 3200K, use neutral density filters for comfort) 4.Close field diaphragm 5.Focus field diaphragm – move condenser up or down 6.Center field diaphragm 7.Open to fill view 8.Observe objectives back focal plane via Ph telescope or by removing ocular 9.Close condenser diaphragm to fill approx. 3/4 of objectives aperture 10.Done!


Download ppt "III Components, Microscope Setup December 2008 Rudi Rottenfusser – Carl Zeiss MicroImaging."

Similar presentations


Ads by Google