Introduction to Light and Electron Microscopy

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Introduction to Light and Electron Microscopy. Introduction to Optics Part I James Bouwer. NEU 259. Spring 2007. Some Length Scales:. 1mm = 1m / 1000 millimeter 1 m = 1mm / 1000 micron 1 nm = 1 m / 1000 nanometer ~10 atoms. Nature of Light:. Particle Nature:

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Introduction to Light andElectron

Microscopy

Introduction to Optics

Part I

James Bouwer

NEU 259

Spring 2007

Some Length Scales:

• 1mm = 1m / 1000 millimeter
• 1 m = 1mm / 1000 micron
• 1 nm = 1 m / 1000 nanometer ~10 atoms

Nature of Light:

• Particle Nature:
• We can treat light as particles that move in straight lines or rays
• Wave Nature of light:
• We can treat light as a propagating electromagnetic wave

Where

 = wavelength of light (visible light ~400 to 700nm)

f = frequency of waves

c = speed of light in a vacuum (3.0 x108 m/sec)

Nature of Particles:

• Particles also have a wavelength when moving
• de Broglie Wavelength:

Where:

h = Planck’s constant (6.63 x 10-34 Kg m/sec)

m = mass of the particle

 = velocity of the particle

Optics I

What is the refractive index n… really?

• n is the ratio of the speed of light in a vacuum to the speed of light
• in the medium
• n determines how much the light is bent as it enters a medium such as glass

North

Wavefront Lensing at Rincon

Refraction:

• Snell’s Law:

n = refractive index of medium

• Examples:
• Ocean waves always approach the shore nearly perpendicular
• Swimming pools always look shallower than they really are…
• belly flops are always safe!

red

n1

n2

green

The refractive index depends of wavelength:

• Light of different colors is bent by different amounts:

Refractive index vs wavelength

• Bending of trajectories is the basis for all lenses including electron magnetic lenses

f

f

Thin Spherical Lenses

• Ability to focus parallel light to a point or a virtual point

Converging Lens:

Diverging Lens:

image

object

Forming Images with Lenses

Lens equation:

Magnification:

Image Formation Using Light Rays:

• How Images are formed:
• An image plane is formed when all the light from a single point on an object
• … here the tip of an arrow... recombines on the othere side of the lens to form
• the image.
• Rules for drawing ray diagrams: (see lens diagrams on previous page)
• 1. Parallel light always goes through the focus on the other side of the lens.
• 2. Light traveling through the center of the lens is undeflected.
• 3. Light traveling through the focus comes out of the lens parallel on the other side.

Types of Images:

• Real images:
• These are images that can be created on a screen…
• Movie Projector
• Virtual images:
• These are images that cannot be projected on a screen
• and must be viewed with the eye…
• Magnifying Glass
• Diverging Lens

Light Collection Efficiency

• Three things most strongly affect light collection efficiency:
• Reflection at the lens surface
• Distance of object from lens
• Size of the lens
• The last two combine to from what is called Numerical Aperture:
• Numerical apertures range from 0.1 to 1.45 for LM objective lenses:
• Oil immersion lenses can increase NA by increasing n of the medium
• Light collection efficiency of 5% is considered extremely good

Limits of the Optical Imaging System:

The diffraction limit:

• As a general rule the best resolution theoretically attainable for
• any imaging system is given by :
• More accurately the resolving power for an optical system is given by:

What Does the Resolving Power or Diffraction Limit Mean:

What is ?

 represents the way each point is transferred to the image

through the lens:

• For an optical system, the best attainable resolution is on the order of
•  = 250 nm

500nm

The Diffraction Limited Spot

A bright point source imaged

on film

Two bright point sources just

separated by the resolvable

diffraction limit 

• This is sometimes called the point spread function (PSF)
• The center of the PSF can be located in an image and a point can be assigned
• to that center. This is the basis of deconvolution software.

Electron Microscope Resolution:

• Wavelength of an electron accelerated by a voltage, V:
• Where:
• V = accelerating Voltage in Volts
•  = relativistic correction factor (typically between 1 and 2 for standard EMs)
• Wavelength of an electron accelerated by a 400,000 volt EM

400KeV = 0.004 nm

• Due to spherical aberration in the lenses the best resolution attainable on modern
• EMs is around 0.10 nm or about the size of an atom.

Spherical Aberration

• Results from using lenses that are ground with spherical surfaces

Focal length depends on the ray position

•  is termed the circle of least confusion
• Spherical aberration tends to worsen the resolution of the microscope beyond
• the diffraction limit… especially true for EM
• Spherical aberration can be corrected with expensive aspherical lenses

n vs. 

A prisim separates colors

Chromatic Aberration

• Chromatic aberration results from the fact that
• the refractive index, n varies with 
• Lenses have the same problem with colors

Chromatic aberration causes serious problems for trying to perform co-localization experiments

blue

white

red

white

red

blue

Converging lens

Diverging Lens

Achromatic Doublet

Fixing Chromatic Aberration

• For a typical system red is bent less than blue:

Object

Image

Distortions

• Two types of lower order distortions:

I) Barrel Distortion:

Further

object

image

Closer

Origin of Pincushion and Barrel Distortions:

Magnification

• These distortions arise from the fact that various points in the object are
• closer or further from the lens and therefore, have different magnification

Correcting Barrel and Pincushion Distortion

• Combinations of lenses can be used to solve many problems

Other Types of Distortion

• Coma: Off-axis aberration resulting from a variation of lens focal lengths
• when moving out from the center of the lens
• Astigmatism: Aberration that results in off-axis light bundles to focus to an ellipse rather than a circle

Optical Instruments:

Simple Compound Microscope

A Real Objective Lens:

A 10 element, 1.3 NA oil immersion objective

Front element

Analogy between Transmission Light

and Electron Microscopes:

Outline of Topics

Part II

• Photon Energy
• Fluorescence Microscopy
• -Two level system
• -Fluorophores and Spectra
• -Epifluorescence
• Interference Optics
• -diffraction grating
• -interferometer
• Polarization of light
• DIC microscopy

Energy of a Photon

the Particle Nature of Light

(the Quantum)

h = 6.62 x 10-34 Joule*sec

f = frequency of light

c = 3.0 x 108 = speed of light (meters/sec)

 = wavelength (meters)

How Energy Depends on Wavelength

1eV = 1.6x 10-19 Joules

S

S

S

S

A

As

A

As

-

O

O

O

h

E2

h

E1

O2-

C

E0

F

l

A

s

H

-

E

D

T

2

g

r

e

e

n

f

l

u

o

r

e

s

c

e

n

c

e

,

F

R

E

T

f

r

o

m

C

F

P

Fluorescence Microscopy

A Typical Fluorescent Molecule

A Two Level Model of Fluorescence

 = 10-10 sec

 = 10-7 sec

Commonly used Fluorophores for

Multi-Color Labeling

Fluorescein goat anti–mouse IgG antibody

DAPI / DNA

Rhodamine Red-X goat anti–mouse IgG antibody

Criteria for Fluorophore Selection

• Excitation Spectrum:
• Damage to Sample? (Shorter Wavelengths are most damaging)
• Do excitation spectra overlap?
• Correct excitation and emission filters
• Emission Spectrum:
• How broad is the emission spectrum
• What is the quantum efficiency of the fluorophore
• - ranges between 0-1 (0-100%)
• describes percent of absorbed photons that emit fluorescence
• Does the fluorophore create singlet state reactive O2

- damaging to live cells

Epi-fluorescene Filter Setup

CCD Camera

Hg or Xe Lamp

Other Methods:

Laser Scanning Confocal

Two-photon excitation

Fluorescence Microscope Image of Rat Cerebellum

Green = GFAP

Red = IPR3 receptor in purkinje neuron

DiA

DiO

DiD

… A Tough Case

Image1

(Green)

The solution…Bleedthrough Subtraction

Image2

(Green Bleedthrough)

+

(Orange)

Orange Image

-

x

=

(%Bleed)

Steps:

1. Detemine bleedthrough percent

2. Subtract fraction of bleedthrough image from other image

Filament image

Kohler Illumination

• Method to ensure uniform illumination
• Across the sample plane
• Ensures that the filament image is in
• the back focal plane of the objective
• (No where near the image plane)
• Ensures matched NA of the illumination
• and cone and the objective lens

Filament Vs. Sample Image Planes for Kohler Illumination

Demonstration later

showing how to align

for Kohler illumination

The Wave Nature of light

Use of Phase Relationships

In Microscopy

What is a phase relationship:

360

2A

• Phase differences
• can create interference
• Constructive
• Destructive

 = 2 - 1

Interference as a Way to Measure

Spectra (Wave Properties of Light)

Two Slit Interference:

Constructive Interference:

(m = 0, 1, 2, 3 …)

Destructive Interference:

(m = 0, 1, 2, 3 …)

Diffraction Grating

m = 1

m = 1

constructive interference:

movable

d

Interferometer

• Used to measure very small translations
• Accurate to ~ 1/20 wavelength of light
• Constructive interference fringes when
• ∆d = ( / 2)/2

Detector Output

Phase Contrast Microscopy

Utilizes differences in the refractive index (n)

of the Sample to Produce Contrast

Frits Zernike (Holland) - 1934

Typically differences in light absorption between cells, membranes, and cellular organelles vs. the surrounding medium (water) are negligible; therefore yielding these entities barely visible with brightfield illumination.

Luckily, differences in the index of refraction (n) of lipid bilayers and water are significant enough to introduce phase differences. These phase differences can be utilized to produce contrast through interference.

Board demo!

The Phase Contrast Optics

Interference

in the image

Side view

Backfocal plane

of the condenser lens

(introduce light from a

specific angle) [surround light]

Backfocal plane of the objective

lens (introduce a 90° phase shift

to the surround light relative to

the diffracted light)

Phase Contrast Images

Positive vs. Negative Phase Shift

Ctenoid Fish Scale

Positive phase shift

in phase plate

Negative phase shift

in phase plate

Polarization of Light

E fields

E fields

Maxwells Eqs determine

the propogation of light:

+

Transmission through a Polarizer

A Microscope that use Polarization:

The Differential Interference Contrast Microscope

(DIC)

• Many Variations on this Basic Design:
• Polarizer:
• Create polarized light at 45to Wallaston prism
• First Wollaston Prism:
• Split light into two linearly polarized beams with slight displacement
• Second Wollaston Prism on Slider:
• Recombine and interfere two polarizations with ability to introduce phase shift between two polarizations by sliding prism

Wollaston / Nomarski Prism

• Made from Calcite or Quartz
• Made from two orthogonal optical axis crystals
• Provides slight (less then ) displacement between the
• polarizations of incident light

Interaction with the Sample through a

Wollaston Prism

moving the prism in x adjusts

the phase shift between two

orthogonal polarized beams

• Two interaction types:
• polarization rotation
• phase shift due to varying index of refraction n

Nomarski’s Differential Interference Contrast Microscope

(DIC)

Using Epi-illumination

Aligns pol. so perp.

beams can interfere

A DIC Image

DIC tends to enhance edges as a

Result of polarized light interaction

with the sample

References:

1. Physics for Scientists and Engineers, Giancoli, Douglas C.

Chapters 35-39

2. The Theory of the Microscope, Martin, L. C.

3. Fundamentals of Light Microscopy, Spencer, Michael

4. Principles and Practice of Electron Microscope Operation, AW. Agar

RH. Alderson, D. Chescoe.

5. Introduction to Microscopy by Means of Light, Electrons, Xrays,

or Ultrasound, Rochow, T. G. and Rochow, E. G.

6. Optics, Welford, W. T.

7. Classical Optics and its Applications, Mansuripur, M.

8. http//www.microscopyu.com

References:

1. Physics for Scientists and Engineers, Giancoli, Douglas C.

Chapters 35-39

2. The Theory of the Microscope, Martin, L. C.

3. Fundamentals of Light Microscopy, Spencer, Michael

4. Principles and Practice of Electron Microscope Operation, AW. Agar

RH. Alderson, D. Chescoe.

5. Introduction to Microscopy by Means of Light, Electrons, Xrays,

or Ultrasound, Rochow, T. G. and Rochow, E. G.

LM Illumination Methods:

… stay tuned

Phase contrast microscopy: Uses differences in sample n and background n to

produce contrast through interference

Laser Scanning Microscopy:

Confocal, 2-photon, 3rd Harmonic

Polarization Optics: Nomarski and DIC

Epi -illumination Fluorescence microscopy: