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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Evaluation
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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
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Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Microscopes
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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”
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Earliest Microscopes
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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.”
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Early Microscopes (Hooke)
1665
© J.Paul Robinson - Purdue University Cytometry Laboratories
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1670-1690
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© J.Paul Robinson - Purdue University Cytometry Laboratories
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
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Screwbarrel Microscope - 1720
• Made by Charles Culpeper
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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.
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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)
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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.
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Secondary Microscopes
• George Adams Sr. made many microscopes from about 17401772 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, 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.
© J.Paul Robinson
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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 - Purdue University Cytometry Laboratories
© J.Paul Robinson
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Pasteur - 1860
Louis Pasteur – his microscope was made in Paris by Nachet in about 1860 and
was brass
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Abbe & Zeiss
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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”.
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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.
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Köhler Illumination
condenser
Field iris
Specimen
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Properties of Light
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Refraction
A Lens
Refractive Index
Numerical Aperture
Resolution
Aberrations
Fluorescence
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Reflection and Refraction
Reflected Beam
r
i
Incident Beam
• Snell’s Law: The angle of
reflection (Ør) is equal to the
Transmitted
angle of incidence (Øi)
(refracted)Beam
regardless of the surface
material
t
• The angle of the transmitted
beam (Øt) is dependent upon
the composition of the
material
n1 sin Øi = n2 sin Øt
The velocity of light in a material
of refractive index n is c/n
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Properties of thin Lenses
f
f
p
q
1
p
Resolution (R) = 0.61 x
(lateral)
(Rayleigh criterion)
+
1
q
l
NA
© J.Paul Robinson - Purdue University Cytometry Laboratories
=
1
f
q
Magnification =
p
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Refraction & Dispersion
rac
Short wavelengths are “bent”
more than long wavelengths
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Refraction
He sees the
fish here….
But it is really here!!
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Control
Absorption
No blue/green light
red filter
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Absorption Chart
Color in white light
Color of light absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
blue
magenta
green
cyan
black
red
red
green
gray
pink
green
© J.Paul Robinson - Purdue University Cytometry Laboratories
blue
blue
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Magnification
1000mm
35 mm slide
24x35 mm
1000 mm
M = 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.
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
Object to
Image
Distance
= 195 mm
Mechanical
tube length
= 160 mm
Focal length
of objective
= 45 mm
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Upright Scope
Epiillumination
Source
Brightfield
Source
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Inverted Microscope
Brightfield
Source
Epiillumination
Source
© J.Paul Robinson - Purdue University Cytometry Laboratories
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© J.Paul Robinson - Purdue University Cytometry Laboratories
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Infinity Optics
Ocular
Primary Image Plane
Tube Lens
Infinite
Image
Distance
Other optics
Other optics
Objective
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)
Modified from “Pawley “Handbook of
Confocal Microscopy”, Plenum Press
Sample being imaged
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Images reproduced from:
http://micro.magnet.fsu.edu/
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 43 t:/classes/BMS602B/lecture 1 602_B.ppt
• Microscope Basics, Magnification, Optical systems
Images reproduced from:
http://micro.magnet.fsu.edu/
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Microscope Components
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Ocular
Objectives
Condensor
Numerical Aperture
Refractive Index
Aberrations
Optical Filters
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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/
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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)
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Microscope Objectives
Immediate above Images reproduced from:
http://micro.magnet.fsu.edu/
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Objectives
 - Infinity
corrected
PLAN-APO-40X 1.30 N.A. 160/0.22
Flat field Apochromat Magnification Numerical Tube Coverglass
Aperture Length Thickness
Factor
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Objectives
Limit for smallest
resolvable distance d
between 2 points is
(Rayleigh criterion):
d = 1.22l

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: lln
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
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A
m
Light cone
NA=n(sin m)
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 52 t:/classes/BMS602B/lecture 1 602_B.ppt
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/
© J.Paul Robinson - Purdue University Cytometry Laboratories
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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).
l
=
NA
.00053
1.00 = 0.53 mm
• With a cone of light filling the entire aperture the theoretical resolution
is…(fully open condenser)..
l
2 x NA
=
© J.Paul Robinson - Purdue University Cytometry Laboratories
.00053
2 x 1.00 = 0.265 mm
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Object Resolution
• Example:
40 x 1.3 N.A. objective at 530 nm light
l
2 x NA
=
.00053
= 0.20 mm
2 x 1.3
40 x 0.65 N.A. objective at 530 nm light
l
2 x NA
=
.00053
= 0.405 mm
2 x .65
R=l/(2NA)
R=0.61 l/NA
R=1.22 l/(NA(obj) + NA(cond))
© J.Paul Robinson - Purdue University Cytometry Laboratories
1
2
3
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Images reproduced from:
http://micro.magnet.fsu.edu/
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Microscope Objectives
60x 1.4 NA
PlanApo
Oil
Microscope
Objective
Stage
Coverslip
Specimen
© J.Paul Robinson - Purdue University Cytometry Laboratories
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Refractive Index
Objective
n = 1.52
n = 1.52
Oil
n=1.52
n=1.52
© J.Paul Robinson - Purdue University Cytometry Laboratories
n = 1.5
n = 1.0
Air
n = 1.52
Coverslip
Specimen
Water
n=1.33
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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
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 59 t:/classes/BMS602B/lecture 1 602_B.ppt
A
m
Light cone
NA=n(sin m)
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 60 t:/classes/BMS602B/lecture 1 602_B.ppt
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/
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 61 t:/classes/BMS602B/lecture 1 602_B.ppt
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).
l
=
NA
.00053
1.00 = 0.53 mm
• With a cone of light filling the entire aperture the theoretical resolution
is…(fully open condenser)..
l
2 x NA
=
© J.Paul Robinson - Purdue University Cytometry Laboratories
.00053
2 x 1.00 = 0.265 mm
Slide 62 t:/classes/BMS602B/lecture 1 602_B.ppt
Object Resolution
• Example:
40 x 1.3 N.A. objective at 530 nm light
l
2 x NA
=
.00053
= 0.20 mm
2 x 1.3
40 x 0.65 N.A. objective at 530 nm light
l
2 x NA
=
.00053
= 0.405 mm
2 x .65
R=l/(2NA)
R=0.61 l/NA
R=1.22 l/(NA(obj) + NA(cond))
© J.Paul Robinson - Purdue University Cytometry Laboratories
1
2
3
Slide 63 t:/classes/BMS602B/lecture 1 602_B.ppt
Images reproduced from:
http://micro.magnet.fsu.edu/
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 64 t:/classes/BMS602B/lecture 1 602_B.ppt
Microscope Objectives
60x 1.4 NA
PlanApo
Oil
Microscope
Objective
Stage
Coverslip
Specimen
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 65 t:/classes/BMS602B/lecture 1 602_B.ppt
Refractive Index
Objective
n = 1.52
n = 1.52
Oil
n=1.52
n=1.52
© J.Paul Robinson - Purdue University Cytometry Laboratories
n = 1.5
n = 1.0
Air
n = 1.52
Coverslip
Specimen
Water
n=1.33
Slide 66 t:/classes/BMS602B/lecture 1 602_B.ppt
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/
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 67 t:/classes/BMS602B/lecture 1 602_B.ppt
Monochromatic Aberration - Spherical aberration
F1
F2
F1
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.
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 68 t:/classes/BMS602B/lecture 1 602_B.ppt
1
Monochromatic Aberrations - Coma
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.
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 69 t:/classes/BMS602B/lecture 1 602_B.ppt
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.
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 70 t:/classes/BMS602B/lecture 1 602_B.ppt
• 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.
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 71 t:/classes/BMS602B/lecture 1 602_B.ppt
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
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 72 t:/classes/BMS602B/lecture 1 602_B.ppt
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
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 73 t:/classes/BMS602B/lecture 1 602_B.ppt
Fluorescent Microscope
Arc Lamp
EPI-Illumination
Excitation Diaphragm
Excitation Filter
Ocular
Dichroic Filter
Objective
Emission Filter
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 74 t:/classes/BMS602B/lecture 1 602_B.ppt
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
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 75 t:/classes/BMS602B/lecture 1 602_B.ppt
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
Modified from Shapiro “Practical Flow Cytometry” Wiley-Liss, p78
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 76 t:/classes/BMS602B/lecture 1 602_B.ppt
Interference
0o 90o 180o 270o 360o
Wavelength
Amplitude
A+B
A
Constructive
Interference
B
C+D
C
D
The frequency does
not change, but the
amplitude is doubled
Here we have a phase difference of
180o (2 radians) so the waves
cancel each other out
Destructive
Interference
Figure modified from Shapiro “Practical Flow
Cytometry” Wiley-Liss, p79
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 77 t:/classes/BMS602B/lecture 1 602_B.ppt
Construction of Filters
Multiple elements
Dielectric filter
components
“glue”
Single Optical
filter
Anti-Reflection Coatings
Coatings are often magnesium fluoride
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 78 t:/classes/BMS602B/lecture 1 602_B.ppt
Anti-Reflection Coatings
Coatings are often magnesium fluoride
Optical Filter
Multiple
Elements
© J.Paul Robinson - Purdue University Cytometry Laboratories
Dielectric filter
components
Slide 79 t:/classes/BMS602B/lecture 1 602_B.ppt
Band Pass Filters
630 nm BandPass Filter
White Light Source
Transmitted Light
620 -640 nm Light
Long Pass Filters
Light Source
520 nm Long Pass Filter
Transmitted Light
>520 nm Light
Short Pass Filters
Light Source
575 nm Short Pass Filter
Transmitted Light
<575 nm Light
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 80 t:/classes/BMS602B/lecture 1 602_B.ppt
Optical Filters
510 LP dichroic Mirror
Dichroic Filter/Mirror at 45 deg
Light Source
Transmitted Light
Reflected light
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 81 t:/classes/BMS602B/lecture 1 602_B.ppt
Filter Properties
Light Transmission
100
50
Bandpass
%T
Notch
0
Wavelength
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 82 t:/classes/BMS602B/lecture 1 602_B.ppt
The intensity of the radiation is inversely proportional to the square of the distance traveled
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 83 t:/classes/BMS602B/lecture 1 602_B.ppt
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
© J.Paul Robinson - Purdue University Cytometry Laboratories
Slide 84 t:/classes/BMS602B/lecture 1 602_B.ppt