Slide 1 t:/classes/BMS602B/lecture 1 602_B.ppt © J.Paul Robinson - Purdue University Cytometry Laboratories Lecture 1 The Principles of Microscopy BME

Slide 1 t:/classes/BMS602B/lecture 1 602_B.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories

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 EngineeringDirector, 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.

UPDATED February 2004

http://www.cyto.purdue.edu/


Evaluation• 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

• Basic Image Analysis• 3D image analysis• Live Cell Studies• Advanced Applications

• Microscopy• Fluorescence• Basic Optics• Confocal Microscopes


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 11. Introduction to Microscopy2. History of Microscopy3. Köhler Illumination4. Basic optical terms5. Light absorption6. Magnification7. Optical properties of microscopes8. Components of the microscope9. Numerical aperture, resolution and refractive index10. Aberrations11. 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

lens.exe


Early Microscopes (Hooke)

1665


1670-1690

• Back: Italian compound microscopes - 1670

• Italian Compound microscopes

• 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!)

“New Improved Compound Microscope, George Adams, 1790Adams 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.

© J.Paul Robinson


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

Specimen Field stopField iris

Conjugate planes for illuminating rays

Specimen Field stopField iris

Conjugate planes for image-forming rays

condenser eyepiece

retina


Some Definitions• Absorption

– 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

t

i

r

Incident Beam

Reflected BeamTransmitted

(refracted)Beam

n1 sin Øi = n2 sin Øt

The velocity of light in a material of refractive index n is c/n


Properties of thin Lensesf

1

p+

1

q=

1

f

f

p q

Resolution (R) = 0.61 xNA

Magnification = q

p(lateral)(Rayleigh criterion)


Refraction & Dispersion

Light is “bent” and the resultant colors separate (dispersion).Red is least refracted, violet most refracted.

dispersion

Short wavelengths are “bent” more than long wavelengths

refraction


Refraction

But it is really here!!

He sees the fish here….


Absorption

Control

No blue/green light red filter


Absorption Chart

Color in white lightColor in white light Color of light absorbedColor of light absorbed

red

blue

green

magenta

cyan

yellow

blue

blue

blue

blue

green

green

green

green

red

red

red

redblack

gray green bluepink


The light spectrumWavelength ---- Frequency

Blue light

488 nm

short wavelength

high frequency

high energy (2 times the red)

Red light

650 nm

long wavelength

low frequency

low energy

Photon as a wave packet of 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


Magnification1000mm

35 mm slide24x35 mm

M = 1000 mm35 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

Focal lengthFocal lengthof objectiveof objective= 45 mm= 45 mm

MechanicalMechanicaltube lengthtube length= 160 mm= 160 mm

Object toImage Distance = 195 mm


The Conventional Microscope

Focal lengthof objective= 45 mm

Object toImage Distance = 195 mm

Mechanicaltube length= 160 mm

Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press


Upright Scope

BrightfieldSource

Epi-illuminationSource


Inverted Microscope

BrightfieldSource

Epi-illuminationSource



Conventional Finite Opticswith Telan system

Sample being imaged

Intermediate Image

Telan Optics

Objective

Other optics

Ocular

45 mm

160 mm195 mm



Infinity Optics

Sample being imaged

Primary Image Plane

Objective

Other optics

Ocular

Other optics

Tube Lens

InfiniteImageDistance

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)



Images reproduced from:

http://micro.magnet.fsu.edu/


• Microscope Basics, Magnification, Optical systems




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:



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:



Objectives

PLAN-APO-40X 1.30 N.A. 160/0.22

Flat field Apochromat Magnification Numerical Tube CoverglassFactor Aperture Length Thickness

- Infinity corrected


Objectives

Limit for smallest resolvable distance d between 2 points is (Rayleigh criterion):

d = 1.22

Thus high NUMERICAL APERTURE is critical for high magnification

In a medium of refractive index n the wavelength gets shorter:n

This defines a “resel” or “resolution element”


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

NA=n(sin )

Light cone


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




Numerical Aperture• 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).

NA

2 x NA

.000532 x 1.00= 0.265 m

.000531.00 = 0.53 m

• With a cone of light filling the entire aperture the theoretical resolution is…(fully open condenser)..

=

=


Object Resolution• Example:

40 x 1.3 N.A. objective at 530 nm light

2 x NA

.000532 x 1.3

= 0.20 m=


2 x NA

.000532 x .65

= 0.405 m=

R=/(2NA) 1R=0.61 /NA 2R=1.22 /(NA(obj) + NA(cond)) 3






SpecimenCoverslip

Oil

MicroscopeObjective

Stage

60x 1.4 NAPlanApo


Refractive Index

Objective

n=1.52

n = 1.52

n = 1.52

Specimen

Coverslip

Oil

n=1.33

n = 1.52

n = 1.0

n = 1.5

Water

n=1.52

Air


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

NA=n(sin )

Light cone


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




Numerical Aperture• 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).

NA

2 x NA

.000532 x 1.00= 0.265 m

.000531.00 = 0.53 m

• With a cone of light filling the entire aperture the theoretical resolution is…(fully open condenser)..

=

=


Object Resolution• Example:


2 x NA

.000532 x 1.3

= 0.20 m=


2 x NA

.000532 x .65

= 0.405 m=

R=/(2NA) 1R=0.61 /NA 2R=1.22 /(NA(obj) + NA(cond)) 3






SpecimenCoverslip

Oil

MicroscopeObjective

Stage

60x 1.4 NAPlanApo


Refractive Index

Objective

n=1.52

n = 1.52

n = 1.52

Specimen

Coverslip

Oil

n=1.33

n = 1.52

n = 1.0

n = 1.5

Water

n=1.52

Air


• Monochromatic Aberrations– Spherical aberration

– Coma

– Astigmatism

– Flatness of field

– Distortion

• Chromatic Aberrations– Longitudinal aberration

– Lateral aberration

Sources of Aberrations




Monochromatic Aberration - Spherical aberration

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.

F1 F2

F1

Corrected lens

Immediate left Image reproduced from:



Monochromatic Aberrations - Coma

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.

1

23




Monochromatic Aberrations - Astigmatism

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 The intensity of light collected decreasesdecreases as the square of the magnificationas the square of the magnification

The intensity of light The intensity of light increases increases as the as the square of the numerical aperturesquare 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

Dichroic Filter

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

EPI-Illumination


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

Wavelength (period T)

Axis of

Magnetic F

ield

Axis of Propagation

Axi

s of

Ele

ctri

c F

ield

Modified from Shapiro “Practical Flow Cytometry” Wiley-Liss, p78


Interference

ConstructiveInterference

DestructiveInterference

A

B

C

D

A+B

C+D

Am

plitude

0o 90o 180o 270o 360o Wavelength

Figure modified from Shapiro “Practical Flow Cytometry” Wiley-Liss, p79

Here we have a phase difference of 180o (2 radians) so the waves cancel each other out

The frequency does not change, but the amplitude is doubled


Construction of Filters

Dielectric filtercomponents

Single Opticalfilter

“glue”

Multiple elements

Coatings are often magnesium fluoride

Anti-Reflection Coatings


Anti-Reflection Coatings

Optical Filter

MultipleElements

Coatings are often magnesium fluoride

Dielectric filtercomponents


Band Pass Filters

Transmitted LightWhite Light Source

630 nm BandPass Filter

620 -640 nm Light

Transmitted LightLight Source520 nm Long Pass Filter

>520 nm Light

Transmitted LightLight Source575 nm Short Pass Filter

<575 nm Light

Long Pass Filters

Short Pass Filters


Optical Filters

Dichroic Filter/Mirror at 45 deg

Reflected light

Transmitted LightLight Source

510 LP dichroic Mirror


Filter Properties Light Transmission

%T

Wavelength

100

0

50

Notch

Ban

dp

ass


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

Documents

Slide 1 t:/classes/BMS602B/lecture 1 602_B.ppt © J.Paul Robinson - Purdue University Cytometry Laboratories Lecture 1 The Principles of Microscopy BME