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III Components, Microscope Setup December 2008. Rudi Rottenfusser – Carl Zeiss MicroImaging. Categories. 25 Nosepieces, Rings, Adapters 28 Compensators, 6x20mm-type Analyzers 30 Fluorescence Reflectors and Filter Sets 31 Other Sliders & Reflectors f/Infinity Space - PowerPoint PPT Presentation
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IIIIII
Components, Microscope SetupComponents, Microscope Setup
December 2008December 2008
Rudi Rottenfusser – Carl Zeiss MicroImaging
Categories1 Stands, Base Plates2 Stereo stands & Accessories3 Fiber Optics Light Sources4 Power Supplies5 Lamp Housings, Coll., Sockets,
Adapters6 Bulbs, Arc Lamps, Burners7 Inserts for Stands, Sliders8 Filters, 42mm & 32mm diameter9 Filters, 25mm & 18mm diameter10 Filters, others12 Condenser- and Illuminator Carriers13 Condensers, Lenses, BF, DF, Ph, Pol,
DIC14 Stage Carriers, Stages, Specimen
holders15 Scanning stages, Spec.hldrs,
Controllers16 Cooling / Heating Stages & Accessories17 Objectives 160mm 18 Objectives ICS (covered specimens)19 Objectives ICS (for non-covered
samples)23 Objectives for SteMi's, Luminars24 Objectives (Spc. e.g. Hoffmann;
McCrone)
25 Nosepieces, Rings, Adapters28 Compensators, 6x20mm-type
Analyzers30 Fluorescence Reflectors and Filter Sets31 Other Sliders & Reflectors f/Infinity
Space32 Components f/ Analyzer slider
receptacle33 Intermediate tubes & modules34 Randomizers, Tube Carriers, Tube
Mounts35 Tubes, Tube panels, Tube heads,
Access. 38 Eyepieces & Projectives - ICS & Stereo40 Eyepiece reticles, Micrometers42 Adapters for Still and Video Cameras43 Attachment Camera Systems44 Digital High Resolution Camera
Systems45 Zeiss Video Camera Systems47 Cases, Dust Covers, Cover Plates,
Cables49 Miscellaneous Micro Items
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
Topics
II. Setting up the Microscope for optimal Performance
– “Contrast”– Basic Setup for Brightfield– Koehler Illumination– Conjugate Image Planes on Microscopes
Cross-section through an ∞ corrected Microscope
Light Source for a typical Laboratory Microscope (late 1800’s to mid 1900’s)
•Perfect even illumination
•Perfect Color Temperature (“Daylight”)
• Evenings? Nights?
• Intensity?
Light Source
Artificial Light Sources (incoherent)
• Tungsten
• Tungsten Halogen
Tungsten – Halogen Principle
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
• Tungsten
• Tungsten Halogen
• Mercury Arc
• Xenon Arc
Arc Lamps
Arc Lamps
Arc Lamps
Courtesy – Michael Davidson
HBO 100
Metal Halide
XBO 75W
• Tungsten
• Tungsten Halogen
• Mercury Arc
• Xenon Arc
• LED’s
Light Sources
LED Light Sources
LED Light Sources
• Long life (10,000+h?)
• Stable Output over time
• Clean spectrum
• Cool
• No lamp alignment
• No need for shutter – no vibration
• Quick switching
Colibri Fast Switching FL Source
• Tungsten
• Tungsten Halogen
• Mercury Arc
• Xenon Arc
• LED’s
• Laser (coherent)
Light Sources
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
Cross-section through an ∞ corrected Microscope
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
Cross-section through an ∞ corrected Microscope
The Condenser
CondenserNANA
Objectivemin
λd
Specimen
Resolution (minimum resolved distance between two points):
Without Condenser:
Objectivemin
λd
NA
With Condenser:
When Condenser NA matches Objective NA Highest Resolution !
Objective
Condenser
Specimen
The Objective
Why do we need a condenser?
More details later…
Numerical Aperture (NA)
c
b
a
sincb
90°
Refractive Index nair = 1 nwater = 1.33 nglycerin = 1.47 noil = 1.518
n
NA = sin · n
How is a sine function defined ?
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°)
3
2
1
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) Objective2) Cover Slip + slide3) Immersion Oil
No Oil“Dry” Oil
Immersion
Plan-Apochromat 40x/0.95 corr. 440654-9902
Plan-Apochromat 100x/1.46 Oil 420792-8000
Cross-section through an ∞ corrected Microscope
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 !
Cross-section through an ∞ corrected Microscope
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
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
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:
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
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)
Cross-section through an ∞ corrected Microscope
Who needs eye-pieces any more?
Eyepieces (Oculars)
• Field of View
• “Presence”
• Detect fine nuances in color shades
• Stereo
• Dynamic Range of the Eye
Magnification
In order to see small objects with the eye the small objects must be magnified to an appropriate size
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 objective’s Numerical Aperture
Rule of Thumb:
1939
Eyepiece Characteristics
Example:
W PL 10x/23 Foc.
ionMagnificationMagnificat
View of Field View of Field
OptovarObjective
Eyepiece
Specimen
W Wide Angle
PL Flat Field (“Plan” > old style: “KPL” or “CPL”)
10x Magnification
23 Field of View diameter in mm
Foc Focusable
Eyepiece Reticles
Useful for:
• Centering Stage (Pol)• Counting• Measuring distances, circles• Discussions (movable pointers)• Setting of Parfocality
Setting your microscope to be “parfocal”
Required: Two focusing eyepieces and/or focusing camera adapter
1Go to highest magnification possible
withyour system
2 Focus carefully via focusing knobs
3Go to the lowest magnification
possible; leave focus alone
4Refocus system with your two focusingeyepieces (or camera focusing
adjustment)
Questions? Short break?
C ONTRAST
50 – 0
/ 50 +
0 =
1
50 – 1
00 / 5
0 +
100 =
-0.3
3
50 – 5
0 / 5
0 +
50 =
0
Background of BrightnessSpecimen of Brightness
Background of Brightness-Specimen of Brightness
50 Units0 Units 100 Units
50 Units 50 50
Examples
Brightfield
DIC Fluorescence
Phase
Contrasting Techniques
BrightfieldBrightfield• For stained or naturally absorbing samples
• True Color Representation
• Proper Technique for Measurements •Spectral•Dimensional
• Koehler Illumination !
specimen
condenser
objective
“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
Necessary components to perform “Koehler” Illumination:
• Adjustable Field Diaphragm
• Focusable and Centerable Condenser
• Adjustable Condenser Aperture Diaphragm
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
Image Planes
Aperture Planes
Conjugate Planes in the Upright Microscope
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
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 !
Let’s Review the steps to achieve Koehler Illumination…
This is how to optimize contrast by Koehler Illumination
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 objective’s back focal plane via Ph telescope or by removing ocular
9. Close condenser diaphragm to fill approx. 3/4 of objective’s aperture
10. Done!
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