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

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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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