Introduction to Light Microscopynic.ucsf.edu/dokuwiki/lib/exe/fetch.php?media=1.microscopy_i.pdf ·...
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(Image: T. Wittman, Scripps)
Introduction to Light Microscopy
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The Light Microscope
• Four centuries of history
• Vibrant current development
• One of the most widely used research tools
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A. Khodjakov et al.
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Major Imaging Functions of the Microscope
• Magnify
• Resolve features
• Generate Contrast
• Capture and Display Images
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An Upright Epifluorescence Microscope
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Waves vs. Photons vs. Rays
• Quantum wave-particle duality
• Rays: photon trajectories
• Rays: propagation direction of waves
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Rays are perpendicular to wavefronts
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Light travels more slowly in matter
n = 1 n > 1 n = 1
v = c/n
The speed ratio is the Index of Refraction, n
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Refractive Index Examples
Depends on wavelength and temperature
• Vacuum 1
• Air 1.0003
• Water 1.333
• Cytoplasm 1.35–1.38 ?
• Glycerol 1.475 (anhydrous)
• Immersion oil 1.515
• Fused silica 1.46
• Optical glasses 1.5–1.9
• Diamond 2.417
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Reflected wave
r
Refraction by an Interface
Refractive index n1 = 1
Speed = c
Refractive index n2
Speed = c/n
Incident wave
1
Refracted wave2
n1 Sin(1) = n2 Sin(2)
Snell’s law:
/n
r = 1
Mirror law:
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Which Direction?
n1
n2 > n1
Refraction goes
towards the normal
in the higher-index medium
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Lenses work by refraction
Incident light
focus
Focal length
f
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Ray Tracing Rules of Thumb(for thin ideal lenses)
f f
Parallel rays converge
at the focal plane
Rays that cross in the focal plane
end up parallel
Rays through the lens center are unaffected
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Imaging
f
The lens law:
1
L11
L21
f
Image
Magnification:
M d2
d1L2
L1
L1 L2
Object
d1d2
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Finite vs. Infinite Conjugate Imaging
Objectf0
f0 f0
Objectf0
>f0 f0
Image• Finite conjugate imaging (older objectives)
Image at infinity• Infinite conjugate imaging (modern objectives).f1
f1
Magnification:
M f1
fo
Image
(uncritical)
Need a tube lens
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Back focal plane
Object
f0
f0 f0
Back focal plane
Rays that leave the object with the same angle
meet in the objective’s back focal plane
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The Compound Microscope
Sample
Objective
Tube lens
Primary or intermediateimage plane
Eyepiece
Back focal plane (pupil)
Exit pupil
Object plane
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The Compound Microscope
Sample
Objective
Tube lens
Intermediate image plane
Eyepiece
Object plane
Back focal plane (pupil)
Exit pupil
Eye
Final image
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Sample
Objective
Tube lens
Intermediate image plane
Eyepiece
Object plane
Back focal plane (pupil)
Exit pupil
Eye
Final image
The Compound Microscope
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The Compound Microscope
Sample
Objective
Tube lens
Intermediate image plane
Eyepiece
Object plane
Back focal plane (pupil)
Exit pupil
Eye
Final image
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The Compound Microscope
Sample
Objective
Tube lens
Intermediate image plane
Eyepiece
Object plane
Back focal plane (pupil)
Exit pupil
Eye
Final image
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The Compound Microscope
Sample
Objective
Tube lens
Intermediate image plane
Projection Eyepiece
Object plane
Back focal plane (pupil)
Secondary pupil
Camera Final image
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Eyepieces (Oculars)
Features
• Magnification (10x typical)
• “High eye point” (exit pupil high
enough to allow eyeglasses)
• Diopter adjust (at least one
must have this)
• Reticle or fitting for one
• Eye cups
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Trans-illumination Microscope
SampleObjective
Tube lens
Intermediate image plane
Projection Eyepiece
Object plane
Back focal plane (pupil)
Secondary pupil plane
Camera Final image plane
Imaging
path
Aperture iris
Field iris
(pupil plane)
(image plane)
(pupil plane)Light source
Illumination
path
Collector
Condenser lens
Field lens
The aperture iris
controls the range of
illumination angles
The field iris
controls the
illuminated
field of view
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Köhler Illumination
Object plane
(pupil plane)
(image plane)
(pupil plane)
Sample
Aperture iris
Field iris
Light source
Critical Illumination
• Each light source point produces a parallel beam
of light at the sample
• Uniform light intensity at the sample even if the light source is “ugly” (e.g. a filament)
• The source is imaged onto the
sample
• Usable only if the light source is perfectly uniform
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Conjugate Planes in A Research Microscope
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Two ways:
• “Eyepiece telescope”
• “Bertrand lens”
How view the pupil planes?
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By far the most important part:
the Objective Lens
Each major manufacturer sells 20-30 different categories of objectives.
What are the important distinctions?
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Working Distance
In general, high NA lenses
have short working
distances
However, extra-long
working distance
objectives do exist
Some examples:
10x/0.3 WD = 15.2mm
20x/0.75 WD = 1.0mm
100x/1.4 WD = 0.13mm
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The focal length of a lens
depends on the refractive index…
Focal length f
f 1/(n-1)
Refractive index n
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… and the refractive index
depends on the wavelength
(“dispersion”)
Glass
types
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Chromatic aberration
• Different colors get focused to different planes
• Not good…
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Dispersion vs. refractive index
of different glass types
Abbe dispersion number
Refractive
index
(Higher dispersion)
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Achromatic Lenses
• Use a weak negative flint glass element
to compensate the dispersion of a
positive crown glass element
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Achromat (2 glass types)
Apochromat (3 glass types)
Achromats and Apochromats
Wavelength
Focal
length
error
Simple lens
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Correction classes of objectives
Achromat
(cheap)Fluor
“semi-apo”
(good correction,
high UV
transmission)
Apochromat
(best correction)
Correction for other (i.e. monochromatic) aberrations
also improves in the same order
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Curvature of Field
Focal planeFocal plane
Focal surface
sampleFocal
surface
objective
Tube lens
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Plan objectives
• Corrected for field curvature
• More complex design
• Needed for most photomicrography
• Plan-Apochromats have the highest performance
(and highest complexity and price)
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InterferenceIn phase
Opposite phase
+
+
=
=
constructive interference
destructive interference
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Diffraction by a periodic structure (grating)
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Diffraction by a periodic structure (grating)
In phase if:
d Sin() = m
for some integer m
d
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Diffraction by an aperture
Larger aperture
weaker diffraction
Light spreads to new angles
drawn as waves
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Diffraction by an aperture
The pure, “far-field”
diffraction pattern
is formed at distance…
…or can be formed
at a finite distance
by a lens…
…as happens in a microscopeObjective pupil
Intermediate
image
Tube lens
drawn as rays
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The Airy Pattern= the far-field diffraction pattern from a round aperture
“Airy disk” diameter
d = 2.44 f/d
(for small angles d/f)
d
f
Height of
first ring
1.75%
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Tube lens
Back focal plane aperture
Intermediate image plane
Diffraction spot
on image plane
= Point Spread Function
Sample
Objective
Aperture and Resolution
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Aperture and Resolution
Tube lens
Back focal plane aperture
Intermediate image plane
Sample
Objective
Diffraction spot
on image plane
= Point Spread Function
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Aperture and Resolution
Tube lens
Back focal plane aperture
Intermediate image plane
Sample
Objective
Diffraction spot
on image plane
= Point Spread Function
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Aperture and Resolution
• Image resolution improves with aperture size
Sample
Objective Tube lens
Back focal plane aperture
Intermediate image plane
Diffraction spot
on image plane
(resolution)
Numerical Aperture (NA)
NA = n sin() = light gathering angle
n = refractive index of samplewhere:
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Numerical Aperture
4X / 0.20 NA
= 11.5°
100X / 0.95 NA
= 71.8°
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Immersion Objectives
Oil immersion:n 1.515
max NA 1.4 (1.45–1.49 for TIRF)
Glycerol immersion:
n 1.45 (85%)
max NA 1.35 (Leica)
Water immersion:
n 1.33
max NA 1.2
NA can approach
the index of the
immersion fluid
NA cannot exceed
the lowest n between the
sample and the objective lens
NA >1 requires fluid immersion
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ResolutionErnst Abbe’s argument (1873)
Sample
Objective lens
Back focal
plane
Condenser
Light source
Consider a striped sample ≈ a diffraction grating
b Diffracted beams
d sin(b) =
d
Consider first
a point light source
Smaller d
larger b
If b > , only one spot makes it through
no interference no image formed
Resolution (smallest resolvable d):
dmin = sample/sin() = /n sin() = /NA
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(Abbe’s argument, continued)
Now consider oblique illumination
(an off-axis source point):
boutd [sin(bin) + sin(bout) ] =
One spot hopelessly lost,
but two spots get through
interference image formed!
Resolution (smallest resolvable d)
with incoherent illumination (all possible illumination directions):
dmin = /(NAobj + NAcondenser )
/2NA if NAcondenser NAobj (“Filling the back focal plane”)
binTwo spots get through if
bout < and bin < .d
![Page 53: Introduction to Light Microscopynic.ucsf.edu/dokuwiki/lib/exe/fetch.php?media=1.microscopy_i.pdf · Introduction to Light Microscopy. The Light Microscope • Four centuries of history](https://reader036.fdocuments.us/reader036/viewer/2022062402/5ee209d9ad6a402d666cb07f/html5/thumbnails/53.jpg)
Aperture, Resolution & Contrast
Can adjust the condenser NA with the aperture iris
Sample
Objective
Tube lens
Imaging
path
Aperture iris
Field iris
Light source
Illumination
path
Collector
Condenser lens
Field lens
Back aperture
Intermed. image
Q: Don’t we always want
it full open??
A: No
Why? Tradeoff:
resolution vs. contrast
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NA and Resolution
High NA Objective Low NA Objective
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Alternate Definitions of Resolution
As the Full Width at Half Max
(FWHM) of the PSF
As the diameter of the Airy disk
(first dark ring of the PSF)
= “Rayleigh criterion”
(Probably most common definition)
Airy disk radius
≈ 0.61 /NA
FWHM
≈ 0.353 /NA
![Page 56: Introduction to Light Microscopynic.ucsf.edu/dokuwiki/lib/exe/fetch.php?media=1.microscopy_i.pdf · Introduction to Light Microscopy. The Light Microscope • Four centuries of history](https://reader036.fdocuments.us/reader036/viewer/2022062402/5ee209d9ad6a402d666cb07f/html5/thumbnails/56.jpg)
Objective Types
Field flatness• Plan or not
Phase rings for phase contrast• Positive or negative
• Diameter of ring (number)
Special Properties• Strain free for Polarization or DIC
Features• Correction collar for spherical aberration
• Iris
• Spring-loaded front end
• Lockable front end
Basic properties• Magnification
• Numerical Aperture (NA)
• Infinite or finite conjugate
• Cover slip thickness if any
• Immersion fluid if any
Correction class• Achromat
• Fluor
• Apochromat
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Further reading
www.microscopyu.com
micro.magnet.fsu.edu
Douglas B. Murphy “Fundamentals of Light Microscopy and
Electronic Imaging”
James Pawley, Ed. “Handbook of Biological Confocal
Microscopy, 3rd ed.”
Acknowledgements
Ron Vale / Mats Gustafsson