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.

1. by
c.Keerthana

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.

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

7. Light source
Annular ring
Condenser
Specimen plate
(interference)
objective lens
phase ring
Image plane

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.

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.