BioImaging &Optics Platform
Basics
in
light microscopy
Dr. Arne Seitz
Swiss Institute of Technology (EPFL)
Faculty of Life Sciences
Head of BIOIMAGING AND OPTICS – BIOP
arne.seitz@epfl.ch
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
BioImaging &Optics Platform
Overview
1.
2.
3.
4.
5.
6.
Motivation
Basic in optics
How microscope works
Illumination and resolution
Microscope optics
Contrasting methods
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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1.Motivation
• Why do we need microscopy?
• Main issues of microscopy
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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The name:
Microscopy
greek
mikros= small
skopein= to observe
“Observation of small objects”
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Human eye
Normal viewing distance - 250 mm
Angular resolution αmin ≈ 1’
Spatial resolution hmin ≈ 80 µm
Nodal distance -17 mm
Average retinal cell distance 1.5 µm
Spectral range 400 nm - 800 nm
Can resolve contrast about 5%
High dynamic range – 10 decades
Max sensitivity at 505 nm (night, rods)
Max sensitivity at 555 nm (day, cones)
More sensitive to color than to intensity
Most perfect sensor for light detection up to now
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Main issues of Microscopy
In order to observe “small objects”, three preconditions have to
be fulfilled
1. Magnification
2. Resolution
3. Contrast
Only fulfillment of these three conditions allows translation of
information as accurately as possible from object into an image
which represents that object.
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Image formation
Light is the messenger and transports the object information from the
specimen through the microscope
Light translates the object information into a microscopic image of the
specimen
The observer observes the microscopic image of the specimen not the
specimen itself !
Only best management of the light allows translation of information as
accurately as possible from object into an image which represents that
object!
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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2.Basics in Optics
• What is light?
• Geometrical optics
• Thin lenses
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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What is light?
Light can be described as an electromagnetic wave
(=electromagnetic radiation).
Light can be described as a particle (photon)
Wave-particle duality
Main properties of light are:
• Intensity
• Frequency or wavelength
• Polarization
• Phase
Study of light in known as optics
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Geometrical optics
• also known as Gaussian optics
•light propagation is explained in terms of “rays”
•an optical axis can be defined and all rays are almost parallel to it
(= paraxial approximation)
•does practically an excellent job
(even under conditions where the paraxial condition is not fulfilled!)
• no wavelength (fails to explain resolution!)
Optical
axis
sin Θ ~ Θ
tan Θ ~ Θ
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
cos Θ ~ 1
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Basics of geometrical optics
h - object height; h’’ - image height
s - object distance; s’’ - image distance
f - effective focal length
Lens formula: 1/f = 1/s’+1/s’’,
m - magnification
m = s”/s’=h”/h’
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Magnifying glass
α2
virtual image
α1
object
250 mm
f
Magnifier increases the angular size of the object
M=α2/α1
Magnification is defined by focal distance of lens
M=250/f
Maximum magnification of magnifying glass is 10x-20x
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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How a thin lens works
Lens focuses
collimated beam of
light parallel to optical
axis into on axis spot
Beams in focus are in
phase
Lens focuses oblique
collimated beam into
an off axis focal spot
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3. How microscope works
Compound microscope
Convergent and infinite beam paths
Components of microscope
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Compound microscope - convergent
beam path
Sample is placed in front of objective focal plane. Intermediate image
is formed by objective and is observed through eyepiece.
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Disadvantage of a convergent beam path
Convergent beam
Parallel beam
Beam is focused differently
More aberrations
Beam is only shifted
Less aberration
Presence of parallel light beam is microscope light path is important
for modern light microscope (for filters, and other optical elements)
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Compound microscope - infinity-corrected
beam path
The sample is placed in the focal plane of the objective. Parallel light beams are
focused by the tube lens. The intermediate image is observed through the eyepiece.
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Objective
Objective are constructed of several high quality lenses.
For infinity corrected objective the specimen is in the focal plane
For not infinity corrected objectives the specimen is in front of the focal plane
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Eyepiece
The eyepiece acts as a magnifier of the intermediate image
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Camera as image detector
When the camera is used, the intermediate image is directly
projected on the camera chip (additionally an intermediate magnifier might be used).
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Microscope Designs
Upright
Inverted
Used in biology mostly for
fixed specimens
Widely used in biology for
living cell imaging
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Main microscope components
field diaphragm (t)
Hal lamp
Hg lamp
condenser
aperture
diaphragm (t)
eyepiece
objective
filter cube
turret
focus
camera
field diaphragm (f)
aperture
diaphragm (f)
stage
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
DIC slider
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Anatomy of microscope
Two independent
illumination paths:
• Transmission
• Fluorescence
Components for
contrasting methods:
• DIC
• Dark field
• Phase contrast
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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How microscope works: summary
Magnifying glass has a limited magnification of 10x-20x
Compound microscope makes two stage magnification
• initial magnification with objective
• further magnification with eyepiece
Compound microscope beam path designs
• finite – old microscopes
• infinity corrected – modern microscopes
There are several microscope types
• inverted
• upright
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4. Illumination and resolution
Koehler illumination
Diffraction of light
Numerical aperture
Resolution
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Light sources
Halogen lamp
• Continuous spectrum: depends on temperature
• For 3400K maximum at 900 nm
• Lower intensity at shorter wavelengths
• Very strong in IR
Mercury Lamp (HBO)
• Most of intensity in near UV
• Spectrum has a line structure
• Lines at 313, 334, 365, 406, 435, 546, and 578 nm
Xenon lamp (XBO)
• Even intensity across the visible spectrum
• Has relatively low intensity in UV
• Strong in IR
Metal halide lamp (Hg, I, Br)
• Stronger intensity between lines
• Stable output over short period of time
• Lifetime up to 5 times longer
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Requirements for illumination
Uniform over whole field of view
Has all angles accepted by objective
Allows optimize image brightness/contrast
Allows continuous change of intensity
Allows continuous change of field of view
Change in illumination and imaging parts do
not effect each other
Realized in Kohler illumination
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Conjugated planes in optical microscopy
Image forming light path
(Observed with eyepiece)
1. Variable field diaphragm
2. Specimen plane
3. Intermediate image plane
4. Image plane (camera, retina)
Illumination light path
(Observed with Bertrand lens)
1. Lamp (filament, arc)
2. Condenser aperture diaphragm
3. Objective rear (back) focal plane
4. Eyepoint (exit pupil of microscope)
Conjugated = imaged onto each other
Has one diaphragm in every path
If light at given plane is focused in one
path, it is parallel in other path
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Collector and condenser
Collector
gathers light from
light source
Condenser
directs light onto
the specimen
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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How to set up Koehler illumination
Transmission
Fluorescence
• Focus on the specimen
• Focus on the specimen
• Close field diaphragm
• Focus condenser until field diaphragm is seen
sharp
• Center field diaphragm
• Close field diaphragm up to 80 – 90 %
• Remove eyepiece, look down to the aperture
diaphragm
• Center (if possible) aperture diaphragm
• Open/Close aperture diaphragm up to 80 – 90 %
• Swing in focusing aid (if available)
• Focus image of arc sharply
• Swing out focusing aid
• Close field diaphragm
• Center field diaphragm
Start with low magnification objective. Repeat for every objective used
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Diffraction of light
A parallel beam falls on the screen with pinholes.
Secondary spherical waves are formed on each
pinhole .
Interference results in several plane waves
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Diffraction orders
d=2λ
1st order (d = 5 λ)
-1
0
+1
d=1λ
for small enough
structures a
first diffraction
maxima is
perpendicular to
the direct light
1st order (d = 1.5 λ)
0
+1
d sin α = mλ
Direction of diffraction maxima depends on wavelength and period
Bigger period results in smaller diffraction angle
Bigger wavelength results in bigger diffraction angle
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Numerical aperture of objective
n =1
NA = n sin α 0
α0
!
!
n = 1.518
The NA defines how much light (brightness) and how many
diffraction orders (resolution) are captured by the objective.
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Role of immersion
NA=nsinα
Refractive indices:
Air - 1.003
Water - 1.33
Glycerol - 1.47
Oil - 1.52
Immersion media
increase the NA of an
objective or a condenser
by bringing the beams
with higher incidence
angle into the light path
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Role of condenser in image formation
NAtot=NAobj+NAcond
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Airy disc
NA=0.7
NA=1.3
Image of a dot is not a dot (PSF)
Airy disc is x-y section of PSF
r = 1.22λ/(NAobj + NAcond)
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Resolution of light microscope
Shortest distance between two points on a
specimen that can still be distinguished by the
observer or camera as separate entities.
Lateral resolution
δ R = 0.61
λ
NA
Axial resolution
δ zR = 2
λn
NA 2
λ=540 nm, NA=1.4, n=1.52: 235 nm - lateral, 838 nm - axial
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Illumination and resolution: summary
Choice of light source depends on application
• transmission – halogen lamp
• fluorescence – HBO, XBO, metal halide
Correct illumination is critical for successful imaging
• always set up Koehler illumination
• condenser as important as objective
Resolution is defined by NA and wavelength
•higher resolution for higher NA
• lower resolution for longer wavelength
Resolution is much better in lateral direction
• NA = 1.4, wavelength = 500 nm
• lateral resolution about 200 nm
• axial resolution about 800 nm
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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5. Microscope optics
Aberrations in optics
Eyepiece engravings
Objective engravings
Choice of magnification
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Optical aberrations
•
•
•
•
•
•
Astigmatism (tangential and meridianal focus are different)
Coma (image of dot is not symmetric)
Distortion (parallel lines are not parallel in image)
Curvature of the field (image of plane is not flat)
Chromatic (different focus for different wavelength)
Spherical (different focus for on and off axis beams)
It is desired to minimize aberrations by proper use of
objectives with good aberration correction
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Chromatic aberration
•
•
•
•
Use of lenses with different dispersion
Achromat (corrected for two colors)
Fluorite (better corrected)
Apochromat (corrected at least for three colors)
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Spherical aberration
Use cover slip 0.17 mm thick or
Use objective with correction ring
Avoid refraction index mismatch of
immersion and mounting media
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Eyepiece engravings
Field number (FN) – diameter of view field in
mm measured in intermediate image plane.
Magnification -10x, 16x, etc.
Eyepiece type
Pl – gives plane image
W - wide field of view
Also indicated: Diopter correction, use with glasses
Field Size = FN/(MobjxMint)
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Working distance and parfocal length
Parfocal distance
Distance from objective shoulder
till specimen plane
45 mm for most manufactures,
60 mm for Nikon CFI 60
Working distance
Distance from front edge of objective
till cover slip
Varies from several mm till several hundreds
micrometers. Special long working distance
objective are available.
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Engravings on objectives
Epi = observation from above (0 = no cover glass)
LD = long (working) distance
plan = minimal curvature in the image plane
APOCHROMAT = especially color corrected
HD = hell/dunkel = bright/dark field
DIC = differential interference contrast (low strain optics for polarized light)
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Objectives with correction collars
NEOFLUAR optics is less color corrected than APOCHROMAT
Range of cover glass thickness
W
W
Glyc
Oil
Ph = phase contrast
(3 specifies matching condenser)
Different immersion media under
various cover glass conditions
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Total microscope magnification
Defined by magnification of objective, eyepiece and intermediate
magnification
Mtot=Mobj x Mint x Meyepiece
Objective magnification defined by focal lengths of tube lens and
objectives
Mobj=ftl/fobj
Tube lens has a standardized value for specific manufacture
Zeiss, Leica, Olympus 165 mm, Nikon 200 mm
Typical magnification rangies:
• Mobj: 2x÷100x
• Mint: 1.5x÷2.5x
• Mobj: 10x÷25x
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Useful magnification range
• Microscope resolution is limited by NA and wavelength.
• Enlargement of image does not necessarily resolve new features.
• Excessively large magnification is called empty magnification.
(The Airy disk on retina/camera should not exceed two
cell/pixel sizes).
Useful magnification = 500-1000 x NA of objective
Mobj
Meyepiece
NAobj
Mtot
Museful
Magnification
10x
10x
0.35
100
175-350
low
40x
10x
0.70
400
350-700
ok
100x
10x
1.40
1000
700-1400
ok
100x
15x
1.40
1500
700-1400
empty
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Light budget in microscope
Microscope has a lot of components in light path
• Microscope optics (T=0.8)
• Dichroic mirror (T=0.8)
• Filters (T=0.8)
• Objective, eyepiece (T=0.9)
• Objective collects light only within NA (T=0.3)
Typically only 10% of light arrives to CCD.
Use optics with antireflection coatings
Use high quality filters, dichroics
Use clean optics
Image brightness (transmission) ~ (NA/M)2
Image brightness (fluorescence) ~ NA4/M2
Use high NA objectives
Do not use unnecessary high magnification
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Microscope optics: summary
Correct choice of microscope optics is the key to successful imaging
Pay attention to the engravings on objective and eyepiece
Optical aberrations can be minimized
• use well corrected optics or use green filter
• use cover slip 0.17 mm thick
• match refractive index of immersion media and specimen
Choose magnification carefully
• excessive magnification does not reveal new details
• moreover it deceases the brightness of the image
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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4. Contrasting methods
Dark field
Phase contrast
DIC
PlasDIC
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Amplitude and phase specimens
Amplitude specimen changes the intensity of incident light
Phase specimen changes the phase of incident light
Most unstained biological specimens are phase ones
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Examples of contrasting methods
Dark field
Bone thin
section
DIC
Neurons
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
Phase contrast
HEK cells
PlasDIC
HEK cells
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Darkfield contrast
5 – iris diaphragm
4 - objective
3 - sample
2 - condenser
1 - phase stop
A - low NA objective
B - high NA objective with iris
Required: special condenser, sometimes
immersion oil
Principle: direct light is rejected or blocked,
only scattered light is observed
Disadvantage: low resolution
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Interference
+
-
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Interference
• Addition of waves
• Amplitude of the resulting wave depends on the pahse relation of two
waves
•Extreme cases:
destructive interference (res. amplitude =0)
positive interference
• With interference a phase difference can be turned into an amplitude
difference
Interference is the basic principle of Phase contrast and DIC.
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Phase Contrast Microscopy
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Phase contrast microscopy
9 - intermediate image
8 - tube lense
7 - indirect light
6 - direct light
5 - phase ring
4 - objective
3 - sample
2 - condenser
1 - phase stop
Required: special objectives and special
condensers.
Principle: direct light is attenuated and its
phase is shifted 90 . Contrast formed due
to interference between direct and
scattered light.
Disadvantages: relatively low resolution,
halos
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Differential interference contrast
9 - intermediate image
8 - tube lens
7 – analyzer
7a - λ-plate
6 - Wollaston prism
5 – objective
4 – sample
3 – condenser
2 – Wollaston prism
1 - polariser
Required: special accessories in light path
(prisms, polarizers).
Principle: specimen is sensed with two
linear polarized slightly shifted (<λ) light
beams. Difference in optical path of the
beams gives a contrast in image.
Disadvantages: accessories are relatively
expensive.
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DIC in details
DIC prism split beam into two perpendicularly polarized.
Shift between beams less that resolution of microscope.
Beams measure difference in optical path in specimen.
If retardation is not zero, they are interfere after
being recombined on the second DIC prism.
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De Seramont compensator
Rotation of polarizer relative to quarter wave plate gives circular polarized light .
This results in phase shift between beams after DIC prism. Thus the contrast of
the DIC image can be adjusted. This method of contrast change is equivalent
to the lateral shift of Wollaston prism but more accurate.
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Adjustment for DIC
(for inverted microscope)
• Set up Koehler illumination in transmission
• Move specimen out of the light path
• Insert polarizer, analyzer, lower Wollaston prism and remove eyepiece
• Shift Wollaston prism to place dark line in the center
• Turn polarizer until the line is seen mostly dark
• Insert eyepiece back into eyetube
• Insert upper Wollaston prism (in condenser)
• Move the specimen back into the light path
• Move lower Wollaston prism to get required contrast
• Rotate the stage to highlight desired area in the sample
• Insert the lambda plate if color staining is required
• Repeat procedure for each objective being used
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PlasDIC
Required: slit diaphragm, prism with
polarizer, analyzer.
Principle: A slit diaphragm creates a
pair of non-polarized light beams that
are λ/4 out-of-phase. The beams get
polarized just before being
recombined into a single beam in the
DIC-prism. The analyzer (linear) sets
a single polarization plane where the
components of the beam can
interfere.
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PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Contrasting techniques: summary
Dark field
Fine structural features at, and even below, the resolution limit of a
light microscope. Highly suitable for metallographic and
crystallographic examinations with reflected light.
Phase contrast
Used for visualizing very fine structural features in tissues and
single cells contained in very thin (< 5 µm), non-stained specimens.
DIC
Method shows optical path differences in the specimen in a relieflike fashion. The method is excellently suited for thick, non-stained
specimens (> 5 µm). Can be used for optical sectioning.
PlasDIC
The same specimen as conventional DIC but in plastic dishes.
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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More about light microscopy
1. Lecture
Biomicroscopy I + II, Prof. Theo Lasser, EPFL
2. Books
a) Digital microscopy, Sluder, G; Wolf, D.E., eds, Elsevier, 2003
b) Optics, 4th ed., Eugene Hecht, Addison-Wesley, 2002
3. Internet
a) http://micro.magnet.fsu.edu
b) b) Web sites of microscope manufactures
Leica
Nikon
Olympus
Zeiss
4. BIOp
EPFL, SV-AI 0241, Sv-AI 0140
http://biop.epfl.ch/
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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Acknowledgments
These slides are based on a lecture given by
Yuri Belyaev
(Advanced Light Microscopy Facility, EMBL Heidelberg)
during a practical course concerning basics of light microscopy. Thus a big thank to
him for providing them and making them available also here at EPFL.
Dr. Arne Seitz
PT-BIOP Course, Basics in Light Microscopy 2010, EPFL
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