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Microscopy Lecture I
Three branches of Microscopy
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Optical
Electron
Scanning Probe
Optical and Electron microscopy measure
refraction, diffraction, and reflection of the source
radiation
– Optical uses white light, fluorescent light, or lasers
– Electron uses electromagnetic radiation/electron beams
• Scanning uses a physical probe to interact with
the surface of the specimen
Imaging Techniques
Technique
Image Formed By
Lowest Resolvable
Unit
Optical Microscopy
Light Rays
Microns (μm)
Confocal Microscopy
Coherent Light Source
(Laser)
Microns (μm)
Electrons
Angstroms (Ǻ)
Electrons
Nanometers (nm) to
Angstroms (Ǻ)
Molecular Mechanical
Probes
Angstroms (Ǻ)
Transmission
Electron Microscopy
(TEM)
Scanning Electron
Microscopy (SEM)
Atomic Force &
Scanning Tunneling
Microscopies
(AFM/STM)
Approx Lower
Limit
1 μm
(monochromatic light)
.1 μm
(X-Y Direction)
2Ǻ
(high resolution TEM)
10 nm
(100 Ǻ)
40 Ǻ
(theoretical)
Units of Measure
• μm - Micrometer
– 1,000,000 micrometers = 1 meter
• Strand of hair has a diameter of ~ 20-180 μm
• 106
• nm - Nanometer
– 1,000,000,000 nanometers = 1 meter
• 109
• Wavelength of visible light (400-700 nm)
• Ǻ - Angstrom
– 10,000,000,000 Angstroms = 1 meter
• 1010
• Used to measure the size of atoms/bond lengths
• Length of a C-H bond in methane is ~1 Angstrom
0.75% Collagen Crosslinked
2% Collagen
Handspun Collagen
Optical Microscopy
Properties of light
• Reflection
• Refraction
• Numerical Aperture
Refraction
• Change in the direction
of a wave (light) due to
a change in speed
• The straw in the picture
looks bent because the
light is bending as it
moves from the water to
the air
Refractive Index (RI)
• RI of a material a measure of
the speed of light in material
• RI is the ratio of the velocity
of light in a vacuum to the
speed of light in the specified
material
• Incident angle (θ1) is related to
the refraction angle (θ2) by
Snell’s Law
• n1sin(θ1)=n2sin(θ2)
• Used in calculating focusing
power of lenses and dispersion
properties of prisms
Reflection
• Reflection is defined as
a change in direction of
a wave at an interface
between 2 different
media so that the
waveform returns to the
media from which it
came
• Used in focusing light
waves to increase
transmitted light
Numerical Aperture
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NA of a microscope objective is a
measure of its ability to gather light
The more light (higher NA) the better
the resolving power of the lens
– Better resolution
NA = (n)sin(θ)
– n = Refractive Index
– θ = ½ the maximum cone of light
than can enter the lens
Usually the NA of an objective
increases with its magnifying power.
The smallest detail that can be
resolved is proportional to:
– λ/NA
Optical Microscope
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Ocular lens
Objective turret
Objective
Coarse Adjustment
Fine Adjustment
Stage
Light source
Condenser
X-Y Control
Phase Contrast
• Uses phase shifted waves
of through transparent
specimens cause changes
in amplitude (contrast) in
structures of the specimen
– One of the most widely
used in biology
– No staining required
Compound Light
Phase Contrast
Fluorescence
• Fluorescence utilizes fluorescent dyes/stains that
fluoresce when radiated with specific wavelengths of
light
– Typically use mercury or xenon lamps
• Fluorescent dyes are extremely useful in
identifying/highlighting specific parts of cells that can
otherwise go undetected using simple phase contrast
• http://www.invitrogen.com/site/us/en/home/support/T
utorials.html
Filter Cube
Live Dead Assay
Confocal Image of Schwann Cells
Green Fluorescent Protein
• Class of proteins that
naturally fluoresce
– First isolated from the
jellyfish
• 238 amino acid long protein
that naturally fluoresces
green (509 nm) in the
presence of blue (488 nm)
light
• Through genetic
engineering, scientists have
artificially engineered many
variations of GFP
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