Cell Biology Application of Microscopy
Class 2: Light, color, and resolution
Paul Marshall,
Carbon nanotubes, post growth (30X)
國立交通大學生物科技學系 黃兆祺老師
References
Optical Microscopy Primer:
http://micro.magnet.fsu.edu/primer/
Zeiss Microscopy Campus:
http://zeiss-campus.magnet.fsu.edu/index.html
iBiology Microscopy Course:
http://www.ibiology.org/ibioeducation/taking-courses/ibiology-microscopy-course.html
國立交通大學生物科技學系 黃兆祺老師 2
What Is A Microscope?
A microscope is an instrument designed to make fine
details visible. The microscope must accomplish
three tasks:
1.  Produce a magnified image of the specimen
(magnification).
2.  Separate the details in the image (resolution).
3.  Render the details visible to the human eye or
camera (contrast).
國立交通大學生物科技學系 黃兆祺老師 3
Microscope
http://micro.magnet.fsu.edu/primer/index.html
國立交通大學生物科技學系 黃兆祺老師 4
Light: Electromagnetic Wave?
ROY G BIV
http://micro.magnet.fsu.edu/primer/index.html
v = c/λ
E = hv = hc/λ
v: frequency in hertz
c: light speed
λ: wavelength
h: Plank’s constant
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Light: Particle?
http://micro.magnet.fsu.edu/primer/index.html
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Important Properties of Light
http://micro.magnet.fsu.edu/primer/index.html
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Laser Pointer Light?
1.  Monochromatic
2.  Polarized
3.  Coherent
4.  Collimated
國立交通大學生物科技學系 黃兆祺老師 8
Microscope Illumination
Four important parameters in determining the suitability of a light source:
1.  Brightness
2.  Wavelength characteristics
3.  Stability
4.  Structure (spatial distribution, source geometry, coherence, alignment)
http://zeiss-campus.magnet.fsu.edu/index.html
國立交通大學生物科技學系 黃兆祺老師 9
Radiant Energy and Power
For a steadily emitting source, that is a radiation source with a
continuous and stable output, radiometric measurement usually implies
measurement of the power of the source.
Radiant energy is the energy emitted, transferred, or received in the form
of electromagnetic radiation.
Symbol: Q
Unit: joule (J)
Radiant power (or radiant flux) is the power (energy per unit time t)
emitted, transferred, or received in the form of electromagnetic radiation.
Symbol: Φ
Unit: watt (W)
國立交通大學生物科技學系 黃兆祺老師 10
where:
L = d2 /{dAproj d }
(W/m2/sr)
Brightness
(Radiance)
dA = dA
cos
Units of
intensity
proj
d
dA
dAproj
adiance
3
A525 – Lecture
Radiance is the ratio of the radiant power (Φ), at an angle θ to the
normal of the surface element, to the infinitesimal elements of both
projected area (A) and solid angle (Ω).
Symbol: L
Unit: watt/steradian meter2 (W sr-1 m2)
http://www.physics.csbsju.edu/370/photometry/etendue.pdf
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Wavelength Characteristics
http://micro.magnet.fsu.edu/primer/index.html
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Typical Fluorescent Lamp
http://en.wikipedia.org/wiki/Fluorescent_lamp
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Blacklight Lamp
http://en.wikipedia.org/wiki/Fluorescent_lamp
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Stability
Power stability
Spectral stability
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Structure
Coherence
A low coherence light source is desirable!
Uniformity
A high uniformity light source is desirable!
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Microscope Illumination
Brightfield Microscopy:
•
Tungsten-Halogen Lamp
Fluorescence Microscopy:
•
•
•
•
•
Mercury Arc Lamp
Xenon Arc Lamp
Metal Halide Arc Lamp
LED
LASER
國立交通大學生物科技學系 黃兆祺老師 17
Tungsten-Halogen Lamp
Temp in K (=°C + 273.15°)
http://micro.magnet.fsu.edu/primer/index.html
國立交通大學生物科技學系 黃兆祺老師 18
Tungsten-Halogen Lamp
Halogen Regenerative Cycle
http://zeiss-campus.magnet.fsu.edu/tutorials/halogencycle/index.html
國立交通大學生物科技學系 黃兆祺老師 19
Mercury Arc (HBO) Lamp
H stands for mercury
B stands for high luminance
O stands for unforced cooling
http://micro.magnet.fsu.edu/primer/index.html
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Mercury Arc Lamp Alignment
http://www.microscopyu.com/tutorials/java/arclamp/index.html
國立交通大學生物科技學系 黃兆祺老師 21
Metal Halide Arc Lamp
國立交通大學生物科技學系 黃兆祺老師
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Arc Lamp Instability
http://zeiss-campus.magnet.fsu.edu/tutorials/arclampinstability/index.html
國立交通大學生物科技學系 黃兆祺老師 23
Light-Emitting Diodes (LED)
http://micro.magnet.fsu.edu/primer/index.html
國立交通大學生物科技學系 黃兆祺老師 24
LASER
Light Amplification by Stimulated Emission of Radiation
http://micro.magnet.fsu.edu/primer/index.html
國立交通大學生物科技學系 黃兆祺老師 25
Light Source Power Levels
http://micro.magnet.fsu.edu/primer/index.html
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Speed of Light
http://micro.magnet.fsu.edu/primer/index.html
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Speed of Light
http://micro.magnet.fsu.edu/primer/index.html
國立交通大學生物科技學系 黃兆祺老師 28
Reflection
國立交通大學生物科技學系 黃兆祺老師
Reflection of Light
Law of Reflection:
θi = θr
http://micro.magnet.fsu.edu/primer/index.html
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Reflection of Light
http://micro.magnet.fsu.edu/primer/index.html
國立交通大學生物科技學系 黃兆祺老師 31
Light-matter Interaction at Interface
Incident wave
Reflected wave
θ1
θr
n1=1
n2>1
θ2
Refracted wave
國立交通大學生物科技學系 黃兆祺老師 32
Refraction
國立交通大學生物科技學系 黃兆祺老師
Refraction of Light
http://micro.magnet.fsu.edu/primer/index.html
國立交通大學生物科技學系 黃兆祺老師 34
Refractive Index
Ratio of velocity of light in a vacuum (or air)
to the velocity of light in any material
n (Refractive Index) = c/v
http://micro.magnet.fsu.edu/primer/index.html
國立交通大學生物科技學系 黃兆祺老師 35
Snell’s Law
normal
n1 x sin(θ1) = n2 x sin(θ2)
http://micro.magnet.fsu.edu/primer/index.html
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Refraction of Light
國立交通大學生物科技學系 黃兆祺老師 37
Critical Angle of Reflection
Total internal reflection
n2 x sin(θ2) = n1 x sin(90º) = n1
sin (θ2) = n1/n2 = 1/n2
國立交通大學生物科技學系 黃兆祺老師 38
Light-matter Interaction at Interface
Normal incident light
Reflected light
~4%
ni=1
nt=1.5
4!!!!
!=
!! + !!
!!
~96%
Transmitted light
% incident light = % transmitted + % reflected + % absorbed
39
Diffraction
國立交通大學生物科技學系 黃兆祺老師
Diffraction of Light
Diffraction is the apparent bending of waves around small obstacles and the spreading out of
waves past small openings. These characteristic behaviors are exhibited when a wave
encounters an obstacle or a slit that is comparable in size to its wavelength.
CD, DVD!!
國立交通大學生物科技學系 黃兆祺老師 41
Diffraction of Light
Diffraction occurs when a wave encounters an obstacle or a slit that is comparable in size to
its wavelength. Similar effects are observed when light waves travel through a medium with a
varying refractive index or a sound wave through one with varying acoustic impedance. 國立交通大學生物科技學系 黃兆祺老師 42
Huygens’ Wavelets
In Christiaan Huygens’ original principle, a wavefront was propagated by generating so-called
wavelets at each point of the old wavefront to construct the new wavefront. A spherical wave
impinges on an aperture. The evolution of the resulting wavefront (red) can be constructed
iteratively using wavelets (gray) with the radius of a wavelength.
http://www.marcuswinter.de/archives/1351
國立交通大學生物科技學系 黃兆祺老師 43
Double Slit Interference
Left: thin black lines represent wavefronts separated by one wavelength. The incoming
monochromatic plane wave is partially blocked at an aperture with two narrow slits (width
smaller than the wavelength). Cylindrical waves emanate from the holes in the aperture and
interfere. Blue dashed lines show areas of constructive interference at which the total intensity
of the compound wave will be high. Right: same construction shown with colors representing
the field amplitude (red for positive values, green for negative). Blue dashed lines again show
the directions of highest intensity.
http://www.marcuswinter.de/archives/1351
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