Confocal microscopy principle & application

1. CONFOCAL MICROSCOPY
K.K.Gupta
kishore_gupta30@yahoo.com
DROSOPHILA CYTOGENETICS & MOLECULAR LABORATORY
UNIVERSITY DEPARTMENT OF ZOOLOGY
VBU. HAZARIBAG

2. Introduction
• Microscope is the instrument used to visualize the cellular structure
and their various physical and biochemical component with respect to
their various structure , organization & functions.
• Every microscope has certain limitation so time to time, scientists
have designed and improved it as per their requirement.
• Optical Microscope like Simple compound to Fluorescence are
confined to imaging the two dimensional structure of tissue section
for which thin section is mandatory.
• More thin section of a tissue more will be the fine visualization.
However, their varies in the staining technique from simple staining
to fluorescence staining for more contrast

3. Limitations
• Simple compound Microscope –suitable for thin fixed & stained
section by non fluorescence staining.
• Fluorescence Microscope – suitable for thin section /live cell
culture suspension with the use of fluorescence dyes under high
contrast condition. Moreover processing of tissue is required .
• Thick tissues can not be scanned and visualized as normal light
unable to pass. So give poor resolution due to back ground noise
by secondary light or fluorescence .
• Only tissue up to 2 micrometer can be imaged .
If thick specimen is viewed by conventional light microscope , the
image obtained by focusing at any one level is degraded by blurred
& out of focus information from the parts above & below the
plane of focus.
Such three dimensional architecture of cell of thick tissues can be
visualized and imaged through confocal microscope as it has
designed to manage the light coming from every plane of focus and
not only single plane
•

4. Advantage of Confocal microscopy
• It is an advance and computer based imaging microscopy technique
where a series of images in different focal planes is obtained and
regarded as new version of fluorescence microscope . It is designed in
such a way that it is possible to focus at any desired /chosen plane and
also ability of rejecting light coming out of focus plane.
Confocal microscopy thus offers several advantages over conventional
optical microscopy like
• provide shallow depth of field,
• eliminate out-of-focus glare/ light
• ability to collect serial optical sections from thick specimens
• Ability for imaging either fixed or living cells and tissues that
have usually been labeled with one or more fluorescent
probes.

5. Principle of confocal Microscopy
• It works on two ideas
• 1. pin to pin illumination of the specimen by the use of light
source pin hole aperture and
• 2. rejection of out focus light
Light source of very high intensity zirconium arc lamp of Minsky’s
design and laser source in modern design is allowed to pass
through pin hole which provide blue excitation light.
The light reflects through a dichoric mirror and is directed through
assembly of vertical and horizontal scanning mirror .
These motor driven scanning mirror scan the lasser beam across
the specimen .
• The specimen is scanned by moving the stage back &forth in the
vertical /horizontal direction while optics is kept constant
stationary.

6. Principle

8. Components & workingConsists
A. laser excitation source – as it has
high frequency and low wave length
of light to illuminate the specimen
B. Light Source pin hole aperture –
allow only selected wave length of
light to pass through the specimen
at some focal plane it is movable
C.Dichoric mirror – allow to reflect
and transmit the selected focus light
and rejecting non focus /out focus
light light
D.Detector pin hole aperture –
allow to pass the focus reflected
light from specimen at a particular
plane of focus

9. Process
• Light from the source passes through pin hole
and fall on the Dichoric mirror
• The scanner scanned the light which is focused
bt the condenser and allow this light to fall on
the specimen at certain point.
• The light entering in to the pin hole can be
change by moving the pin hole so that light may
be focused at different focal plane.
• The focused light excite the specimen an a light
reflecting from the specimen enters through
dichoric mirror and allow it to pass through
second pin hole from where it goes to detector
and forms digital image

10. Advantage and application
• The specimen is illuminated every where
axially,rather tha at different angles ,therby
avoiding optical abberrration
• Entire view of field is illuminated uniformly
• The field of view can be made larger than that of
the static objective by controlling the amplitude
of the stage movement
• Better resolution
• Cell can be aliveor fixed
• Serial optical section can be collected stage

11. Limitations of confocal microscope
• Resolution –it has inherent limitation of
resolution due to diffraction .maximum best
resolution of cofocal microscope is 200nm
• Pin hole size –strength of optical sectioning
depends upon the size of pin hole.