Systems Biology Microscopy Workshop Si l LT itt d Li ht Simple Lenses, Transmitted Light Optical

McGill University Life SciencesMcGill University Life SciencesComplex Imaging Facility

Systems BiologyMicroscopy WorkshopMicroscopy Workshop

Tuesday December 7th, 2010

Si l L T itt d Li htSimple Lenses, Transmitted Light Optical Train, Köhler Illuminationp ,

What Does a Microscope Need To Do?

Three things need to be accomplished:

1. M A G N I F YProduce a magnified image of the specimen.g g p

2. C O N T R A S TRender the details within the specimen visible to the imagingRender the details within the specimen visible to the imaging device: e.g. eye, camera, photomultiplier tube

3. R E S O L V EDistinguish between objects or features within the specimen.

All three things need to be accomplished.

Magnification and Contrast – but Low ResolutionLow Resolution

http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Image-Gallery.html

Magnification and Resolutionbut Low Contrast– but Low Contrast


Contrast and Resolution but Low Magnification- but Low Magnification


Magnification, Contrast, Resolution


MagnificationMagnify: Produce a magnified image of the specimen.

Magnification = Mag Objective lens x Mag Eyepiece LensMagnification = Mag. Objective lens x Mag. Eyepiece Lens20x lens X 10x eyepiece = 200x magnification60x lens X 10x eyepiece = 600x magnification60x lens X 10x eyepiece = 600x magnification

http://media.thestar.topscms.com/images/3a/7d/fe044496470cbcd585a2307b6ab0.jpeg

http://www.astrographics.com/GalleryPrints/Display/GP2129.jpghttp://www5.pbrc.hawaii.edu/microangela/mhflyo.jpg

Simple Biconvex Lens

Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html


Focal Points: R F l P i t (F’) P i t h li ht i f i fi itRear Focal Point (F’): Point where light coming from an infinite distance is focused to a fine point.



Focal Points: R F l P i t (F’) P i t h li ht i f i fi itRear Focal Point (F’): Point where light coming from an infinite distance is focused to a fine point.Front Focal Point (F’): Point where light rays collected from a i l i t d t i t ll lsingle point source spread out into parallel rays.



Focal Points: R F l P i t (F’) P i t h li ht i f i fi itRear Focal Point (F’): Point where light coming from an infinite distance is focused to a fine point.Front Focal Point (F): Point where light rays collected from a single

i t d t i t ll lpoint source spread out into parallel rays.Optical Axis: Imaginary line through center of lens and focal points.



Focal Points: R F l P i t (F’) P i t h li ht i f i fi itRear Focal Point (F’): Point where light coming from an infinite distance is focused to a fine point.Front Focal Point (F’): Point where light rays collected from a i l i t d t i t ll lsingle point source spread out into parallel rays.

Optical Axis: Imaginary line through center of lens and focal points.Focal Length: Distance along the optical axis between the center of the

lens and either focal point.



Focal Planes (f and f’): planes that are one focal length away from either side of the lenseither side of the lens.




2F Points: points along the optical axis that lie at 2x the focal length.




2F Points: points along the optical axis that lie at 2x the focal length.2f Planes: Planes that are 2 focal lengths away from either side of the

llens.



If an object is at 2F in front of the lens then an intermediate image is formed at 2F’ behind the lens and itintermediate image is formed at 2F’ behind the lens and it is a real image. This image is formed at the intermediate image plane.

Real Images:1. Need to cross through a focal point2 Are inverted

IntermediateImage

Intermediate2. Are inverted3. Can be projected onto a surface, captured on film or

a digital detector. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

IntermediateImagePlane


• When an object is an infinite distance from the lens, aWhen an object is an infinite distance from the lens, atiny real image is formed at the rear focal point (F’).

• Used in portrait photography.


Magnification and LensesObject at Infinity – Scenic Photography

Ray Diagram

Object at Infinity Scenic Photography

Real image formed at focal plane.

Focal Point

http://www.olympusmicro.com/primer/anatomy/magnification.html


• When an object is moved closer to 2F, the real imageforms at the intermediate image plane behind F’.• The image is smaller than the object.• Typically used in photography.



• When the object lies in the 2F Plane, the real image isformed at the 2F’ Plane behind the lensformed at the 2F’ Plane behind the lens.

• The object and the image are the same size.



• When the object lies between F and 2F, the real imageis formed behind 2F’.is formed behind 2F .

• The image is larger than the object.• Used for image and film projection.



What happens when the object is placed at the front focal plane?



What happens when the object is placed at the front focal plane?

No real image can be formed behind the lens since light rays fromevery point in the object plane leave the lens in parallel.


Magnification and LensesObject at the front focal plane

Ray Diagram

Object at the front focal plane

No Image Formed

Front Focal Point

Infinity Space



What happens when the object is placed closer than the focal point?

A virtual image is perceived behind the object when looking throughthe lens.

Virtual Images:1. Image is upright2. Image can’t be projected onto a surface, film or digital detectors.3. Image appears on same side of the lens as the object.


Magnifying Glass or a Mirror


Two Lens Systems

Modern microscopes use a two lens system with the objective lens being one and the tube lens being the second

http://micro.magnet.fsu.edu/primer/java/components/twolenssystem/index.html

being one and the tube lens being the second.

Finite vs Infinity Corrected Optics

Davidson M.W and Abramowitz M., Optical Microscopy, review article, Olympus America Inc.

Infinity Corrected Optics1. Can add in additional optics between the objective lens and the tube lens.

2. Can focus by moving the objective and not the specimen (stage).

3 Eli i t " h t i " th t b d b fl ti f th3. Eliminate "ghost images" that can be caused by reflections from the surfaces of lenses in finite systems.

http://micro.magnet.fsu.edu/primer/images/objectives/infinitypathways.jpg

Abbe’s Theory of Image FormationErnst Karl Abbe (1840-1905)

A German physicist who created the firstA German physicist who created the first mathematical formulation for microscope design. Prior to his work microscopes components and design was done primarily by trial and error.design was done primarily by trial and error.

He worked with Carl Zeiss to develop early scientific research microscopes.p

He derived the formula for the theoretical resolution of the microscope.

Depends on the wavelength of light, numerical aperture of the plens, refractive index of the imaging medium.

Interference & Diffraction Patterns

Destructive Interference

Slit ort

No Interference

aperture

No Interference.

Constructive Interferencehttp://micro.magnet.fsu.edu/primer/java/imageformation/airydiskbasics/index.html

Modified by Claire Brown, McGill Imaging Facility using http://en.wikipedia.org/wiki/Diffraction

Interference & Diffraction Patterns

DestructiveInterference

1st orderDefracted

2nd orderDefracted

http://micro.magnet.fsu.edu/primer/lightandcolor/diffractionintro.html

Zero Order not Defracted

DefractedConstructiveInterference

DefractedConstructiveInterference

Imaging a Line GratingDiffraction pattern depends on colour of light.act o patte depe ds o co ou o g t

Larger spacing with longer wavelength.

Red light is diffracted more than blue light

http://www.olympusmicro.com/primer/java/imageformation/gratingdiffraction/index.html

White Light Diffraction•• See a spectrum of light when white light is diffracted by a grid

pattern.

• Blue less diffraction than red.Blue less diffraction than red.

• Higher order diffraction is due to smaller features in the image.

• Smaller features cause a bigger difference in the amount of diffraction between the different colours.

Increasing Diffraction

http://www.olympusmicro.com/primer/anatomy/image.html

Imaging a Line GratingDiffraction Pattern depends on line spacing.

Closer lines are together more diffraction.

http://www.olympusmicro.com/primer/java/imageformation/gratingdiffraction/index.htmlDiffraction Grating Tutorial

Imaging a Line GratingDiffraction Pattern depends on line spacing.

Closer lines are together more diffraction.g

http://www.olympusmicro.com/primer/java/imageformation/diffractionpaths/index.htmlDiffraction Paths Tutorial

Imaging a Line Grating

N G ti 10 L 40 L 60 LNo Grating 10x Lens 40x Lens 60x Lens

Diffraction seen at the back aperture of the objective lens.


Imaging Complex Patterns

Vertical Lines Horizontal LinesHorizontal Lines


Complex Patterns Tutorial

Complex Diffraction PatternsInterference of two circular waves

Interference: Addition of two or more waves resulting in a new wave form.

IncreasingIncreasingWavelength Interference

Simulation

Increasing distance between wave centers.http://en.wikipedia.org/wiki/Interference

Microscope Image

Diffraction patterns from light interacting with all aspect of the specimen.

Large Features

Small Features

Random Features

Ordered FeaturesClaire Brown, McGill Imaging Facility

Upright Microscope

Illumination system below the ispecimen

Objective lenses above the specimen

Fixed location of objectives stage moves.

Good for tissue or small organism imaging with water dipping lenses.

http://micro.magnet.fsu.edu/primer/anatomy/components/componentsfigure1.jpg

Upright Transmitted Light PathwayOptical TrainOptical Train

1) Light from the halogen lamp is f d b ll t l dfocused by collector lenses and sent to the condenser.

http://micro.magnet.fsu.edu/primer/lightandcolor/lensesintro.html



2) Diffusers and filters are in the2) Diffusers and filters are in the light path to ensure even illumination of the field of view and to select or block certainand to select or block certain wavelengths of light.




2) Diffusers and filters are in the2) Diffusers and filters are in the light path to ensure even illumination of the field of view and to select or block certainand to select or block certain wavelengths of light.

3) The field diaphragm is used to ) p gselect only the central portion of light from the lamp and to collimate the light.



4) The condenser is attached to th i b ththe microscope by the condenser carrier it provides focused even illumination across the field of view for aacross the field of view for a wide range of magnifications.




5) The specimen is placed on the5) The specimen is placed on the microscope stage.




5) The specimen is placed on the5) The specimen is placed on the microscope stage.

6) The objective lenses are held in ) jplace by the nosepiece which can be turned putting different lenses in place.



7) The eyepieces (10x) magnify a i t l i f th l th tvirtual image of the sample that

generates a real image on the detector.



7) The eyepieces (10x) magnify a i t l i f th l th tvirtual image of the sample that

generates a real image on the detector.

8) The light can be redirected with a beam splitter to a detector, such as a CCD camerasuch as a CCD camera.


Inverted Transmitted Light Pathway

Illumination system above the specimen.

Objective lenses below the specimen.

Fixed location of stage and objectives move.

Good for living cells that need environmental control.

Modified by Aleks Spurmanis from http://zeiss-campus.magnet.fsu.edu/tutorials/axioobserver/index.html

Inverted Microscope Optical Train1) Light from the halogen lamp

is focused by collector lenses and sent to the condenser.


Inverted Microscope Optical Train1) Light from the halogen lamp

is focused by collector lenses and sent to the condenser.

2) Diff d filt i2) Diffusers and filters are in the light path to ensure even illumination of the field of view and to select orof view and to select or block certain wavelengths of light.

3) The field diaphragm is used to select only the central portion of light from the


p glamp and to collimate the light.

Inverted Microscope Optical Train4) The condenser is attached

to the microscope by the d i itcondenser carrier it

provides focused even illumination across the field of view for a wide range ofof view for a wide range of magnifications.


Inverted Microscope Optical Train4) The condenser is attached

to the microscope by the d i itcondenser carrier it

provides focused even illumination across the field of view for a wide range ofof view for a wide range of magnifications.

5) The specimen is placed on5) The specimen is placed on the microscope stage.

6) The objective lenses are ) jheld in place by the nosepiece which can be turned putting different


lenses in place.

Inverted Microscope Optical Train

7) The eyepieces (10x) if i t l i fmagnify a virtual image of

the sample that generates a real image on the detector.





8) The light can be redirected with a beam splitter to a detector such as a CCDdetector, such as a CCD camera.






9) Camera front port.) p






9) Camera front port.) p

10) Camera side port.


Tutorial

Köhler Illumination

August Köhler (1866-1948): A German f h fi t d ib dprofessor who first described a new

way to illuminate microscope samples in 1893. He later went on to work for Carl Zeiss in Germany as a PhysicistCarl Zeiss in Germany as a Physicist for 45 years.

Köhl Ill i ti U d t tKöhler Illumination: Used to create even illumination over the specimen field of view without having an image of the light source in the field of viewthe light source in the field of view.

Conjugate Image PlanesConjugate Image Planes: A set of image planes that are optically linked and whose images are superimposed.

A collector lens focuses the light source at the back focal plane of the objective putting the front focal plane of the condenser and the filament i i j t l Th f th fil t i i limage in conjugate planes. Therefore, the filament image is no longer conjugate to the image plane, and is no longer visible.

Conjugate IMAGE PlanesC j t I Pl Th f IMAGE l th h t thConjugate Image Planes: There are four IMAGE planes throughout the optical train of the microscope that are all simultaneously in focus.

4. Retina/Detector EyeIris Diaphragm

3. Intermediate ImageBack FP of ObjectiveBack Aperture of EyepieceEyepiece Lens

2. Specimen PlaneCondenser Lens

Objective LensStage

Back FP of Objective

1. Field Stop Diaphragm

Front FP of Condenser

Lamp

Collector Lens

Claire Brown, McGill Imaging Facility

Conjugate IMAGE Planes

http://www.microscopyu.com/articles/formulas/formulasconjugate.html

Conjugate APERTURE PlanesConjugate Aperture Planes: There are four aperture planes throughout the optical train of the microscope that are all simultaneously in focus.

E4. Pupil of the Eye Eye

Back Aperture of Eyepiece

3. Back FP of ObjectiveObjective LensBack FP of Objective

Back Aperture of Eyepiece

2. Front FP of CondenserCondenser Lens

jStage

Front FP of Condenser

Collector Lens

1. Lamp Filament Lamp


Conjugate APERTURE Planes


IMAGE and APERTUREPlanes are InterlacedPlanes are Interlaced

4. Retina/Detector

4. Pupil of the Eye

3. Intermediate Image3. Back FP of Objective

2. Specimen Plane

2. Front FP of Condenser

1. Field Stop Diaphragm

1. Lamp Filament


Image PlanesImage Planes

1. Field Diaphragmp g2. Specimen3. Intermediate Image4 Detection Plane4. Detection Plane

(Eye, CCD Camera)


Aperture PlanesAperture planes

1. Lamp Filamentp2. Condenser Aperture3. Objective Back Focal Plane4 Pupil of the Eye4. Pupil of the Eye


Image and Aperture Planes

Filament is unfocused where image is focusedwhere image is focused.


Image and Aperture Planes

Image is unfocused where filament is focusedfilament is focused.


Conjugate Planes

Three simultaneously focused conjugate image planes


Tutorial

Köhler Illumination Alignment

1. Image forming light rays cross over at specimen and field diaphragm.

2. Optimal illumination enters the objective and is focused at the specimen.

Modified by Aleks Spurmanis from the Molecular Expressions Web Page

Condenser Too Low

1. Light is focused below the specimen.

2. The field diaphragm is not in a conjugatenot in a conjugate image plane with the specimen.

3. Illumination is weak.

4. Diffraction artifacts compromise contrast and resolutionand resolution.


Condenser Too High

1. Light is focused above the specimenspecimen.

2. The field diaphragm is no p glonger in a conjugate image plane with the specimen.

3. Illumination is weak.

4. Diffraction artifacts compromise contrast and resolution.



1. Focus on the specimen.

McGill Imaging Facility


1. Focus on the specimen.2. Close down the field diaphragm.



1. Focus on the specimen.2. Close down the field diaphragm.3 Adjust the condenser height until3. Adjust the condenser height until

the field diaphragm is in focus.




the field diaphragm is in focus.4. Center the condenser.


Condenser Not Centered

1. Illumination is weak AND non-uniform.

2. Reduces contrast and resolution.resolution.

3. Can interfere with image analysisanalysis.




the field diaphragm is in focus.4. Center the condenser.5 Fine tune the centering5. Fine tune the centering.



Best contrast when condenser is set to ~80% of the size of the objective back japerture.



• Aperture set too small.•• Reduced resolution.• Image too dark.• Diffraction artifacts.



• Aperture set too large.•• Reduced resolution.• Too much stray light.• Low contrast.


Microscope Alignment

Köhler Aligned Microscope Mis-Aligned Microscope


The Lenses, Transmitted Light Optical Train and Köhler Alignment Talk wasTrain and Köhler Alignment Talk was

Clearly Presented

1. Strongly Disagreeg y g2. Disagree3 Neutral3. Neutral4. Agree5. Strongly Agree

The Lenses, Transmitted Light Optical Train and Köhler Alignment Talk wasTrain and Köhler Alignment Talk was

Relevant to My Work

1. Strongly Disagree2. Disagree3. Neutral4. Agree5 Strongly Agree5. Strongly Agree

The Lenses, Transmitted Light Optical T i d K hl Ali T lkTrain and Köhler Alignment Talk was:

1. Too Short2. Too Long3. A Good Lengthg

Systems Biology Microscopy Workshop Si l LT itt d Li ht Simple Lenses, Transmitted Light Optical

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