Download Bright Field Microscopy

Modern Inverted Microscope Design
In critical illumination, the image
of the light source falls in the
same plane as the image of the
specimen, i.e. the bulb filament is
visible in the final image.
In Kohler illumination, the collector lens collects light from the bulb
and focus it at the plane of the condenser diaphragm. The
condenser projects this light, without focusing it, through the
sample. This illumination scheme creates two sets of conjugate
image planes, one with the light source (aperture) and one with the
specimen.
Aperture planes: Lamp, condenser diaphragm, objective back focal
plane, pupil
Image planes: Field diaphragm, Specimen, the eyepiece diaphragm,
retina or camera
Conjugate Planes of a Microscope
Infinity Optics
Microscope systems with finite tube
length were produced in the past.
When objectives and tube lengths are
mismatched, image quality often
suffers due to the introduction of
spherical aberrations because the
optical tube length is changed
When optical element is inserted in
the light path between the back of
the objective and the eyepiece, the
mechanical tube length changes
infinity-corrected objectives
generate emerging parallel rays and
provide flexibility to the user to
modify the path.
Diversity of Cells
What is Inside Cells?
E. Coli (model prokaryotic cell)
What is Inside Cells?
Fibroblast (model higher eukaryotic cell)
What is Inside Organelles?
Mitochondria (power plant of a cell, model organelle)
Play Power Plant of A Cell Movie
Biological Scale
http://learn.genetics.utah.edu/content/begin/cells/scale/
Bright Field Microscopy
Some specimens are considered amplitude objects because they absorb light
partially or completely, and can thus be readily observed using conventional
brightfield microscopy.
Limitations of standard optical microscopy: (bright
field microscopy) lie in three areas;
• The technique can only image dark or strongly
refracting objects effectively.
• Diffraction limits resolution to approximately 0.2
micron.
• Out of focus light from points outside the focal
plane reduces image clarity.
Live cells in particular generally lack sufficient contrast to be
studied successfully, internal structures of the cell are colorless
and transparent.
Solution: Kill the cell, stain it with dark material to generate
contrast
What do you see?
Overall cell shape,
membrane, nucleus and
large organelles.
Most of the internal
structures of the cell are
invisible.
Most biological specimens do not absorb much light to produce contrast,
but they diffract the light and cause a phase shift of the transmitted wave.
Phase Contrast Microscopy
•Other specimens do not absorb light and are referred to as phase objects. Because the
human eye can only detect intensity and color differences, the phase changes due to objects
must be converted to intensity differences.
•The phase contrast microscope is designed to take advantage of phase differences between
objects in a specimen and in the surrounding medium. However, it is not simply a phase
difference that is necessary, but also diffraction from the specimen must occur for the phase
contrast microscope to work.
•One of the major advantages of phase contrast microscopy is that living cells can be examined
in their natural state without previously being killed, fixed, and stained.
• As a result, the dynamics of ongoing biological processes can be observed and recorded in
high contrast with sharp clarity of minute specimen detail.
Phase Specimens
Phase is generated by the refractive index and thickness
Optical Path Difference (OPD) = Δ = (t × ns - t × nm) = t × (ns - nm)
phase shift (δ) δ = 2πΔ/λ
If Δ is λ/2, two waves become “out of phase”
Interaction of Light Through Phase Specimens
• An incident light is divided into two components upon passing through a phase
specimen.
• The primary component is undiffracted surround (S) wave, which passes through and
around the specimen, but does not interact with it.
• A diffracted D-wave is also produced, which is scattered in many directions. Typically,
only a minority of incident waves are diffracted by cellular objects.
• Both S and D waves are collected by the objective lens and focused in the image plane at,
where they undergo interference and generate a resultant particle wave (P wave).
•P=S+D
• Only when the amplitudes of the P and S waves are significantly different in the image
plane can we see the object in the microscope.
Phase Differences in Biological Specimens
• For individual cells in tissue culture, the optical path difference is relatively small.
• A typical cell in monolayer culture has a thickness around 5 micrometers and a
refractive index of approximately 1.36.
• The cell is surrounded by a nutrient medium having a refractive index of 1.335
• A typical optical path difference is 0.125 micrometer, or about a quarter
wavelength (of green light).
• D is retarded by ~λ/4 (first order
diffraction), amplitude is low
• Amplitudes of P and S are similar
• P is shifted by only ~λ/20
Since the amplitudes of P and S are the same, the phase variations do not create a contrast
and these objects remain invisible in the bright field microscopy
Optical Design of the Phase Contrast Microscope
The key elements of the optical design are:
1. separation of the direct zeroth order light (S wave) from the first order
diffracted light (D wave) at the rear focal plane of the objective. (Condenser
annulus)
2. Introduce a phase shift so that the S-wave gives rise to completely destructive or
constructive interference. (Phase Plate)
3. Decrease the intensity of the S wave to make it comparable to D wave (ND filter)
4. Combine the direct and diffracted light so that the resulting interference
generates contrast
Under conditions of Koehler
illumination, the objective’s back focal
plane is conjugate to the condenser’s
front aperture
Plane.
S waves (nondiffracted, 0th-order) that
do not interact with the specimen are
focused as a bright ring in
the back focal plane of the objective
(the diffraction plane).
•In some phase contrast objectives, the phase plate is a plate of glass with an etched
ring of reduced thickness to selectively advance the phase of the S wave by λ/4.
•The same ring is coated with a partially absorbing metal film to reduce the amplitude
of the light by 70–75%.
λ/4 phase shift to S wave is
introduced positively (S and D
are out of phase, λ/2) or
negatively (S and D are in phase).
Only S wave is observed for
nondiffracted regions (water)
Positive: Objects with high nd look
darker from the environment
Negative: Environment looks darker
than the high nd object
Comparison of Phase Contrast with Brightfield
Bright Field
Phase Contrast
Interpreting the Phase Contrast Image
What can we see?
• Objects with high density (Cytoplasm,
nucleus and nucleolus) can be seen as
progressively darker objects
• Objects with low density (some vesicles,
vacuoles and lipid droplets) look brighter than
cytoplasm
• Live cells!
Limitations:
• Relies on index of refraction
• Sample thickness changes the phase
• Small object with high nd have same
intensity with large object with low nd
• Objects with large phase retardations (phase
shift of the D wave ~λ/2), interference
becomes constructive, making the objects
appear brighter than the background.
In order to create a sharp edge in the
image, all of the spatial frequencies
diffracted by the specimen must be
represented in the final image.
Image Artifacts in Phase Contrast
Halos:
• Phase ring transmits some diffracted light.
• A problem accentuated by the fact that the width of the annulus generated
by the 0th-order surround waves is smaller (~25%) than the actual width of the
annulus of the phase plate.
• Low spatial frequency diffracted light remain 90 degree out of phase (e.g. no
destructive interference).
• As a result, these waves cause a localized contrast reversal—that is, a halo—
around the object.
• Halos are especially prominent around large, low-spatial-frequency objects
such as nuclei and cells.
• Dark objects have a bright surrounding ring, vice versa.
Shade Off
frequently observed on large, extended objects such as extended or flattened cells, flattened
nuclei, and planar slabs of materials, for example, mica or glass.
• Center and the edges of an object
diffract light differently.
• In central regions, the amount of
diffraction and the angle of
scattering are greatly reduced.
• Object rays, although retarded in
phase, deviate only slightly from the
0th-order component, and fall
within the annulus of the phase
plate.
• As a result, the amplitude and
intensity of the central region are
essentially the same as the
background.
Dark Field Microscopy
• For unstained transparent specimens, the component of nondiffracted
background light is very large, resulting in bright, low-contrast images.
• In dark-field microscopy, the nondiffracted rays are removed altogether so that the image is
composed solely of diffracted wave components.
• This technique is very sensitive because images based on small amounts of diffracted light
from minute phase objects are seen clearly against a black or very dark background.
Image Interpretation
• Dark-field microscopy is most commonly used for imaging small light-diffracting
specimens such as diatoms, bacteria and bacterial flagella, isolated organelles and
polymers such as cilia, flagella, microtubules, and actin filaments, and silver grains and
gold particles in labeled cells and tissues.
• Edges diffract the greatest
amount of the light, dominate
the image
• Provides high contrast
• Background is black
• Lysosomes, flagella, fibrins and
even microtubules (24 nm
width) are visible
Requires high laser power,
potential damage of the sample
www.arizonahomeopathic.org
Polarization Microscopy
The bulk light from most illuminators is nonpolarized, the E vectors of different rays
vibrating at all possible.
In a ray or beam of linearly polarized light, the E vectors of all waves vibrate in the same
plane.
Absorptive Polarizer
Metallic wire grid
Distance is less than the wavelength
Parallel beam oscillates the electrons, and
reflected back
Perpendicular beam travels through the grid
Beam Splitting Polarizer
Brewster Angle
• Electric dipoles in the media respond to p-polarized light.
• Light incident on the surface is absorbed, and then reradiated by oscillating electric
dipoles at the interface between the two media.
• The polarization of freely propagating transmitted light is always perpendicular to the
direction in which the light is travelling.
• These oscillating dipoles also generate the reflected light. However, dipoles do not
radiate any energy in the direction of the dipole moment.
• Consequently, if the direction of the refracted light is perpendicular to the direction in
reflected light, the dipoles cannot create any reflected light.
Solving for θB gives:
For a glass medium (n2 ≈ 1.5) in air
(n1 ≈ 1), Brewster's angle for visible
light is approximately 56°, while for
an air-water interface (n2 ≈ 1.33), it
is 53°.
Polarizing Sunglasses
• Polarizing sunglasses mount sheets of polarizing material with the transmission axis of
the Polaroids oriented perpendicularly in the field of view.
• Bright reflections off horizontal surfaces, such as the roofs of cars or water on a lake,
are efficiently blocked, while the random light is partially blocked.
Polarizing Crystals
The most efficient polarizers are made of transparent crystals such as calcite
The term anisotropy refers to a non-uniform spatial distribution of properties,
which results in different values being obtained when specimens are probed from
several directions within the same material.
Anisotropic crystals (i.e. quartz or calcite) exhibit
double refraction (or birefringence)
Light polarized parallel to the crystal orientation has a
different index of refraction (that is to say it travels at
a different velocity) than perpendicular polarized light.
Double Refraction: Birefringence
Ordinary ray (O ray) observes the law of refraction
Extraordinary ray (E ray ) follows a different path.
Electric fields of O and E rays are perpendicular.
Birefringence (B) = |ne - no|
Birefringence can be either positive or negative
depending on the crystal orientation, and is not a
fixed value.
Unique angles in birefringence
a) When light enters the unique optical axis of anisotropic crystals, it behaves in a
manner similar to the interaction with isotropic crystals, and passes through at a
single velocity.
b) Incident beams that are perpendicular to the optic axis are split into O and E rays,
but the trajectories of these rays are coincident. The O and E rays emerge at the
same location on the crystal surface, but have different optical path lengths and are
therefore shifted in phase. This geometry pertains to most biological specimens that
are examined in a polarizing microscope.
c) Incident rays that follow trajectories parallel to this axis behave as ordinary rays and
are not split into O and E rays. B = 0.
Wave Plates
• Optical axis is perpendicular to incident beam.
• O and e rays are phase shifted (n2-n1)t
• Thickness of the crystal generates λ/4 phase shift
• At 45°, intensities of E and O rays become equal
• Resulting wave of E and O rays has a constant intensity
but Electric vector rotates around its own axis.
(circularly polarized light)
Half Wave Plate
• λ/2 retardation
• Useful for rotating the polarization of the incoming
light
http://optics.byu.edu/animation/polarwav.mov
Relative Phase Shift
π = λ/2
Birefringence in Microscopy
Optical Path Difference Γ = (ne - no) • t (Thickness)
1. Non-polarized white light enters the polarizer and is linearly polarized
2. The polarized light enters the anisotropic crystal where it is refracted and
divided into two separate components vibrating parallel to the
crystallographic axes and perpendicular to each other
3. The polarized light waves travel through the analyzer, which allows the
components of parallel waves to pass.
Polarization Microscopy
Polarized light microscopy can
distinguish between isotropic
and anisotropic materials
90 percent of all solid substances
are anisotropic and have optical
properties that vary with the
orientation of incident light with
the crystallographic axes.
compensator
Polarizer and analyzer
orientations are perpendicular
to each other (extinction).
Compensator (birefringent
crystal) is optional and can
increase the contrast
http://academic.brooklyn.cuny.edu/geology/powell/
Alignment
• Koehler illumination.
• Insert the fixed polarizer.
• Insert the rotatable analyzer, and rotate it until the
two polars are crossed and maximum extinction is
obtained.
• Insert a telescope eyepiece and focus on the back
aperture of the objective lens.
• A dark polarization cross is observed at extinction
with brighter regions of intensity between the
horizontal and vertical arms of the cross.
• The horizontal and vertical arms are broader in
the center and narrower at the periphery. This is
caused by the depolarizing effects of spherical lens
surfaces.
• Perfect alignment with respect to an azimuth at 0°
is essential if quantitative measurements of
azimuthal angles or birefringence are to be made
with a compensator.
• Using a Bertrand lens, partially close the
condenser aperture diaphragm to block bright
outer regions of depolarized light.
Compensators
Compensator makes polarization microscope an analytical instrument.
Since the angle of rotation of the
analyzer at extinction is equal to onehalf of the full phase shift between the
O and E rays, the relative retardation is
given as Γ = 2θ.
• de Senarmont compensator is a λ/4 plate.
• Linearly polarized light passes through a
specimen, whose optical axis is at 45º relative
to the polarizer.
• Elliptical or circularly polarized light from
the specimen is converted back into linearly
polarized light by the compensator.
• The azimuth is rotated 45º relative to that
of excitation light.
• When the analyzer transmission axis is
crossed, half of the light is transmitted.
• Rotating the analyzer clockwise by 45º
produces maximum transmission, while
rotating the analyzer counterclockwise by 45
degrees yields maximum extinction.
Other Uses of Compensators
•The image background looks very dark, approaching black. Small (λ/20)
retardation can be introduced with a compensator to see the background by a
small amount.
•The compensator can also be used to increase or decrease the amount of phase
displacement between the O and E rays to improve the visibility of details in the
object image.
What can be Seen?
• Mitotic Spindle
• Actin and myosin filaments
• Condensed DNA
• Lipid Bilayers
• Isotropic specimen (i.e. gases, liquids and cubic crystals)
are not visible since they do not rotate the polarization of
incoming beam, which is blocked by analyzer.
• Birefringent objects look bright or dark
depending on their molecular orientation or
geometry of the object
Spherical objects with radially symmetric
structures will show four bright quadrants.
Linear objects (striated muscle) will appear
bright at 45° 135° 225° 315° and dark at 0° 90°
180° 270°.
http://www.olympusmicro.com/primer/virtual/polarizing/index.html
Full Wave Retardation Plates
When placed in front of the analyzer, oriented 45° with respect to the crossed polars, the
plate introduces a relative retardation between O and E rays of exactly one wavelength for
green wavelengths of 551 nm.
Green wavelengths therefore emerge from the retardation plate linearly polarized in the
same orientation as the polarizer and are blocked at the analyzer.
O and E waves of all other wavelengths experience relative phase retardations of less than
1 λ; they emerge from the plate as elliptically polarized waves and are only partially blocked
by the analyzer.
Full-wave plates introduce vivid colors to birefringent objects and are useful for making
quantitative assessments of relative retardation, and the orientation of index ellipsoids.
Differential Interference Contrast Microscopy
• The DIC microscope employs a mode of dualbeam optics that transforms
local gradients in optical path length in an object
into regions of contrast in the object image.
•DIC microscopy is a beam-shearing interference
system in which the reference beam is sheared
by less than the diameter of an Airy disk.
•The technique produces a monochromatic
shadow-cast image that effectively displays the
gradient of optical paths in the specimen.
•Those regions of the specimen where the optical
paths increase along a reference direction appear
brighter, while regions where the path
differences decrease appear in reverse contrast.
Wollaston Prism
• Wollaston Prism generates parallel coherent wave bundles from all of the incoming
polarized light.
• The separation of the two beams depends on the shear axis.
• The interference fringes appear to lie inside the prism at a location termed the beam
splitting plane. This makes it difficult to use a conventional Wollaston prism for certain
objective lenses, where the interference plane of the prism must lie within the back focal
plane (the diffraction plane) of the lens.
• Modified Wollaston prisms where the beam splitting plane is displaced several milimeters
away from the center of the prism.
• The condenser prism acts as a beam
splitter
• The objective prism recombines the
beams and regulates the amount of
retardation between O and E wavefronts.
•Koehler illumination is required to
correctly position the interference planes
of the DIC prisms in the conjugate aperture
planes of the condenser and objective
lenses.
Optical Axes in DIC
• Polarizer and Analyzer are perpendicular, to eliminate the background
• Nomarski prisms are parallel to each other (to recombine E and O rays)
• Nomarski prisms are fixed at 45° to the polarizer to obtain same intensity
for E and O rays.
Formation of DIC Image
Physical separation of the distance (shear axis) can be as low as 180 nm.
In the absence of OPD, the beam is blocked by analyzer.
If E and O rays encounter a phase object, they become differentially shifted in phase,
resulted in elliptically polarized light.
• DIC image of human red blood cells having thick edges and thin center.
• E and O rays pass through a phase object and are differentially shifted in phase.
• The shear axis is the axis of lateral displacement of the O and E wavefronts at the
specimen.
• Amplitude of the transmitted wave is the derivative of OPD as a function of distance.
• As the shear distance is reduced, resolution improves, at some expense to contrast,
until the shear distance is about one half the objective’s maximum resolution.
Typical DIC Image
a) Phase shift between the E and O rays. Difference is only observed at the
edges of the sample, in the direction of the shear axis.
b) Amplitude is the derivative of the OPD.
c) DIC image of oil drops.
Introducing bias retardation makes objects much easier to see, because phase gradients in
the specimen are now represented by bright and dark patterns on a gray background.
The resultant image exhibits a shadow-cast, three-dimensional appearance and makes
objects look like elevations or sunken depressions depending on the orientation of phase
gradients.
de Senarmont Retardation
A phase displacement between the Oand E-ray wavefronts was introduced
by a retardation plate.
Microscope contains a λ/4 plate.
The objective DIC prism is fixed.
The bias adjustment is made by rotating the
polarizer.
The amount of displacement between the O
and E rays caused by the objective DIC prism is
small, usually λ /10.
Image Orientation
shadow and highlight intensity is greatest along the shear axis
of the microscope
Comparison of DIC and Phase Contrast
• Phase contrast yields an image as a function of specimen optical path length
magnitude, with regions having large path lengths appear darker.
• In DIC, optical path length gradients are primarily responsible for introducing
contrast into specimen images.
• In DIC, steep gradients in path length generate excellent contrast, and images
display a pseudo three-dimensional relief shading that is characteristic of the DIC
technique.
• Extended flat object look like background with high contrast edges.
• Phase contrast is not susceptible to orientation effects
the nucleus, cytoplasmic inclusions, and
numerous bacteria on the upper surface.
DIC
Phase
Contrast
cheek cell
Pronounced halos around the cellular periphery and
nucleus, which are absent in DIC image. Optical
sectioning DIC microscopy investigations (not
illustrated) reveal that the bacteria are present on
the membrane surface as opposed to lying on the
underside of the cell. This fact cannot be
unambiguously determined with phase contrast.
differential interference contrast reveals a
bundle of cells enclosed within a tubule.
A phase contrast image of the same area is
confusing and disturbed by the presence of
phase halos outside the plane of focus.
However, several of the cellular nuclei appear
visible in the phase contrast image, which are
not distinguishable in DIC.
thick section of murine kidney tissue
Phase
Contrast
DIC
Halos are absent in DIC, but images have shadow-cast
appearance oriented along the shear axis.
Phase
Contrast
DIC
Phase contrast images become ambiguous when optical path length fluctuates over a large
range, the technique should be restricted to specimens having a path difference of one-tenth
wavelength or less. Thicker specimens can be imaged with DIC by using optical sectioning
techniques.
http://www.olympusmicro.com/primer/java/dic/dicphaseos/index.html
Phase
Contrast
DIC
Because DIC relies on polarized light, differential absorption of ordinary and
extraordinary waves leads to confusing images. Phase contrast does not require the
use of polarized light, and is free of optical disturbances generated by birefringent
specimens.