UNIT-2
Brightfield microscopy
TECHNOLOGY:
Brightfield imaging is the simplest form of microscopy where light is
either passed through, or reflected off, a specimen. Illumination is not
altered by devices that alter the properties of light (such as polarizers
or filters).
APPLICATIONS:
In biological applications, brightfield observation is widely used
for stained or naturally pigmented or highly contrasted
specimens mounted on a glass microscope slide.
The specimen is illuminated from below and observed from
above. The specimen appears bright, but darker than the bright
background. This technique is widely used in pathology to view
fixed tissue sections or cell films / smears.
Brightfield imaging is not very useful for unstained living cells
or unstained tissue sections as, in most cases, the light passes
through transparent or translucent samples with little or no
definition of structure.
Light is reflected from opaque samples and this is exploited in
industrial environments where brightfield imaging is used for
wafer inspection and liquid crystal board inspection.
MICROSCOPE CONFIGURATION:
All light microscopes are capable of brightfield imaging.
,Darkfield microscopy
TECHNOLOGY:
Darkfield microscopy creates contrast in transparent unstained
specimens such as living cells. It depends on controlling specimen
illumination so that central light which normally passes through and
around the specimen is blocked. Rather than light illuminating the
sample with a full cone of light. the condenser forms a hollow cone
with light travelling around the cone rather than through it.
This form of illumination allows only oblique rays of light to strike
the specimen on the microscope stage and the image is formed by
rays of light scattered by the sample and captured in the objective
lens. When there is no sample on the microscope stage the view is
completely dark.
Care should be taken in preparing specimens as features above and
below the plane of focus can also scatter light and compromise image
quality . In general, thin specimens are better because the possibility
of diffraction artifacts is reduced.
APPLICATIONS:
In darkfield microscopy, contrast is created by a bright specimen
on a dark background. It is ideal for revealing outlines, edges,
boundaries, and refractive index gradients but does not provide a
great deal of information about internal structure. Ideal subjects
include living, unstained cells, although fixed stains cells can
also be imaged successfully.
Darkfield imaging is particularly useful in haematology for the
examination of fresh blood.
Non-biological specimens include minerals, chemical crystals,
colloidal particles, inclusions and porosity in glass, ceramics,
and polymer thin sections.
,MICROSCOPE CONFIGURATION:
Almost any brightfield laboratory microscope can be easily converted
for use with darkfield illumination using special darkfield condensers
(dry or oil type).
Phase contrast microscopy
TECHNOLOGY:
In phase contrast imaging, small changes in optical path length are
optically translated into corresponding changes in light intensity,
which can be observed as differences in image contrast.
Light from a tungsten-halogen lamp is directed through a collector
lens and focused on a specialized annulus positioned in the substage
condenser.
Wavefronts passing through the annulus illuminate the specimen and
either pass straight through or are diffracted and retarded by phase
gradients in the specimen. Undeviated and diffracted light collected
by the objective is separated by a phase plate and focused at the
intermediate image plane to form the final image.
One drawback of phase contrast imaging is the occurrence of "halos"
around areas of high phase shift.
APPLICATIONS:
Phase contrast imaging is applicable to many transparent
subjects, such as living cells in culture, micro-organisms, thin
tissue slices, lithographic patterns, fibres, latex dispersions,
glass fragments, and subcellular particles .where the technique
reveals structure that is not visiblein brightfield imaging.
An advantage of phase contrast microscopy is that living cells
can be imaged in detail without the need for staining or use of
fluorophores.
MICROSCOPE CONFIGURATION:
Phase contrast capability can be added to almost any brightfield
microscope, provided the specialized phase objectives conform to the
, tube length parameters and the condenser will accept an annular phase
ring of the correct size.
Fluorescence microscopy
TECHNOLOGY:
Fluorescence imaging uses high intensity illumination to excite
fluorescent molecules in the sample. When a molecule absorbs
photons, electrons are excited to a higher energy level. As electrons
'relax' back to the ground-state, vibrational energy is lost and, as a
result, the emission spectrum is shifted to longer wavelengths.
Fluorescence emanates from the sample.In epi-fluorescence
microscopes, the objective both focuses the excitation light and
collects light returning to the eyepiece or detector.
Fluorescence is separated from excitation light by a dichroic mirror
and appropriate filters: excitation light is reflected back into the
objective while fluorescence is transmitted. Filters, excluding and / or
transmitting selected wavelengths of light, optimize fluorescence and
reduce unwanted 'background noise'.
Brightfield microscopy
TECHNOLOGY:
Brightfield imaging is the simplest form of microscopy where light is
either passed through, or reflected off, a specimen. Illumination is not
altered by devices that alter the properties of light (such as polarizers
or filters).
APPLICATIONS:
In biological applications, brightfield observation is widely used
for stained or naturally pigmented or highly contrasted
specimens mounted on a glass microscope slide.
The specimen is illuminated from below and observed from
above. The specimen appears bright, but darker than the bright
background. This technique is widely used in pathology to view
fixed tissue sections or cell films / smears.
Brightfield imaging is not very useful for unstained living cells
or unstained tissue sections as, in most cases, the light passes
through transparent or translucent samples with little or no
definition of structure.
Light is reflected from opaque samples and this is exploited in
industrial environments where brightfield imaging is used for
wafer inspection and liquid crystal board inspection.
MICROSCOPE CONFIGURATION:
All light microscopes are capable of brightfield imaging.
,Darkfield microscopy
TECHNOLOGY:
Darkfield microscopy creates contrast in transparent unstained
specimens such as living cells. It depends on controlling specimen
illumination so that central light which normally passes through and
around the specimen is blocked. Rather than light illuminating the
sample with a full cone of light. the condenser forms a hollow cone
with light travelling around the cone rather than through it.
This form of illumination allows only oblique rays of light to strike
the specimen on the microscope stage and the image is formed by
rays of light scattered by the sample and captured in the objective
lens. When there is no sample on the microscope stage the view is
completely dark.
Care should be taken in preparing specimens as features above and
below the plane of focus can also scatter light and compromise image
quality . In general, thin specimens are better because the possibility
of diffraction artifacts is reduced.
APPLICATIONS:
In darkfield microscopy, contrast is created by a bright specimen
on a dark background. It is ideal for revealing outlines, edges,
boundaries, and refractive index gradients but does not provide a
great deal of information about internal structure. Ideal subjects
include living, unstained cells, although fixed stains cells can
also be imaged successfully.
Darkfield imaging is particularly useful in haematology for the
examination of fresh blood.
Non-biological specimens include minerals, chemical crystals,
colloidal particles, inclusions and porosity in glass, ceramics,
and polymer thin sections.
,MICROSCOPE CONFIGURATION:
Almost any brightfield laboratory microscope can be easily converted
for use with darkfield illumination using special darkfield condensers
(dry or oil type).
Phase contrast microscopy
TECHNOLOGY:
In phase contrast imaging, small changes in optical path length are
optically translated into corresponding changes in light intensity,
which can be observed as differences in image contrast.
Light from a tungsten-halogen lamp is directed through a collector
lens and focused on a specialized annulus positioned in the substage
condenser.
Wavefronts passing through the annulus illuminate the specimen and
either pass straight through or are diffracted and retarded by phase
gradients in the specimen. Undeviated and diffracted light collected
by the objective is separated by a phase plate and focused at the
intermediate image plane to form the final image.
One drawback of phase contrast imaging is the occurrence of "halos"
around areas of high phase shift.
APPLICATIONS:
Phase contrast imaging is applicable to many transparent
subjects, such as living cells in culture, micro-organisms, thin
tissue slices, lithographic patterns, fibres, latex dispersions,
glass fragments, and subcellular particles .where the technique
reveals structure that is not visiblein brightfield imaging.
An advantage of phase contrast microscopy is that living cells
can be imaged in detail without the need for staining or use of
fluorophores.
MICROSCOPE CONFIGURATION:
Phase contrast capability can be added to almost any brightfield
microscope, provided the specialized phase objectives conform to the
, tube length parameters and the condenser will accept an annular phase
ring of the correct size.
Fluorescence microscopy
TECHNOLOGY:
Fluorescence imaging uses high intensity illumination to excite
fluorescent molecules in the sample. When a molecule absorbs
photons, electrons are excited to a higher energy level. As electrons
'relax' back to the ground-state, vibrational energy is lost and, as a
result, the emission spectrum is shifted to longer wavelengths.
Fluorescence emanates from the sample.In epi-fluorescence
microscopes, the objective both focuses the excitation light and
collects light returning to the eyepiece or detector.
Fluorescence is separated from excitation light by a dichroic mirror
and appropriate filters: excitation light is reflected back into the
objective while fluorescence is transmitted. Filters, excluding and / or
transmitting selected wavelengths of light, optimize fluorescence and
reduce unwanted 'background noise'.
