Introduction to The Light Microscope

Light Microscopy
• Four centuries of history
• Vibrant current development
• One of the most widely used research tools

(Image: T. Wittman, Scripps)

Major Imaging Functions of the Microscope

• Magnify

• Resolve features

A. Khodjakov et al.
• Generate Contrast

• Capture and Display Images

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 An Upright Epifluorescence Microscope
Waves vs. Photons vs. Rays

• Quantum wave-particle duality

• Rays: photon trajectories

• Rays: propagation direction of waves

Rays are perpendicular to wavefronts Light travels more slowly in matter

The speed ratio is the Index of Refraction, n
v = c/n

n=1 n>1 n=1

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 Refraction by an Interface
Refractive Index Examples θr
Incident wave Reflected
• Vacuum 1 wave
θ1
• Air 1.0003
Refractive index n1 = 1
• Water 1.333 Speed = c
λ

• Cytoplasm 1.35–1.38 ?
• Glycerol 1.475 (anhydrous)
• Immersion oil 1.515
Refractive index n2
• Fused silica 1.46 Speed = c/n
θ2 Refracted wave
• Optical glasses 1.5–1.9 λ/n

• Diamond 2.417
⇒ Snell’s law: Mirror law:
Depends on wavelength and temperature n1 Sin(θ1) = n2 Sin(θ2) θr = θ1

Which Direction? Lenses work by refraction

n1 Incident light

focus
n2 > n1

Focal
Refraction goes length f
towards the normal
in the higher-index medium

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 Ray Tracing Rules of Thumb Imaging
(for thin ideal lenses)

Parallel rays converge Rays that cross in the focal plane

at the focal plane end up parallel f
Image
Object

d1 d2

f f L1 L2

Rays through the lens center are unaffected

The lens law: Magnification:

Finite vs. Infinite Conjugate Imaging Back focal plane

• Finite conjugate imaging (older objectives) Back
Image focal
f0 plane
Object
f0

Object

>f0 f0

• Infinite conjugate imaging (modern objectives). Image at infinity

f1 ⇒ Need a tube lens
Image
f0 f0 f0
Object

Rays that leave the object with the same angle

(uncritical)
f0 f0 f1
meet in the objective’s back focal plane
Magnification:

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The Compound Microscope The Compound Microscope
Final image
Eye

Exit pupil Exit pupil

Eyepiece Eyepiece

Primary or intermediate Intermediate

image plane image plane

Tube lens Tube lens

Back focal plane (pupil) Back focal plane (pupil)

Objective Objective
Sample Object plane Sample Object plane

The Compound Microscope The Compound Microscope

Final image Final image
Eye Eye

Exit pupil Exit pupil

Eyepiece Eyepiece

Intermediate Intermediate
image plane image plane

Tube lens Tube lens

Back focal plane (pupil) Back focal plane (pupil)

Objective Objective
Sample Object plane Sample Object plane

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The Compound Microscope The Compound Microscope
Final image Camera Final image
Eye

Exit pupil
Secondary pupil

Eyepiece Projection Eyepiece

Intermediate Intermediate
image plane image plane

Tube lens Tube lens

Back focal plane (pupil) Back focal plane (pupil)

Objective Objective
Sample Object plane Sample Object plane

Eyepieces (Oculars) Trans-illumination Microscope

Camera Final image plane

Secondary pupil plane

Features Projection Eyepiece
Imaging
• Magnification (10x typical) path
Intermediate image
• “High eye point” (exit pupil high plane
enough to allow eyeglasses) Tube lens
• Diopter adjust (at least one
must have this) Back focal plane (pupil)
• Reticle or fitting for one Objective
• Eye cups Sample Object plane
Condenser lens The aperture iris
Aperture iris (pupil plane) controls the range of
illumination angles
Illumination Field lens
path The field iris
Field iris (image plane)
controls the
illuminated
Collector field of view
Light source (pupil plane)

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 Köhler Illumination Critical Illumination
Conjugate Planes in A Research Microscope
Sample Object plane

Aperture iris (pupil plane)

Field iris (image plane)

Light source (pupil plane)

• Each light source point produces a parallel beam • The source is imaged onto the
of light at the sample sample
• Uniform light intensity at the sample even if the • Usable only if the light source
light source is “ugly” (e.g. a filament) is perfectly uniform

How view the pupil planes? By far the most important part:
the Objective Lens

Two ways:

• “Eyepiece telescope”
• “Bertrand lens”

Each major manufacturer sells 20-30 different categories of objectives.

What are the important distinctions?

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 Working Distance The focal length of a lens
depends on the refractive index…

Refractive index n
In general, high NA lenses
have short working
distances
However, extra-long
working distance objectives
do exist
Focal
length f
Some examples:
10x/0.3 WD = 15.2mm
20x/0.75 WD = 1.0mm
f ∝ 1/(n-1)
100x/1.4 WD = 0.13mm

… and the refractive index

depends on the wavelength ⇒ Chromatic aberration
(“dispersion”)
• Different colors get focused to different planes
• Not good…

Glass
types

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 Dispersion vs. refractive index
Achromatic Lenses
of different glass types Refractive
index
• Use a weak negative flint glass element
to compensate the dispersion of a
positive crown glass element

Abbe dispersion number (Higher dispersion→)

Achromats and Apochromats Correction classes of objectives

Focal
length
error

Wavelength

Apochromat (≥3 glass types) Achromat Fluor Apochromat

(cheap) “semi-apo” (best correction)
(good correction,
Achromat (2 glass types) high UV
transmission)

Simple lens
Correction for other (i.e. monochromatic) aberrations
also improves in the same order

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 Curvature of Field Plan objectives
• Corrected for field curvature
Focal plane • More complex design
Focal surface
• Needed for most photomicrography

Tube lens

objective

sample
Focal
surface • Plan-Apochromats have the highest performance
(and highest complexity and price)

Interference Diffraction by a periodic structure (grating)

In phase
constructive interference

=
+

Opposite phase

destructive interference
=
+

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Diffraction by a periodic structure (grating) Diffraction by an aperture drawn as waves

Light spreads to new angles

θ
θ
d
Larger aperture
⇔
weaker diffraction
)
in(θ
dS

In phase if:
d Sin(θ) = m λ
for some integer m

Diffraction by an aperture drawn as rays The Airy Pattern

= the far-field diffraction pattern from a round aperture
The pure, “far-field”
diffraction pattern
is formed at ∞ distance…
Height of
first ring
≈ 1.75%

“Airy disk” diameter

d = 2.44 λ f/d
(for small angles d/f)
Tube lens

…or can be formed

at a finite distance
by a lens… d

f
Intermediate
…as happens in a microscope
Objective pupil image

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 Aperture and Resolution Aperture and Resolution
Diffraction spot Diffraction spot
on image plane on image plane
= Point Spread Function = Point Spread Function
Intermediate Intermediate
Objective Tube lens image plane Objective Tube lens image plane

Sample Sample

Back focal plane aperture Back focal plane aperture

Aperture and Resolution Aperture and Resolution

Diffraction spot Diffraction spot
on image plane on image plane
= Point Spread Function (resolution)
Intermediate Intermediate
Objective Tube lens image plane Objective Tube lens image plane

α
Sample Sample

Back focal plane aperture Back focal plane aperture

• Image resolution improves with aperture size Numerical Aperture (NA)

α = light gathering angle

NA = n sin(α) where:
n = refractive index of sample

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 Numerical Aperture Immersion Objectives

100X / 0.95 NA
α = 71.8°

⇒ NA cannot exceed Oil immersion:

the lowest n between the n ≈ 1.515
sample and the objective lens max NA ≈ 1.4 (1.45–1.49 for
⇒ NA >1 requires fluid immersion TIRF)
Glycerol immersion:
4X / 0.20 NA
n ≈ 1.45 (85%)
α = 11.5°
NA can approach max NA ≈ 1.35 (Leica)
Water immersion:
the index of the n ≈ 1.33
immersion fluid max NA ≈ 1.2

Resolution (Abbe’s argument, continued)

Ernst Abbe’s argument (1873)
Now consider oblique illumination
Consider a striped sample ≈ a diffraction grating (an off-axis source point):
Back focal
plane
One spot hopelessly lost,
but two spots get through
Objective lens → interference → image formed!

β Diffracted beams
βout
d sin(β) = λ d [sin(βin) + sin(βout) ] = λ
Smaller d
Sample
→ larger β
d Two spots get through if
d βin
Condenser βout < α and βin < α.

Light source
Consider first
a point light source If β > α, only one spot makes it through
Resolution (smallest resolvable d)
⇒ no interference ⇒ no image formed
with incoherent illumination (all possible illumination directions):
Resolution (smallest resolvable d): dmin = λ/(NAobj + NAcondenser )
dmin = λ sample/sin(α) = λ/n sin(α) = λ/NA λ/2 NA if NAcondenser ≥ NAobj (“Filling the back focal plane”)

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 Aperture, Resolution & Contrast Alternate Definitions of Resolution
Can adjust the condenser NA with the aperture iris

Imaging Intermed. image As the Full Width at Half Max

path (FWHM) of the PSF
Tube lens
FWHM
Back aperture ≈ 0.353 λ /NA
Objective Q: Don’t we always want
Sample it full open?? As the diameter of the Airy disk
Condenser lens (first dark ring of the PSF)
Aperture iris A: No = “Rayleigh criterion”
Airy disk diameter
Field lens ≈ 0.61 λ /NA
Illumination Why? Tradeoff: (Probably most common definition)
path Field iris resolution vs. contrast

Collector

Light source

Objective Types
Basic properties
• Magnification
• Numerical Aperture (NA)
• Infinite or finite conjugate
• Cover slip thickness if any
• Immersion fluid if any
Further reading
www.microscopyu.com
Field flatness
• Plan or not micro.magnet.fsu.edu
Douglas B. Murphy “Fundamentals of Light Microscopy and
Phase rings for phase contrast
• Positive or negative
Electronic Imaging”
• Diameter of ring (number)
James Pawley, Ed. “Handbook of Biological Confocal
Microscopy, 3rd ed.”
Special Properties
• Strain free for Polarization or DIC

Features
Correction class • Correction collar for spherical aberration
• Achromat • Iris Acknowledgements
• Fluor • Spring-loaded front end
• Apochromat • Lockable front end
Ron Vale / Mats Gustafsson

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