Phase contrast microscopy is a type of light microscopy technique that produces high contrast images of transparent specimens without staining. It works by interfering surrounding light waves and sample-diffracted waves to create an image with the inner regions of the sample appearing darker than the outer regions. Fluorescence microscopy uses fluorescent dyes excited by high-energy light to emit lower-energy light, allowing specific structures within samples to be imaged. It is used to study living cells and view genetic material.
2. First
described by Dutch physicist frits
Zernike in 1934.
It is a type of light microscopy.
It is a contrast enhancing optical technique
that produces high contrast images of
transparent specimens.
Specimen- unstained and alive.
3.
4. ANNULAR
RING : is between condenser and
light source.
PHASE RING : is between objective lens and
image plane.
SPL PROPERTY : both the rings allow partial
light to pass through it and the rest is
blocked.
5. Basic mechanism is interference of light
beams.
INTERFENRENCE:
Interaction of two light waves which leads to
the formation of resultant wave.
TYPES OF INTERFERENCE:
constructive interference
destructive interference
8. Light
passes through the condenser via
annular ring.
After reaching the specimen plate two types
of beams are formed.
IF THERE IS NO SPECIMEN IN LENS:
1.Surrounding wave (S)
2.particle wave (p)
P=S
NO INTERFERENCE
9. IF
LENS CONTAINS SAMPLE:
Light beam gets diffracted because of
different density at different regions of
sample.
1.surrounding wave (S)
2.diffaracted wave (D)
P=S+D
Either constructive interference or
destructive interference may occur.
10. Positive
phase contrast produces
Constructive interference.
Thus, the image of the specimen obtained is
Inner region of the sample – darker
Outer region of the sample– bright
Surrounding lens – opaque
11. Negative
phase contrast microscopy produces
destructive interference.
Thus, the image obtained is
Inner region of the sample – bright
Outer region of the sample– darker
Surrounding lens – opaque
12. Fluorescence
microscope is
an one of the light microscope.
It refers to any microscope
that uses fluorescence to
generate an image.
It produces 3d image.
The technique is used to
study specimens, which
can be made to fluorescence.
13.
14. Fluorescence
is a
phenomenon that takes place
when a substance absorbs
light at a given wavelength
and emits light at another
wavelength.
Fluorescence occurs as an
electron, which has been
excited to a higher, and more
unstable energy state,
relaxes to its ground state
and gives off a photon of
light.
15. The
sample to be analyzed Is placed on a
lens. And the sample is coated with a
fluorescence material.
The light is illuminated through the lens with
the higher energy source. The illumination
light is absorbed by the fluorophores.
The sample causes them to emit a longer
lower energy wavelength light.
This fluorescent light can be separated from
the surrounding radiation with filters.
16. The light from the light
source is passed through
the excitation filter.
The specific wavelength of
light is passed through the
sample via dichronic filter.
The objective lens focuses
the light to the specimen.
The light emitted from the
specimen is filtered by
barrier filter.
17. Imaging
structural components of small
specimens, such as cells.
Conducting viability studies on cell
populations (are they alive or dead).
Imaging the genetic material within a cell
(DNA and RNA).
Viewing specific cells within a larger
population with techniques such as FISH.
To differentiate different type of cell.