Download Microscope = from the Greek words μικρόν (micron) meaning "small

10/22/2012
Optical Microscopy 2012
Arto Koistinen,
UEF / SIB-labs
22.10.2012
Microscope = from the Greek words
μικρόν (micron) meaning "small,"
and σκοπεῖν (skopein) meaning "to
look at”
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Why microscopy?
— Optical microscopy is a useful tool for various kinds of
analysis. Some not so familiar topics:
— Recognition of minerals/crystallinity (geology)
— Diagnostics of deceases (medicine)
— Structural or failure analysis (engineering)
— Typically, further analyses using e.g. spectroscopy,
fluorescence microscopy or electron microscopy
— However, microscope is only a tool; know-how is
always needed for interpretation
Short history of microscopy
14th century: eye glasses for farsightedness
16h century: convex lenses for nearsightedness
1590: Janssen bros – first compound microscopes
1624: Galileo Galilei – perfected a compound microscope
1660: Malpighi - ”father of embryology” observed capillaries
1665: Hooke – Book ”Micrographia”, named the cell
1632-1723: Antoni van Leeuwenhoek – magnification of 270x
1810-1882: A. Schwann – ”theory”of cell biology
1839 Royal Microscopy society was established
1846 Carl Zeiss began producing optical equipment
1849 Carl Kellner started producing oculars
Photo: © J. Paul Robinson
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Short history of microscopy
1872 Abbe & Zeiss manufacture the first microscopy by theory of Abbe
1880 Leitz attaches a photographic device to a microscope
1886 O. Schott - the first color-corrected objective
1904 Köhler developed the famous method for illumination
1925 A.Coons – fluorescense stains
1933 E. Ruska – development of electron microscopy
1919-1997: Nomarski – invention of differential interface contrast (DIC)
1953: First principles for confocal microscopy
Field iris
conde
nser
Specimen
eyepiece
Field stop
retin
a
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
Galileo Galilei (1564-1642)
— 1610 - he began publicly supporting the heliocentric view, which
placed the Sun at the centre of the universe
— Galileo has been variously called
— the "father of modern observational astronomy
— the "father of modern physics
— the "father of science
— The name "telescope" was coined for Galileo's instrument by a
Greek mathematician, Giovanni Demisiani, at a banquet held in
1611 by Prince Federico Cesi to make Galileo a member of his
Accademia dei Lincei
•
•
•
•
•
Telescope was derived from the Greek tele = 'far' and skopein = 'to look or see'. In
1610, he used a telescope at close range to magnify the parts of insects.
Denounced to the Roman Inquisition early in 1615
1624 he had perfected a compound microscope
The Linceans played a role again in naming the "microscope" a year later when fellow
academy member Giovanni Faber coined the word for Galileo's invention from the
Greek words μικρόν (micron) meaning "small," and σκοπεῖν (skopein) meaning "to
look at."
Published “Dialogue Concerning the Two Chief World Systems” in 1632, and was
tried by the Inquisition, found "vehemently suspect of heresy," forced to recant, and
spent the rest of his life under house arrest (to 1642)
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Robert Hooke (1635-1703)
•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. Named the term “Cell”
The Royal Society of London founded in 1616 during the reign of King James I
Antioni van Leeuwenhoek (1632-1723)
• 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
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Carl Zeiss 1816-1888
— Carl Zeiss opens his workshop in Jana,
Germany to make eyeglasses and microscopes
for the University in 1846
— 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
— 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
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August Karl Johann Valentin
Köhler (1866-1948)
— Early 20th Century Professor Köhler developed
the method of illumination still called “Köhler
Illumination”
— In 1900, he was invited to join the Zeiss Optical
Works company in Jena, Germany, by Siegfried
Czapski based on his earlier work on improving
microscope illumination. He stayed with Zeiss
as a physicist for 45 years and became
instrumental to the development of modern
light microscope design.
— Köhler recognized that using shorter
wavelength light (UV) could improve
resolution
— The driving force for Köhler’s even illumination
invention was the use of gas lamps and similar
uneven light sources that created serious
problems in trying to gain even and constant
illumination
Image:
http://en.wikipedia.org/wiki/File:Augu
st_Koehler.jpg
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The light
— Some background:
— Speed of light c = 299 726 000 m/s (in vacuum)
— The wavelength (l) is defined by speed and frequency (f)
l=
c
f
— The speed is affected by the medium
v=
c
n
n = refractive index, v = speed in medium
— In another medium, the wavelength changes BUT
frequency remains
c
l=
n× f
Nature of light
— Light is a type of electro-
magnetic radiation that
has two types of nature:
particle-like and wavelike nature
— The electric (E) and
magnetic (B)
components are
perpendicular (90 deg).
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Interaction of light: Refraction
dispersion
Light is “bent” and the resultant colors separate (dispersion).
Red is least refracted, violet most refracted.
Refraction
He sees
the fish
here….
But it is really here!!
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Interaction of light: Absorbtion
Control
Absorption
B & G absorbed
No blue/green light
red filter
Light in microscopy
— In light microscopy, mostly visible light is used
— Wavelength approx. 400 – 700 nm
— For UV applications, wavelength less than 400 nm
— In spectroscopy, wavelengths more than 700 nm
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Interaction of radiation
Type of radiation
Wavelengthrange
Type of transition
Gamma rays
10-12 - 10-16 m
nuclear
x- rays
1 nm - 1 pm
inner electrons
Ultraviolet light
400 - 1 nm
outer electrons
Visible light
700 - 400 nm
outer electrons
Infrared light
2.5 mm - 700 nm
vibrations
Microwaves
1 mm - 2.5 mm
rotations
Radiowaves
108 - 1 m
spin flips
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
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Image from a microscope
— As the light confronts the specimen, electrical field of
the light interacts with the atoms (i.e. electrons) of the
specimen
— The interactions can be observed and visualized by the
microscopes and spectroscopes
— We detect the change in light à understand the reason
Factors in image formation
— Common factors affecting image formation:
— Absorbtion
— Creates amplitude contrast
— Main factor in light microscopy
— Interference
— Creates phase differences; invisible for the eye
— In phase-contrast microscopy, this is transformed to amplitude
— Diffraction
— In many applications, reduces the image quality
— Can be used to increase contrast (but will reduce resolution)
— Scattering
— Minor factor in light microscopy
— Main factor in transmission electron microscopy
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Interaction between light and matter
— Various interactions between visible light and matter
— Reflection; diffuse reflection
— Trasmittance, partial absorbtion (àutilized in spectroscopy)
— Absorbtion à change in wavelength due to staining
— Absorbtion of energy (excitation) à release of energy
(emission) à fluorescensce microscopy
— Refraction à e.g. lenses
Propagation of light;
refractive index
— Speed of light changes in different medium
— Change is caused by interaction between the electric
component of light and parts of an atom
c
— Refractice index = ratio of speeds of light
n=
— Snell’s law:
v
n1 sin q 2
=
n2 sin q1
— At the air-water interface, the
light refracts towards the
normal of the surface when
propagated from air to water
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Propagation of light; lenses
— Light refracts in a lens and forms an image of the
specimen (concave lens)
— Lens equation defines the position of the image:
—
1 1 1
= +
f a b
f = focus of the lens, a = distance of the
specimen and b = distance of the image
— Magnification generated by the lens is defined:
M=
b
a
Image formed by the lenses
— ”Typically" a real but upside down image is formed
— However, if the specimen is closer than the focus a
false image is formed.
— In microscopy, this kind of image is visualized through
the oculars.
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Simplified lens system of a
microscope
Ocular
Condenser Objective
Intermediate
image
Sample
— E = esine, obj = objektiivi, Ok = okulaari, kond = kondensori
Final image
Behaviour of light;
Interference
— The phase of two or more
wavefronts (”wave up or
down”) affects the total
intensity of light in each
position
— Application; phase contrast
and DIC-microscopy
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Behaviour of light;
Diffraction
— As a wavefront propagates throught hole(s), it takes
shape of arch à round pattern having the brightest
spot in the middle
— The pattern is caused by interference; the wavefronts
create a constructive interference in bright spots
— The phenomenon limits the resolution in microscopy
Abbe’s theory for image formation
— Carl Zeiss hired Ernst Abbe to develop lens systems for
microscopes
— Abbe defined a theory for image formation (1873):
— The specimen act as a grid causing diffraction
— Lens forms images of several order; the furthest consist
information of the finest details
à Challenge to collect all information
— Theory is utilized in the
present Microscopes!
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Numeriral aperture of an objective
— The resolution is dependent on the amount of light
collected by the objective (remember Abbe’s theory)
— Term "Numerical aperture (NA)" is a product of
physical properties of the objective:
— q = half of the solid angle and n refractive index
NA = n sin q
—
E.g. for the best oil immersion
objectives n = 1.515 and regular n= 1.000
Basics of micriscopy;
Resolving power
— Resolving power (or; resolution)
—
Minimum distance between two objects that can be
imaged as separate
—
—
—
Human eye: 0.1 mm = 100 μm = 100000 nm
Light microscope: 0.0002 mm = 0.2 μm = 200 nm
Electron microscope: 0.0000001 mm = 0.0001 μm = 0.1 nm
silmä
Eye
Light
microscope
valomikroskooppi
läpäisyelektronimikroskooppi
Transmission electron microscope
0
1
10
100
0,01
0,1
1000
1
10000
100000
10
100
0,01
0,1
1000000 nm
1000
1
um
mm
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Basics of microscopy;
Resolving power
— Formed image is not a point-like but circular (so called Airy disc)
Point-like
object
Formed image
Aperture /
specimen
causes
diffraction and
interference
r
Basics of microscopy;
Resolving power
— When Airy discs can be separated for two point-like
sources
à distance (r) between sources = resolving power
— Abbes’s law:
RP =
0.61l
NA
— E.g. purple light (400 nm) and a good objective (NA = 1.4):
RP = (0.61 x 400nm)/(1.4)= 174 nm = 0.174µm)
(compare; red blood cell approx. 5-7 µm)
— NB! Resolving power is affected by several factors; optical
components, alingment, specimen etc.
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Factors affecting resolving power
— NAcondenser > NAobjective ; resolving power defined by objective
RP =
0.61l
NAobjective
— NAcondenser > NAobjective ; resolving power defined as
RP =
1.22l
NAobjective + NAcondenser
Lens distortions i.e. aberrations 1
— Two types of lens distortions; chromatic and geometric
(monochromatic)
— Chromatic aberration:
— Light of different wavelengths focused at different
distances (i.e. refractive index depenfs on wavelength)
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Lens distortions i.e. aberrations 2
— Geometric aberration;
— Spherical aberration:
—
—
—
Light is refracted more on the edge of the lens than in the
middle
Reduces resolution significantly
Prevented by complex lens systems and by preventing light
from passing the edges of the lenses
Lens distortions i.e. aberrations 3
— Geometric aberrations, continued:
— Coma = image distorted away from the center axis
—
Corrected with sperical aberration
— Curvature of field = a flat subject plane being imaged as the
surface of a sphere instead of a flat plane
—
Corrected objectives with prefix ”plan” (i.e. "Plan-achromat")
— Astigmatism = light in the vertical plane being focused
differently to light in the horizontal plane
—
Corrected with curvature of field
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Effect of some aberrations
Images from: micro.magnet.fsu.edu
Depth of field
— Depth of field (DOF), i.e. how thick slice
can be in focus
nl
DOF =
NA2
— E.g. green light (555 nm):
— Hint: depth of field can be improved by
closing aperture stop. However, resolving
power is decreased
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Microscope components
— Light source (15)
— Luminous stop (11)
— Condensor with lenses, optics and
stop (8)
— Sample stage (7)
— Objectives (6)
— Imaging system focused on the
image formation plane (2,3)
— Various additional components;
polarizers, prisms, etc.
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Microscope components; objective
— Most important component in a microscope!
— Objectives in a microscope should be parfocal
— The higher the magnification (i.e. higher NA), the
smaller the working distance
Objectives
— Even the simpliest objective contains several lenses for
compensating lens distortions
— Plan; cheapest, flat field corrected
— Achromat; chromatic aberration is corrected for red and
blue light and spherical aberration for green light
— Fluorite;"semi-apochromat", better than the previous,
spherical aberration corrected for green and blue light
— Apochromat; the best, chromatic aberration corrected
for four wavelenghts and spherical aberration corrected
for three wavelengths
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Structure of an objective
Some principles
— Rule of thumb 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
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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
Thus when possible, use low magnification
or high NA objectives
Magnification with the microscope
— In practice, the achieved magnification is a product of
magnifications of each optical component
— E.g. 10x objective, 10x oculars (intermediate lens 1.25x)
--> total magnification 100x (125x)
—
NB! This is magnification for your observation.
In publications/presentations, show always the scale bar!
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Other types of light microscope
— Physical effects to increase contrast;
— Suitable for observation of unstained specimens
(i.e. live cells, particles, material science).
— Typically, special objectives, condensors, prisms
or polarizers are needed.
Dark field microscopy
— Only the of light rays at the edge of the light are used
for illumination
--> scattered and/or diffracted light from the specimen
is visualized as bright on dark background
— E.g. unstained biological specimens, particle-like
specimens
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Polarized light microscopy
— 2 polarizers (grids) in perpendicular
orientation are positioned below and
above the specimen
Analyzer
— Note: like two polaroid sunglasses
à image is black
— If the specimen is anisotropic the light is
rotated due to the specimen à part of the
light is trasmitted through the analyzer à
image
— In biology; cartilage, bone (collagen)
Specimen
Polarizer
Light source
Other techniques
— Phase contrast microscopy, differential inteference
contrast microscopy (DIC)
— Lectures later…
— Epi-fluorescence microscopy, confocal microscopy
— Lectures later…
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Other techniques 2
— Stereo zoom microscopy
— 3-dimensional visualization
— Relatively low magnification
— Large depth-of-view
— Motorized microscope
— E.g. applications of
”epi-illumination”
—
An example…
Example of ”epi-illumination”
— Surface topography of rough (uneven) specimen can
be reconstructed
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”Image slices" by epi-fluorescense
— Apotome system can be attached to a Zeiss microscope
à increase in resolution along z-axis
— ”Poor man’s confocal microscope"
With Apotome
Without apotome
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The use of microscope
— Remember to adjust Köhler illumination !
— E.g. Diffused illumination is prevented, performance is
optimized and the effect of dist particles in the optics
are minimized.
— Adjust the light intensity by using neutral density
filters and not by adjusting the lamp voltage!
— Clean all components after using immersion oil.
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Köhler illumination
— 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
Köhler Illumination
condenser
Specimen
Field iris
eyepiece
Field stop
retina
Conjugate planes for image-forming rays
Field iris
Specimen
Field stop
Conjugate planes for illuminating rays
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The use of microscope
KÖHLER ILLUMINATION (Zeiss AxioImager M2):
1. Choose 10x objective and adjust suitable light intensity.
2. Place a sample on the stage and focus.
3. Adjust eyepieces and their optics suitable for your eyes.
4. Close partially the luminous field stop (aperture) so that an image of luminous
field stop is visible (polygon).
5. Focus the luminous field stop image by adjusting the height of condenser.
6. Center the luminous field stop imafe with condenser centering screws.
7. Open the luminous field stop until its image disappears outside the field of view.
8. Adjust the aperture stop (in condenser) :
a.
b.
c.
Remove another eyepiece (ocular).
Look into the empty tube (à you will again see the polygon) and fully open the
aperture stop.
Close the aperture stop until approximately 1/3 of the FOV is not illuminated.
NB! Steps 7 and 8 must be repeated after each objective change
to achieve optimal illumination.
Cleaning of the microscope
— Optical components can be cleaned with…
— Special cleaning solution (50ml distilled water + 49ml
isopropanol + 1ml 25% ammonia)
— 70% or 100% ethanol
— DON’T USE ACETONE!
— Remove dust or dirt with pressurized air
— Clean the components from immersion oil immediately
— Remember to cover the microscope with the hood!
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”Troubleshooting"
— No illumination: Check lamps and fuses
— Illumination, but no image: Check the optical path; do
—
—
—
—
apertures, filters, polarizers etc. block the path
Undefined disturbance in the image: Check Köhler
illumination
Dust particles in image: Locate the dust and clean
(also Köhler illumination)
Blurred image: Clean the objectives
Something else: Call for help
Literature
— Resources:
— Lounatmaa K.& Rantala I. (1996) Biologisen valomikroskopian perusteet.
—
—
—
—
Yliopistopaino.
Chandler & Roberson: Bioimaging; Current concepts in light and electron
microscopy. Jones and Bartlett Publishers 2009
Bell S. & Morris K.: An Introduction to microscopy. Taylor and Francis 2010
http://micro.magnet.fsu.edu
http://www.microscopyu.com
— Software:
— ImageJ: http://rsb.info.nih.gov/ij/
— BioimageXD: http://www.bioimagexd.net/
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