1. 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.
UPDATED February 2004

2. 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

3. 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

4. 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

5. 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”

6. Earliest Microscopes
Hans & Zacharias Janssen of Middleburg, Holland manufactured the first compound microscopes
1590 – Galileo – one of the earliest microscopists (naming of term “microscope”
Marcello Malpighi circa 1660, was one of the first great microscopists, considered the father embryology and early histology - observed capillaries in 1660
Robert Hooke ( )- 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.”

7. Earliest Microscopes
Antioni van Leeuwenhoek ( ) 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 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

8. Early Microscopes (Hooke)
1665

9. 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 Only opaque objects can be viewed.
Front: Guiseppe Campani, Rome 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

10. Screwbarrel Microscope - 1720
Made by Charles Culpeper

11. Secondary Microscopes
George Adams Sr. made many microscopes from about 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.

12. 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)

13. 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.

14. Secondary Microscopes
George Adams Sr. made many microscopes from about 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 The mechanism allowed freedom of movement. The specimen could be viewed in direct light or in light reflected from a large mirror.

15. Giovanni Battista Amici
In 1827 Giovanni Battista Amici, built high quality microscopes and introduced the first matched achromatic microscope in 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

16. 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

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

18. 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”.

19. 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

20. 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.

21. 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

22. 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

23. 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

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

25. 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

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

27. 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.

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

29. Control
Absorption
No blue/green light
red filter

30. 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

31. 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

32. 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

33. 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.

34. 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

35. 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

36. 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

37. 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

38. Upright Scope
Brightfield
Source
Epi-
illumination

39. Inverted Microscope
Brightfield
Source
Epi-
illumination

41. 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

42. 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

43. Images reproduced from:

44. Microscope Basics, Magnification, Optical systems
Images reproduced from:

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

46. 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:

47. 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)

48. Microscope Objectives
Immediate above Images reproduced from:

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

50. 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

51. 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

52. A m
Light cone
NA=n(sin m)

53. 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:

54. 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 =
= m
2 x NA
2 x 1.00

55. 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 =
= 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

56. Images reproduced from:

57. Microscope Objectives
60x 1.4 NA
PlanApo
Oil
Stage
Coverslip
Specimen

58. 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

59. 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

60. A m
Light cone
NA=n(sin m)

61. 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:

62. 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 =
= m
2 x NA
2 x 1.00

63. 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 =
= 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

64. Images reproduced from:

65. Microscope Objectives
60x 1.4 NA
PlanApo
Oil
Stage
Coverslip
Specimen

66. 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

67. Sources of Aberrations
Monochromatic Aberrations
Spherical aberration
Coma
Astigmatism
Flatness of field
Distortion
Chromatic Aberrations
Longitudinal aberration
Lateral aberration
Images reproduced from:

68. Monochromatic Aberration - Spherical aberrationF1 F2
Corrected lens
Immediate left Image reproduced from:
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.

69. Monochromatic Aberrations - Coma1
Images reproduced from: 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.

70. Monochromatic Aberrations - Astigmatism
Images reproduced from:
If a perfectly symmetrical image field is moved off axis, it becomes either radially or tangentially elongated.

71. 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.

72. 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

73. 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

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

75. 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

76. 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

77. 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

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

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

80. Band Pass Filters Long Pass Filters Short Pass Filters
Transmitted Light
White Light Source
630 nm BandPass Filter
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

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

82. Filter Properties Light Transmission
100
Bandpass
Notch %T 50
Wavelength

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

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