Systems Biology. Optical Train, Köhler Illumination

McGill University Life Sciences Complex Imaging Facility Systems Biology Microscopy Workshop Tuesday December 7 th, 2010 Simple Lenses, Transmitted Light Optical Train, Köhler Illumination

What Does a Microscope Need To Do? Three things need to be accomplished: 1. M A G N I F Y Produce a magnified image of the specimen. 2. C O N T R A S T Render the details within the specimen visible to the imaging device: e.g. eye, camera, photomultiplier tube 3. R E S O L V E Distinguish between objects or features within the specimen. All three things need to be accomplished.

Magnification and Contrast but Low Resolution http://www.invitrogen.com/site/us/en/home/support/research-tools/image-gallery.html

Magnification and Resolution but Low Contrast http://www.invitrogen.com/site/us/en/home/support/research-tools/image-gallery.html

Contrast and Resolution - but Low Magnification http://www.invitrogen.com/site/us/en/home/support/research-tools/image-gallery.html

Magnification, Contrast, Resolution http://www.invitrogen.com/site/us/en/home/support/research-tools/image-gallery.html

Magnification Magnify: Produce a magnified image of the specimen. Magnification = Mag. Objective lens x Mag. Eyepiece Lens 20x lens X 10x eyepiece = 200x magnification 60x lens X 10x eyepiece = 600x magnification http://media.thestar.topscms.com/images/3a/ 7d/fe044496470cbcd585a2307b6ab0.jpeg http://www5.pbrc.hawaii.edu/microangela/mhflyo.jpg http://www.astrographics.com/ GalleryPrints/Display/GP2129.jpg

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

Simple Biconvex Lens Focal Points: Rear Focal Point (F ): Point where light coming from an infinite it distance is focused to a fine point. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens Focal Points: Rear Focal Point (F ): Point where light coming from an infinite it distance is focused to a fine point. Front Focal Point (F ): Point where light rays collected from a single point source spread out into parallel l rays. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens Focal Points: Rear Focal Point (F ): Point where light coming from an infinite it distance is focused to a fine point. Front Focal Point (F): Point where light rays collected from a single point source spread out into parallel l rays. Optical Axis: Imaginary line through center of lens and focal points. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens Focal Points: Rear Focal Point (F ): Point where light coming from an infinite it distance is focused to a fine point. Front Focal Point (F ): Point where light rays collected from a single point source spread out into parallel l 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. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens Focal Planes (f and f ): planes that are one focal length away from either side of the lens. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens Focal Planes (f and f ): planes that are one focal length away from either side of the lens. 2F Points: points along the optical axis that lie at 2x the focal length. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens Focal Planes (f and f ): planes that are one focal length away from either side of the lens. 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 lens. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens If an object is at 2F in front of the lens then an intermediate 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 point 2. Are inverted 3. 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 Intermediate Image Intermediate Image Plane

Simple Biconvex Lens When an object is an infinite distance from the lens, a tiny real image is formed at the rear focal point (F ). Used in portrait photography. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Magnification and Lenses Object at Infinity Scenic Photography Ray Diagram Real image formed at focal plane. Focal Point http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens When an object is moved closer to 2F, the real image forms at the intermediate image plane behind F. The image is smaller than the object. Typically used in photography. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens When the object lies in the 2F Plane, the real image is formed at the 2F Plane behind the lens. The object and the image are the same size. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens When the object lies between F and 2F, the real image is formed behind 2F. The image is larger than the object. Used for image and film projection. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens What happens when the object is placed at the front focal plane? Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens What happens when the object is placed at the front focal plane? No real image can be formed behind the lens since light rays from every point in the object plane leave the lens in parallel. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Magnification and Lenses Ray Diagram No Image Formed Object at the front focal plane Front Focal Point Infinity Space http://www.olympusmicro.com/primer/anatomy/magnification.html

Simple Biconvex Lens What happens when the object is placed closer than the focal point? A virtual image is perceived behind the object when looking through the lens. Virtual Images: 1. Image is upright 2. Image can t be projected onto a surface, film or digital detectors. 3. Image appears on same side of the lens as the object. Modified by Aleks Spurmanis from http://www.olympusmicro.com/primer/anatomy/magnification.html

Magnifying Glass or a Mirror http://www.olympusmicro.com/primer/anatomy/magnification.html

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

Finite vs Infinity Corrected Optics Davidson M.W and Abramowitz M., Optical Microscopy, review article, Olympus America Inc.

Infinity Corrected Optics 1. 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. Eliminate i "ghost images" that t 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 Formation Ernst Karl Abbe (1840-1905) A 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. He worked with Carl Zeiss to develop early scientific research microscopes. He derived the formula for the theoretical resolution of the microscope. Depends on the wavelength of light, numerical aperture of the lens, refractive index of the imaging medium.

Interference & Diffraction Patterns Destructive Interference Slit or aperture No Interference. Modified by Claire Brown, McGill Imaging Facility using http://en.wikipedia.org/wiki/diffraction Constructive Interference http://micro.magnet.fsu.edu/primer/java/imageformation/airydiskbasics/index.html

Interference & Diffraction Patterns Destructive Interference 1 st order Defracted Constructive Interference http://micro.magnet.fsu.edu/primer/lightandcolor/diffractionintro.html Zero Order not Defracted 2 nd order Defracted Constructive Interference

Imaging a Line Grating Diffraction pattern depends ds on colour ou of light. 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. 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 Grating Diffraction Pattern depends on line spacing. Closer lines are together more diffraction. http://www.olympusmicro.com/primer/java/imageformation/gratingdiffraction/index.html Diffraction Grating Tutorial

Imaging a Line Grating Diffraction Pattern depends on line spacing. Closer lines are together more diffraction. http://www.olympusmicro.com/primer/java/imageformation/diffractionpaths/index.html Diffraction Paths Tutorial

Imaging a Line Grating No Grating 10x Lens 40x Lens 60x Lens Diffraction seen at the back aperture of the objective lens. http://www.olympusmicro.com/primer/anatomy/image.html

Imaging Complex Patterns Vertical Lines Horizontal Lines http://www.olympusmicro.com/primer/anatomy/image.html Complex Patterns Tutorial

Complex Diffraction Patterns Interference: Addition of two or more waves resulting in a new wave form. Interference of two circular waves Increasing Wavelength Interference Simulation http://en.wikipedia.org/wiki/interference Increasing distance between wave centers.

Microscope Image Diffraction patterns from light interacting with all aspect of the specimen. Claire Brown, McGill Imaging Facility Large Features Small Features Random Features Ordered Features

Upright Microscope Illumination system below the specimen 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 Pathway Optical Train 1) Light from the halogen lamp is focused by collector lenses and sent to the condenser. http://micro.magnet.fsu.edu/primer/lightandcolor/lensesintro.html

Upright Transmitted Light Pathway Optical Train 1) Light from the halogen lamp is focused by collector lenses and sent to the condenser. 2) Diffusers and filters are in the light path to ensure even illumination of the field of view and to select or block certain wavelengths of light. http://micro.magnet.fsu.edu/primer/lightandcolor/lensesintro.html

Upright Transmitted Light Pathway Optical Train 1) Light from the halogen lamp is focused by collector lenses and sent to the condenser. 2) Diffusers and filters are in the light path to ensure even illumination of the field of 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 lamp and to collimate the light. http://micro.magnet.fsu.edu/primer/lightandcolor/lensesintro.html

Upright Transmitted Light Pathway Optical Train 4) The condenser is attached to the microscope by the condenser carrier it provides focused even illumination across the field of view for a wide range of magnifications. 5) The specimen is placed on the microscope stage. http://micro.magnet.fsu.edu/primer/lightandcolor/lensesintro.html

Upright Transmitted Light Pathway Optical Train 4) The condenser is attached to the microscope by the condenser carrier it provides focused even illumination across the field of view for a wide range of magnifications. 5) The specimen is placed on the microscope stage. 6) The objective lenses are held in place by the nosepiece which can be turned putting different lenses in place. http://micro.magnet.fsu.edu/primer/lightandcolor/lensesintro.html

Upright Transmitted Light Pathway Optical Train 7) The eyepieces (10x) magnify a virtual it limage of fthe sample that t generates a real image on the detector. http://micro.magnet.fsu.edu/primer/lightandcolor/lensesintro.html

Upright Transmitted Light Pathway Optical Train 7) The eyepieces (10x) magnify a virtual it limage of fthe sample that t generates a real image on the detector. 8) The light can be redirected with a beam splitter to a detector, such as a CCD camera. http://micro.magnet.fsu.edu/primer/lightandcolor/lensesintro.html

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 Train 1) Light from the halogen lamp is focused by collector lenses and sent to the condenser. Modified by Aleks Spurmanis from http://zeiss-campus.magnet.fsu.edu/tutorials/axioobserver/index.html

Inverted Microscope Optical Train 1) Light from the halogen lamp is focused by collector lenses and sent to the condenser. 2) Diffusers and filters are in the light path to ensure even illumination of the field of 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 lamp and to collimate the light. Modified by Aleks Spurmanis from http://zeiss-campus.magnet.fsu.edu/tutorials/axioobserver/index.html

Inverted Microscope Optical Train 4) The condenser is attached to the microscope by the condenser carrier it provides focused even illumination across the field of view for a wide range of magnifications. 5) The specimen is placed on the microscope stage. 6) The objective lenses are held in place by the nosepiece which can be turned putting different lenses in place. Modified by Aleks Spurmanis from http://zeiss-campus.magnet.fsu.edu/tutorials/axioobserver/index.html

Inverted Microscope Optical Train 7) The eyepieces (10x) magnify a virtual image of the sample that generates a real image on the detector. Modified by Aleks Spurmanis from http://zeiss-campus.magnet.fsu.edu/tutorials/axioobserver/index.html

Inverted Microscope Optical Train 7) The eyepieces (10x) magnify 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 CCD camera. Modified by Aleks Spurmanis from http://zeiss-campus.magnet.fsu.edu/tutorials/axioobserver/index.html

Inverted Microscope Optical Train 7) The eyepieces (10x) magnify 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 CCD camera. 9) Camera front port. Modified by Aleks Spurmanis from http://zeiss-campus.magnet.fsu.edu/tutorials/axioobserver/index.html

Inverted Microscope Optical Train 7) The eyepieces (10x) magnify 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 CCD camera. 9) Camera front port. 10) Camera side port. Tutorial Modified by Aleks Spurmanis from http://zeiss-campus.magnet.fsu.edu/tutorials/axioobserver/index.html

Köhler Illumination August Köhler (1866-1948): A German professor who first described d a new way to illuminate microscope samples in 1893. He later went on to work for Carl Zeiss in Germany as a Physicist for 45 years. Köhler Illumination: Used to create even illumination over the specimen field of view without having an image of the light source in the field of view.

Conjugate Image Planes Conjugate 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 image in conjugate planes. Therefore, the filament image is no longer conjugate to the image plane, and is no longer visible.

Conjugate IMAGE Planes Conjugate Image Planes: There are four IMAGE planes throughout h t the optical train of the microscope that are all simultaneously in focus. 4. Retina/Detector 3. Intermediate Image 2. Specimen Plane 1. Field Stop Diaphragm Claire Brown, McGill Imaging Facility Eye Iris Diaphragm Eyepiece Lens Back Aperture of Eyepiece Back FP of Objective Objective Lens Stage Condenser Lens Front FP of Condenser Collector Lens Lamp

Conjugate IMAGE Planes http://www.microscopyu.com/articles/formulas/formulasconjugate.html

Conjugate APERTURE Planes Conjugate Aperture Planes: There are four aperture planes throughout the optical train of the microscope that are all simultaneously in focus. 4. Pupil of the Eye Eye Back Aperture of Eyepiece 3. Back FP of Objective 2. Front FP of Condenser Back FP of Objective Objective Lens Stage Condenser Lens Front FP of Condenser 1. Lamp Filament Lamp Collector Lens Claire Brown, McGill Imaging Facility

Conjugate APERTURE Planes http://www.microscopyu.com/articles/formulas/formulasconjugate.html

IMAGE and APERTURE Planes are Interlaced 4. Retina/Detector 4. Pupil of the Eye 3. Intermediate Image 3. Back FP of Objective 2. Specimen Plane 2. Front FP of Condenser 1. Field Stop Diaphragm 1. Lamp Filament Claire Brown, McGill Imaging Facility

Image Planes Image Planes 1. Field Diaphragm 2. Specimen 3. Intermediate Image 4. Detection Plane (Eye, CCD Camera) http://www.microscopyu.com/articles/formulas/formulasconjugate.html

Aperture Planes Aperture planes 1. Lamp Filament 2. Condenser Aperture 3. Objective Back Focal Plane 4. Pupil of the Eye http://www.microscopyu.com/articles/formulas/formulasconjugate.html

Image and Aperture Planes Filament is unfocused where image is focused. http://www.microscopyu.com/articles/formulas/formulasconjugate.html

Image and Aperture Planes Image is unfocused where filament is focused. http://www.microscopyu.com/articles/formulas/formulasconjugate.html

Conjugate Planes Three simultaneously focused conjugate image planes Tutorial http://www.microscopyu.com/articles/formulas/formulasconjugate.html

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

1. Light is focused below the specimen. 2. The field diaphragm is not in a conjugate image plane with the specimen. 3. Illumination is weak. 4. Diffraction artifacts compromise contrast and resolution. Condenser Too Low Modified by Aleks Spurmanis from the Molecular Expressions Web Page

Condenser Too High 1. Light is focused above the specimen. 2. The field diaphragm is no longer in a conjugate image plane with the specimen. 3. Illumination is weak. 4. Diffraction artifacts compromise contrast and resolution. Modified by Aleks Spurmanis from the Molecular Expressions Web Page

Köhler Illumination Alignment 1. Focus on the specimen. McGill Imaging Facility

Köhler Illumination Alignment 1. Focus on the specimen. 2. Close down the field diaphragm. McGill Imaging Facility

Köhler Illumination Alignment 1. Focus on the specimen. 2. Close down the field diaphragm. 3. Adjust the condenser height until the field diaphragm is in focus. McGill Imaging Facility

Köhler Illumination Alignment 1. Focus on the specimen. 2. Close down the field diaphragm. 3. Adjust the condenser height until the field diaphragm is in focus. 4. Center the condenser. McGill Imaging Facility

Condenser Not Centered 1. Illumination is weak AND non-uniform. 2. Reduces contrast and resolution. 3. Can interfere with image analysis. Modified by Aleks Spurmanis from the Molecular Expressions Web Page

Köhler Illumination Alignment Best contrast when condenser is set to ~80% of the size of the objective back aperture. Modified by Aleks Spurmanis from the Molecular Expressions Web Page

Köhler Illumination Alignment Aperture set too small. Reduced resolution. Image too dark. Diffraction artifacts. Modified by Aleks Spurmanis from the Molecular Expressions Web Page

Köhler Illumination Alignment Aperture set too large. Reduced resolution. Too much stray light. Low contrast. Modified by Aleks Spurmanis from the Molecular Expressions Web Page

Microscope Alignment Köhler Aligned Microscope Mis-Aligned Microscope Claire Brown, McGill Imaging Facility

The Lenses, Transmitted Light Optical Train and Köhler Alignment Talk was Clearly Presented 1. Strongly gydisagree 2. Disagree 3. Neutral 4. Agree 5. Strongly Agree

The Lenses, Transmitted Light Optical Train and Köhler Alignment Talk was Relevant to My Work 1. Strongly Disagree 2. Disagree 3. Neutral 4. Agree 5. Strongly Agree

The Lenses, Transmitted Light Optical Train and Köhler Alignment Talk was: 1. Too Short 2. Too Long 3. A Good Length