Advantages:

·       
It provides high contrast imaging because
it only provides us the idea of which region of the cell contain fluorescent
dye and which is not.

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·       
High specificity since we tag the
fluorescent dye with our interested cellular components.

·       
Quantitative imaging and live cell
imaging

Disadvantage:

·       
Photo bleaching

 

Confocal
Microscopy:

Confocal microscopy is
an optical microscopic technique for increased resolution and contrast. The
reconstruction of 3D structure from the obtained images can be done. The
special feature here is the pinhole, which only selects the light coming from a
special focal point of the sample and blocks any other scattered lights that
are coming from the different planes of the specimen. It allows only particular
point to be focused and only detect light or emission that is coming from the
specific point which eventually gives crystal clear images.

The idea about the
fluorescence is same as the fluorescence microscopy, the pinhole is placed
which only allows the emission coming from the particular plane of the specimen
to pass and block any other excitation. Concise and contrast image from the
particular location can be seen. Pinhole provides the optical sectioning which
allows blocking the emission light from out of plane regions of the sample and
only select one particular focal point and light coming from that point passes
through the pinhole to the detector.

Working
Principle:

When the laser light
from the laser module is focused onto the specimen and illuminated, it enters
the excitation filter and hits the dichroic mirror. Then the laser passes
through the objective and hits in different focal plane. The emission beam
again passes through the objective and dichroic mirror and finally there is a
emission filter which will prevent any other further scattering there, the
laser light pass through it and reaches pinhole, which is a unique feature
about the confocal microscopy. Selected emission light will break through the
pinhole aperture and hits the detector. We can precisely focus particular
region of the cell which we are interested.

Advantages
of Confocal microscopy:

·       
Possible to get 3D image and 3D
reconstruction

·       
Optical sectioning can be done without
physical contact

·       
High resolution image (0.1-0.2?m)

·       
We can choose the suitable excitation
wavelength of the laser source.

Limitations
of Confocal microscopy:

·       
If it is a thick sample, tissue depth
problem occurs. When the laser hits the sample, intensity of the light/
penetration power depends on the length/depth of the tissue. If it is thicker,
the light will not able to penetrate the tissue. Lot of energy loss occurs.

·       
Choice of fluorophores is important

·       
It is largely time consuming

·       
Diffraction limits the image resolution.