Lecture 1
The Principles of Microscopy
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BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”
Purdue University Department of Basic Medical Sciences,
School of Veterinary Medicine
J.Paul Robinson, Ph.D.
• Professor of Immunopharmacology
• 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. Textbook for this lecture series in Jim
Pawley’s “Handbook of Confocal Microscopy” Plenum Press which has been used extensively
for material and ideas to support the class.
UPDATED December 27, 1998
J.Paul Robinson - Purdue University Cytometry Laboratories
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Evaluation
• End of term quiz - 100% grade
J.Paul Robinson - Purdue University Cytometry Laboratories
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Introduction to the Course
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Microscopy
Fluorescence
Basic Optics
Confocal Microscopes
Basic Image Analysis
3D image analysis
Live Cell Studies
Advanced Applications
J.Paul Robinson - Purdue University Cytometry Laboratories
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Introduction to Lecture 1
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Early Microscopes
Modern Microscopes
Magnification
Nature of Light
Optical Designs
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" a scientific
society which included Galileo
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
• 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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Secondary Microscopes
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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!)
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Simple microscopes could attain around 2 micron resolution, while the best
compound microscopes were limited to around 5 microns because of chromatic
aberration
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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 Dolland who copied the idea in 1759 and patented the achromatic lens.
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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 - Purdue University Cytometry Laboratories
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Lister, Abbe, Zeiss & Schott
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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
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”.
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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
• Köhler illumination creates an evenly
illuminated field of view while illuminating
the specimen with a very wide cone of light
• Two conjugate image planes are formed
– one contains an image of the specimen and the
other the filament from the light
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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Refraction
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.
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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Light absorption
white light
blue light
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red light
green light
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Absorption Chart
Color
Colorin
inwhite
whitelight
light
Color
Colorof
oflight
lightabsorbed
absorbed
red
blue
green
blue
green
red
red
green
yellow
blue
magenta
blue
green
black
red
red
green
gray
pink
green
cyan
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
Object to
Image
Distance
= 195 mm
Mechanical
tube length
= 160 mm
Focal length
of objective
= 45 mm
• Currently we use the ISO
standard
J.Paul Robinson - Purdue University Cytometry Laboratories
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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
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Inverted Microscope
Brightfield
Source
Epiillumination
Source
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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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Summary Lecture 1
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Simple versus compound microscopes
Achromatic aberration
Spherical aberration
Köhler illumination
Refraction, Absorption, dispersion,
diffraction
• Magnification
• Upright and inverted microscopes
• Optical Designs - 160 mm and Infinity optics
J.Paul Robinson - Purdue University Cytometry Laboratories
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