MSE 333 Characterization Techniques

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MSE 333Characterization Techniques
UNIVERSITY OF WASHINGTON
Spr. 2008
M. Sarikaya
Specialized Light Optical Microscopy Techniques
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PHASE CONTRAST MICROSCOPY - Intro

• - Phase contrast microscopy A contrast-enhancing
  optical technique, utilized to produce
  high-contrast images of transparent specimens
  such as living cells, microorganisms, thin tissue
  slices, lithographic patterns,
• - It employs an optical mechanism to translate
  minute variations in phase into corresponding
  changes in amplitude, which can be visualized as
  differences in image contrast.
• - One of the major advantages of phase contrast
  microscopy is that living cells can be examined
  in their natural state without being killed,
  fixed, and stained. As a result, the dynamics of
  ongoing biological processes in live cells can be
  observed and recorded in high contrast with sharp
  clarity of minute specimen detail.

Essential parameters optical path length
index of refraction
Phase contrast microscopy interprets differences
in specimen optical path length as fluctuations
in light intensity, which are readily observed as
variations in contrast through the microscope.
This interactive tutorial explores the effects of
refractive index and thickness changes on the
apparent overall optical path length, and
demonstrates how two specimens can have different
combinations of these variables but still display
the same path length.
Example
Figure 2 is a comparison of living cells in
culture imaged in both brightfield and phase
contrast illumination. The cells are human glial
brain tissue grown in monolayer culture bathed
with a nutrient medium containing amino acids,
vitamins, mineral salts, and fetal calf serum. In
brightfield illumination (Figure 2(a)), the cells
appear semi-transparent with only highly
refractive regions, such as the membrane,
nucleus, and unattached cells (rounded or
spherical), being visible. When observed using
phase contrast optical accessories, the same
field of view reveals significantly more
structural detail (Figure 2(b)). Cellular
attachments become discernable, as does much of
the internal structure. In addition, the contrast
range is dramatically improved.
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Phase Contrast Microscopy

• Show ray paths
• Show image contrast
• Any change of resolution
• What is the magnification range
• Examples

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Phase contrast microscopy contd
The Principle The most important concept
underlying the design of a phase contrast
microscope is the segregation of surround and
diffracted wavefronts emerging from the specimen,
which are projected onto different locations in
the objective rear focal plane (the diffraction
plane at the objective rear aperture). In
addition, the amplitude of the surround
(undeviated) light must be reduced and the phase
advanced or retarded (by a quarter wavelength) in
order to maximize differences in intensity
between the specimen and background in the image
plane. The mechanism for generating relative
phase retardation is a two-step process, with the
diffracted waves being retarded in phase by a
quarter wavelength at the specimen, while the
surround waves are advanced (or retarded) in
phase by a phase plate positioned in or very near
the objective rear focal plane. Only two
specialized accessories are required to convert a
brightfield microscope for phase contrast
observation. A specially designed annular
diaphragm, which is matched in diameter and
optically conjugate to an internal phase plate
residing in the objective rear focal plane, is
placed in the condenser front focal plane.
Optical Path Length (OPL) n t Phase shift
d 2pD/l Optical Path Difference (OPD) D
(n2 - n1) t
Ray Diagram
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CONFOCAL MICROSCOPY - Intro
Principle Illumination is achieved by scanning
one or more focused beams of light, usually from
a laser or arc-discharge source, across the
specimen. This point of illumination is brought
to focus in the specimen by the objective lens,
and laterally scanned using some form of scanning
device under computer control. The sequences of
points of light from the specimen are detected by
a photomultiplier tube (PMT) through a pinhole
(or in some cases, a slit), and the output from
the PMT is built into an image and displayed by
the computer. Although unstained specimens can be
viewed using light reflected back from the
specimen, they usually are labeled with one or
more fluorescent probes.
A flow diagram of a generic laser scanning
confocal microscope showing the locations of a
number of the "39" features mentioned in the text
is shown in Figure 1. The system illustrated is
based on an inverted optical microscope
configured for live cell imaging. Individual
components referred to in the following text are
labeled in the figure with the letters (a)
through (g). The illustration includes three
optical sections, taken at different levels along
the z-axis through an antibody-labeled fruit fly
embryo, and designated Z1, Z2, and Z3.
Example
Advantages and Applications Confocal microscopy
offers several advantages over conventional
optical microscopy, including controllable depth
of field, the elimination of image degrading
out-of-focus information, and the ability to
collect serial optical sections from thick
specimens. The key to the confocal approach is
the use of spatial filtering to eliminate
out-of-focus light or flare in specimens that are
thicker than the plane of focus. There has been a
tremendous explosion in the popularity of
confocal microscopy in recent years, due in part
to the relative ease with which extremely
high-quality images can be obtained from
specimens prepared for conventional optical
microscopy, and in its great number of
applications in many areas of current research
interest.
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Confocal microscopy

• Show ray paths
• How contrast arises
• Image reconstruction
• Resolution
• Range of magnification
• Give examples

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Confocal Microscopy Basics
Fluorescent agents (organic dyes, Qdots) are used
for labeling the desired targets.
FM
CM
In Biology, -imaging either fixed or living
tissue or cells, -sharper imaging comparing to FM.
What does the technique offer - Elimination of
out-of-focus glare - Ability to collect serial
optical sections from thick specimens. -
Reconstruction and 3D imaging
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Confocal Microscopy Examples
The Sullivan laboratory uses confocal microscopy
to examine at the cellular level the effects of
the bacteria Wolbachia  on reproductive
mechanisms of the fruit fly D. melanogaster. In
this image, DNA is labeled green, and Wolbachia
are red.
Fluorescent and laser confocal images of a neuron
in hippocampal slice gene gun transfected with
DCX DsRed and µ1A GFP (courtesy of McNeil lab.).
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FLOURESCENCE MICROSCOPY-Intro
Principle The basic concept of total internal
reflection fluorescence is schematically
illustrated in Figure 1, in which specimen cells
incorporating fluorescent molecules (green
fluorophores in the figure) are supported on a
glass microscope slide. The refractive indices of
the glass slide (1.518) and the aqueous specimen
medium (approximately 1.35) are appropriate to
support total internal reflection within the
glass slide. With adjustment of the laser
excitation incidence angle to a value greater
than the critical angle, the illuminating beam is
entirely reflected back into the microscope slide
upon encountering the interface, and an
evanescent field is generated in the specimen
medium immediately adjacent to the interface. The
fluorophores nearest the glass surface are
selectively excited by interaction with the
evanescent field, and secondary fluorescence from
these emitters can be collected by the microscope
optics.
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Fluorescence Microscopy, Contd
Example
At the biomolecular level, TIRFM techniques have
been utilized to image single molecules of the
mutant protein GFP-Rac trafficking along thin
filopodia of cells growing on a substrate (Figure
6). This protein is involved in cell motility,
and knowledge of the dynamics of its interactions
at the cell membrane are crucial to understanding
the process. The visualization of single-molecule
fluorescence with sufficient temporal resolution
for dynamic studies is possible with TIRFM
because of the outstanding signal-to-noise ratio
afforded by the evanescent wave excitation.
Figure 6 presents four sequential time lapse
frames taken at 200-millisecond intervals,
illustrating the movement of a GFP-Rac fusion
protein molecule (arrows) through a fine
filopodium of a Xenopus cell growing out on a
substrate.
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Fluorescence Microscopy

• Show ray paths
• Demonstrate how contrast arises
• Image resolution
• Magnification range
• Examples

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Principle of Fluorescence
Excitation Light (?exc)
Emission Light (?em)
Excited state

• Fluorophores Fluorescence emitting materials
• Fluorescent dye (FITC, Alexa, SYTO..)

FITC (490/520nm)

• Nanostructures Quantum Dots - QDots

QDots Size (10-20nm)
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Imaging
Conventional Optical Microscope (with additional
features and components) -Source of excitation
light -Fluorescent filters -Imaging software
BF reflected light collected DF scattered light
collected FM emitted light collected
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Specimen Preparation
Examples
AuBP-FliTrx cells labeled with SYTO9
(DNA-binding dye)
(Invitrogen, CL Instrument, Nikon, Zeiss)
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Fluorescence Microscopy- More Basics Examples
20µm
Qdot605-Strep immobilization via Bio-QBP on glass
Ab-FITC labeled AP-Enzyme on gold
Labeling agents -Qdots (e.g.gt_at_565,605nm) -Organic
dyes (e.g. Fluorescein , rhodamine)
Specimen
Objective Lens
Excitation filter
Dichroic beam splitter
light
Illumination source
Barrier filter
Eyepiece
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Dark-Field Microscopy
Gold NP immobilization on glass via
bifunctional GEPI
Specimen
Specimen
Background scattering
NP scattering
DF Objective Lens
Illumination source
Illumination
50/50 Mirror
Eyepiece