Lecture 1 The Principles of Microscopy BME 695Y / BMS 634 Confocal Microscopy: Techniques and Application Module Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine & Department of Biomedical Engineering, Schools of Engineering J.Paul Robinson, Ph.D. Professor of Immunopharmacology & Biomedical Engineering Director, Purdue University Cytometry Laboratories These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these graphics. The intent is to have the student NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All material copyright J.Paul Robinson unless stated. A useful textbook for this lecture series is Jim Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively for material and ideas to support the class. http://www.cyto.purdue.edu/ UPDATED February 2004

Evaluation Overview of the Course Microscopy Basic Image Analysis Required 100% attendance at all lectures and Practicals Completion of all practical material Demonstrated competence in practical classes Complete laboratory notebook at end of class (these will be graded) Overview of the Course Microscopy Fluorescence Basic Optics Confocal Microscopes Basic Image Analysis 3D image analysis Live Cell Studies Advanced Applications

Learning Goals of this Course At the end of this course you will: Have a good background in 2 D image analysis Understand the basics of image structure Know the basics of 3D image analysis Understand the operation and function of a transmitted light microscope, fluorescence microscope and confocal microscope Learn about preparation techniques and assay systems Learn about many applications of the technologies of confocal imaging Practical Aspects Learn to use a microscope, a fluorescence microscope, a confocal microscope Learn to do basic digital imaging, image manipulation, 3D analysis Learn to use several image analysis packages

Introduction to Lecture 1 1. Introduction to Microscopy 2. History of Microscopy 3. Köhler Illumination 4. Basic optical terms 5. Light absorption 6. Magnification 7. Optical properties of microscopes 8. Components of the microscope 9. Numerical aperture, resolution and refractive index 10. Aberrations 11. Interference and optical filters

Microscopes Upright Inverted Köhler Illumination Fluorescence Illumination "Microscope" was first coined by members of the first "Academia dei Lincei" (Academy of the Lynx} scientific society which included Galileo. It was not Galileo tho’ who came up with the word, it was Johannes Faber, an entomologist and member of the same society that gave the magnifying instrument the name “microscope”

Earliest Microscopes 1590 - Hans & Zacharias Janssen of Middleburg, Holland manufactured the first compound microscopes 1590 – 1609 - Galileo – one of the earliest microscopists (naming of term “microscope” 1660 - Marcello Malpighi circa 1660, was one of the first great microscopists, considered the father embryology and early histology - observed capillaries in 1660 1665 - Robert Hooke (1635-1703)- book Micrographia, published in 1665, devised the compound microscope most famous “microscopical” observation was his study of thin slices of cork. He wrote: “. . . I could exceedingly plainly perceive it to be all perforated and porous. . . these pores, or cells, . . . were indeed the first microscopical pores I ever saw, and perhaps, that were ever seen, for I had not met with any Writer or Person, that had made any mention of them before this.”

Earliest Microscopes 1673 - Antioni van Leeuwenhoek (1632-1723) Delft, Holland, worked as a draper (a fabric merchant); he is also known to have worked as a surveyor, a wine assayer, and as a minor city official. Leeuwenhoek is incorrectly called "the inventor of the microscope" Created a “simple” microscope that could magnify to about 275x, and published drawings of microorganisms in 1683 Could reach magnifications of over 200x with simple ground lenses - however compound microscopes were mostly of poor quality and could only magnify up to 20-30 times. Hooke claimed they were too difficult to use - his eyesight was poor. Discovered bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells, microscopic nematodes In 1673, Leeuwenhoek began writing letters to the Royal Society of London - published in Philosophical Transactions of the Royal Society In 1680 he was elected a full member of the Royal Society, joining Robert Hooke, Henry Oldenburg, Robert Boyle, Christopher Wren

Early Microscopes (Hooke) 1665

1670-1690 Back: Italian compound microscopes - 1670 Back: 1670 (probably Campani) This microscope was formerly at the University of Bologna - it contains a field lens which was the first optical advance about 1660. Only opaque objects can be viewed. Front: Guiseppe Campani, Rome - 1690 - Campani was the leading Italian telescope and microscope maker in the late `17th century - he probably invented the screw focusing mechanism shown on this scope - the slide holder in the base allows transparent and opaque objects to be viewed

Screwbarrel Microscope - 1720 Made by Charles Culpeper

Secondary Microscopes George Adams Sr. made many microscopes from about 1740-1772 but he was predominantly just a good manufacturer not inventor (in fact it is thought he was more than a copier!) Simple microscopes could attain around 2 micron resolution, while the best compound microscopes were limited to around 5 microns because of chromatic aberration In the 1730s a barrister names Chester More Hall observed that flint glass (newly made glass) dispersed colors much more than “crown glass” (older glass). He designed a system that used a concave lens next to a convex lens which could realign all the colors. This was the first achromatic lens. George Bass was the lens-maker that actually made the lenses, but he did not divulge the secret until over 20 years later to John Dollond who copied the idea in 1759 and patented the achromatic lens.

George Adams Toymaker to Kings This microscope made by George Adams, Mathematical Instrument maker to King George III around 1763, It was probably intended for the Prince of Wales, the future King George IV. The instrument is based on the design of the “Universal Double Microscope" (London Museum of Science)

The famous patent of 1758 George Bass was the lens-maker that actually made the lenses, but he did not divulge the secret until over 20 years later to John Dollond who copied the idea in 1757 and patented the achromatic lens in 1758.

Secondary Microscopes George Adams Sr. made many microscopes from about 1740- 1772 but he was predominantly just a good manufacturer not inventor (in fact it is thought he was more than a copier!) © J.Paul Robinson “New Improved Compound Microscope, George Adams, 1790 Adams described this instrument in his “Essays on the Microscope” in 1787. The mechanism allowed freedom of movement. The specimen could be viewed in direct light or in light reflected from a large mirror.

Giovanni Battista Amici In 1827 Giovanni Battista Amici, built high quality microscopes and introduced the first matched achromatic microscope in 1827. He had previously (1813) designed “reflecting microscopes” using curved mirrors rather than lenses. He recognized the importance of coverslip thickness and developed the concept of “water immersion” © J.Paul Robinson © J.Paul Robinson

Joseph Lister In 1830, by Joseph Jackson Lister (father of Lord Joseph Lister) solved the problem of Spherical Aberration - caused by light passing through different parts of the same lens. He solved it mathematically and published this in the Philosophical Transactions in 1830 © J.Paul Robinson

Pasteur - 1860 Louis Pasteur – his microscope was made in Paris by Nachet in about 1860 and was brass

Abbe & Zeiss Ernst Abbe together with Carl Zeiss published a paper in 1877 defining the physical laws that determined resolving distance of an objective. Known as Abbe’s Law “minimum resolving distance (d) is related to the wavelength of light (lambda) divided by the Numeric Aperture, which is proportional to the angle of the light cone (theta) formed by a point on the object, to the objective”.

Abbe & Zeiss Abbe and Zeiss developed oil immersion systems by making oils that matched the refractive index of glass. Thus they were able to make the a Numeric Aperture (N.A.) to the maximum of 1.4 allowing light microscopes to resolve two points distanced only 0.2 microns apart (the theoretical maximum resolution of visible light microscopes). Leitz was also making microscope at this time. Zeiss student microscope 1880

Abbe, Zeiss & Schott Abbe and Zeiss developed oil immersion systems by making oils that matched the refractive index of glass. Thus they were able to make the a Numeric Aperture (N.A.) to the maximum of 1.4 allowing light microscopes to resolve two points distanced only 0.2 microns apart (the theoretical maximum resolution of visible light microscopes). Leitz was also making microscope at this time. Dr Otto Schott formulated glass lenses that color-corrected objectives and produced the first “apochromatic” objectives in 1886.

Modern Microscopes Early 20th Century Professor Köhler developed the method of illumination still called “Köhler Illumination” Köhler recognized that using shorter wavelength light (UV) could improve resolution

Köhler Illumination eyepiece Specimen Field iris Field stop retina condenser Specimen Field iris Field stop retina Conjugate planes for image-forming rays Specimen Field iris Field stop Conjugate planes for illuminating rays

Some Definitions Absorption Refraction Diffraction Dispersion When light passes through an object the intensity is reduced depending upon the color absorbed. Thus the selective absorption of white light produces colored light. Refraction Direction change of a ray of light passing from one transparent medium to another with different optical density. A ray from less to more dense medium is bent perpendicular to the surface, with greater deviation for shorter wavelengths Diffraction Light rays bend around edges - new wavefronts are generated at sharp edges - the smaller the aperture the lower the definition Dispersion Separation of light into its constituent wavelengths when entering a transparent medium - the change of refractive index with wavelength, such as the spectrum produced by a prism or a rainbow

Properties of Light Refraction A Lens Refractive Index Numerical Aperture Resolution Aberrations Fluorescence

Reflection and Refraction Snell’s Law: The angle of reflection (Ør) is equal to the angle of incidence (Øi) regardless of the surface material The angle of the transmitted beam (Øt) is dependent upon the composition of the material Transmitted (refracted)Beam Reflected Beam r t i Incident Beam n1 sin Øi = n2 sin Øt The velocity of light in a material of refractive index n is c/n

Properties of thin Lenses q 1 1 1 + = p q f Resolution (R) = 0.61 x l NA q Magnification = (lateral) p (Rayleigh criterion)

Refraction & Dispersion Short wavelengths are “bent” more than long wavelengths dispersion Light is “bent” and the resultant colors separate (dispersion). Red is least refracted, violet most refracted.

Refraction He sees the fish here…. But it is really here!!

Control Absorption No blue/green light red filter

Absorption Chart Color in white light Color of light absorbed blue red green blue red green green red blue blue yellow magenta green cyan red black red green blue gray pink green blue

The light spectrum Wavelength ---- Frequency Blue light 488 nm short wavelength high frequency high energy (2 times the red) Photon as a wave packet of energy Red light 650 nm long wavelength low frequency low energy

Magnification An object can be focussed generally no closer than 250 mm from the eye (depending upon how old you are!) this is considered to be the normal viewing distance for 1x magnification Young people may be able to focus as close as 125 mm so they can magnify as much as 2x because the image covers a larger part of the retina - that is it is “magnified” at the place where the image is formed

p Magnification 1000mm 1000 mm = 28 M = 35 mm 35 mm slide 24x35 mm M = 1000 mm 35 mm = 28 p The projected image is 28 times larger than we would see it at 250 mm from our eyes. If we used a 10x magnifier we would have a magnification of 280x, but we would reduce the field of view by a factor of 10x.

Some Principles Rule of thumb is is not to exceed 1,000 times the NA of the objective Modern microscopes magnify both in the objective and the ocular and thus are called “compound microscopes” - Simple microscopes have only a single lens

Basic Microscopy Bright field illumination does not reveal differences in brightness between structural details - i.e. no contrast Structural details emerge via phase differences and by staining of components The edge effects (diffraction, refraction, reflection) produce contrast and detail

Microscope Basics Originally conformed to the German DIN standard Standard required the following real image formed at a tube length of 160mm the parfocal distance set to 45 mm object to image distance set to 195 mm Currently we use the ISO standard Object to Image Distance = 195 mm Mechanical tube length = 160 mm Focal length of objective = 45 mm

The Conventional Microscope Mechanical tube length = 160 mm Object to Image Distance = 195 mm Focal length of objective = 45 mm Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press

Upright Scope Brightfield Source Epi- illumination

Inverted Microscope Brightfield Source Epi- illumination

Conventional Finite Optics with Telan system Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press Ocular Intermediate Image 195 mm 160 mm Telan Optics Other optics Objective 45 mm Sample being imaged

Infinity Optics Ocular Primary Image Plane Tube Lens Other optics The main advantage of infinity corrected lens systems is the relative insensitivity to additional optics within the tube length. Secondly one can focus by moving the objective and not the specimen (stage) Tube Lens Infinite Image Distance Other optics Other optics Objective Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press Sample being imaged

Images reproduced from: http://micro.magnet.fsu.edu/

Microscope Basics, Magnification, Optical systems Images reproduced from: http://micro.magnet.fsu.edu/

Microscope Components Ocular Objectives Condensor Numerical Aperture Refractive Index Aberrations Optical Filters

Ocular - Eyepiece Essentially a projection lens (5x to 15x magnification) Note: there is usually an adjustment call the inter-pupillary distance on eyepieces for personal focusing Huygenian Projects the image onto the retina of the eye your eye should not be right on the lens, but back from it (eyecups create this space) Compensating designed to work with specific apochromatic or flat field objectives - it is color compensated and cannot be mixed with other objectives (or microscopes) Photo-adapter designed to project the image on the film in the camera - usually a longer distance and lower magnification from 0.5x to 5x Immediate above Images reproduced from: http://micro.magnet.fsu.edu/

Condensor Has several purposes must focus the light onto the specimen fill the entire numerical aperture of the objective (i.e. it must match the NA of the objective) Most microscopes will have what is termed an “Abbe” condenser (not corrected for aberrations) Note: If you exceed 1.0 NA objective, you probably will need to use oil on the condensor as well (except in inverted scopes)

Microscope Objectives Immediate above Images reproduced from: http://micro.magnet.fsu.edu/

Objectives PLAN-APO-40X 1.30 N.A. 160/0.22  - Infinity corrected Flat field Apochromat Magnification Numerical Tube Coverglass Factor Aperture Length Thickness

Objectives This defines a “resel” or “resolution element” Limit for smallest resolvable distance d between 2 points is (Rayleigh criterion): d = 1.22  This defines a “resel” or “resolution element” Thus high NUMERICAL APERTURE is critical for high magnification In a medium of refractive index n the wavelength gets shorter:n

Numerical Aperture Resolving power is directly related to numerical aperture. The higher the NA the greater the resolution Resolving power: The ability of an objective to resolve two distinct lines very close together NA = n sin u (n=the lowest refractive index between the object and first objective element) (hopefully 1) u is 1/2 the angular aperture of the objective

A m Light cone NA=n(sin m)

Numerical Aperture The wider the angle the lens is capable of receiving light at, the greater its resolving power The higher the NA, the shorter the working distance Images reproduced from: http://micro.magnet.fsu.edu/

Numerical Aperture   .00053 = = 0.53 m 1.00 NA .00053 = = 0.265 m For a narrow light beam (i.e. closed illumination aperture diaphragm) the finest resolution is (at the brightest point of the visible spectrum i.e. 530 nm)…(closed condenser).  .00053 1.00 = 0.53 m = NA With a cone of light filling the entire aperture the theoretical resolution is…(fully open condenser)..  .00053 = = 0.265 m 2 x NA 2 x 1.00

40 x 0.65 N.A. objective at 530 nm light Object Resolution Example: 40 x 1.3 N.A. objective at 530 nm light  .00053 = = 0.20 m 2 x NA 2 x 1.3 40 x 0.65 N.A. objective at 530 nm light  .00053 = = 0.405 m 2 x NA 2 x .65 R=l/(2NA) 1 R=0.61 l/NA 2 R=1.22 l/(NA(obj) + NA(cond)) 3

Images reproduced from: http://micro.magnet.fsu.edu/

Microscope Objectives 60x 1.4 NA PlanApo Oil Stage Coverslip Specimen

Refractive Index Water Objective n = 1.52 n = 1.5 n = 1.52 Oil n = 1.0 Air n = 1.0 n=1.52 n = 1.52 Coverslip n=1.33 n=1.52 Specimen Water

Numerical Aperture Resolving power is directly related to numerical aperture. The higher the NA the greater the resolution Resolving power: The ability of an objective to resolve two distinct lines very close together NA = n sin u (n=the lowest refractive index between the object and first objective element) (hopefully 1) u is 1/2 the angular aperture of the objective

A m Light cone NA=n(sin m)

Numerical Aperture The wider the angle the lens is capable of receiving light at, the greater its resolving power The higher the NA, the shorter the working distance Images reproduced from: http://micro.magnet.fsu.edu/

Numerical Aperture   .00053 = = 0.53 m 1.00 NA .00053 = = 0.265 m For a narrow light beam (i.e. closed illumination aperture diaphragm) the finest resolution is (at the brightest point of the visible spectrum i.e. 530 nm)…(closed condenser).  .00053 1.00 = 0.53 m = NA With a cone of light filling the entire aperture the theoretical resolution is…(fully open condenser)..  .00053 = = 0.265 m 2 x NA 2 x 1.00

40 x 0.65 N.A. objective at 530 nm light Object Resolution Example: 40 x 1.3 N.A. objective at 530 nm light  .00053 = = 0.20 m 2 x NA 2 x 1.3 40 x 0.65 N.A. objective at 530 nm light  .00053 = = 0.405 m 2 x NA 2 x .65 R=l/(2NA) 1 R=0.61 l/NA 2 R=1.22 l/(NA(obj) + NA(cond)) 3

Images reproduced from: http://micro.magnet.fsu.edu/

Microscope Objectives 60x 1.4 NA PlanApo Oil Stage Coverslip Specimen

Refractive Index Water Objective n = 1.52 n = 1.5 n = 1.52 Oil n = 1.0 Air n = 1.0 n=1.52 n = 1.52 Coverslip n=1.33 n=1.52 Specimen Water

Sources of Aberrations Monochromatic Aberrations Spherical aberration Coma Astigmatism Flatness of field Distortion Chromatic Aberrations Longitudinal aberration Lateral aberration Images reproduced from: http://micro.magnet.fsu.edu/

Monochromatic Aberration - Spherical aberration F1 F2 Corrected lens Immediate left Image reproduced from: http://micro.magnet.fsu.edu/ Generated by nonspherical wavefronts produced by the objective, and increased tube length, or inserted objects such as coverslips, immersion oil, etc. Essentially, it is desirable only to use the center part of a lens to avoid this problem.

Monochromatic Aberrations - Coma 1 Images reproduced from: http://micro.magnet.fsu.edu/ 3 2 Coma is when a streaking radial distortion occurs for object points away from the optical axis. It should be noted that most coma is experienced “off axis” and therefore, should be less of a problem in confocal systems.

Monochromatic Aberrations - Astigmatism Images reproduced from: http://micro.magnet.fsu.edu/ If a perfectly symmetrical image field is moved off axis, it becomes either radially or tangentially elongated.

Monochromatic Aberrations Flatness of Field Distortion Lenses are spherical and since points of a flat image are focused onto a spherical dish, the central and peripheral zones will not be in focus. Complex Achromat and PLANAPOCHROMAT lenses partially solve this problem but at reduced transmission. DISTORTION occurs for objects components out of axis. Most objectives correct to reduce distortion to less than 2% of the radial distance from the axis.

Useful Factoids The intensity of light collected decreases as the square of the magnification The intensity of light increases as the square of the numerical aperture

Fluorescence Microscopes Cannot view fluorescence emission in a single optical plane Generally use light sources of much lower flux than confocal systems Are cheaper than confocal systems Give high quality photographic images (actual photographs) whereas confocal systems are restricted to small resolution images

Fluorescent Microscope Arc Lamp EPI-Illumination Excitation Diaphragm Excitation Filter Ocular Dichroic Filter Objective Emission Filter

Interference in Thin Films Small amounts of incident light are reflected at the interface between two material of different RI Thickness of the material will alter the constructive or destructive interference patterns - increasing or decreasing certain wavelengths Optical filters can thus be created that “interfere” with the normal transmission of light Interference and Diffraction: Gratings Diffraction essentially describes a departure from theoretical geometric optics Thus a sharp objet casts an alternating shadow of light and dark “patterns” because of interference Diffraction is the component that limits resolution

Polarization and Phase: Interference Electric and magnetic fields are vectors - i.e. they have both magnitude and direction The inverse of the period (wavelength) is the frequency in Hz Axis of Electric Field Wavelength (period T) Magnetic Field Axis of Axis of Propagation Modified from Shapiro “Practical Flow Cytometry” Wiley-Liss, p78

Interference A+B A B C+D C D Wavelength Amplitude Constructive The frequency does not change, but the amplitude is doubled A Amplitude B Constructive Interference Here we have a phase difference of 180o (2 radians) so the waves cancel each other out C+D C D Destructive Interference Figure modified from Shapiro “Practical Flow Cytometry” Wiley-Liss, p79

Construction of Filters Multiple elements Dielectric filter components “glue” Single Optical filter Anti-Reflection Coatings Coatings are often magnesium fluoride

Anti-Reflection Coatings Coatings are often magnesium fluoride Optical Filter Multiple Elements Dielectric filter components

Band Pass Filters Long Pass Filters Short Pass Filters Transmitted Light White Light Source 630 nm BandPass Filter 620 -640 nm Light Long Pass Filters Transmitted Light Light Source 520 nm Long Pass Filter >520 nm Light Short Pass Filters Transmitted Light Light Source 575 nm Short Pass Filter <575 nm Light

Optical Filters Dichroic Filter/Mirror at 45 deg 510 LP dichroic Mirror Dichroic Filter/Mirror at 45 deg Light Source Transmitted Light Reflected light

Filter Properties Light Transmission 100 Bandpass Notch %T 50 Wavelength

The intensity of the radiation is inversely proportional to the square of the distance traveled

Summary Lecture 1 History, simple versus compound microscopes Köhler illumination Refraction, Absorption, dispersion, diffraction, Magnification Upright and inverted microscopes Optical Designs - 160 mm and Infinity optics Components of the microscope Numerical Aperture (NA) Refractive Index/refraction (RI), Aberrations Fluorescence microscope Properties of optical filters