Confocal Laser Scanning Microscopy (CLSM)

1. Confocal Laser Scanning
Microscopy (CLSM)
Nethravathi R

2. The optical details of the confocal microscope are complex, but
the basic idea is simple.
CLSM is classified under Single beam scanning microscopy, as
the specimen s illuminated and scanned using only 1 light source
MARVIN MINSKY awarded confocal patent focal scanning
microscope in 1957, US PATENT 301467

3. PRINCIPLE
 CLSM is generally used with fluorescence optics.
 but instead of illuminating the whole specimen at once, in the usual way,
the optical system at any instant focuses a spot of light onto a single point
at a specific depth in the specimen.
 It requires a very bright source of pinpoint illumination that is usually
supplied by a laser whose light has been passed through a pinhole.
 The fluorescence emitted from the illuminated material is collected and
brought to an image at a suitable light detector.
 A pinhole aperture is placed in front of the detector, at a position that is
confocal with the illuminating pinhole-that is, precisely where the rays
emitted from the illuminated point in the specimen come to a focus.
 Thus, the light from this point in the specimen converges on this aperture
and enters the detector.

4. Preparation of Samples
1. Fixation
2. Staining
a. Direct Method: Fluorescently labeled primary antibody or chemicals –
Fluorescent.
b. Indirect Method: Binding of Primary antibody + Fluorescently labelled
secondary antibody – Fluorescent.
3. Mounting
Biomedia corporation aqueous mounting medium

5. Schematic diagram of CLSM

6. PROCEDURE
In confocal scanning microscopy, exciting light from a
focused LASER beam illuminates only a single small part of a
sample for an instant and then rapidly moves to different
spots in the sample focal plane.
The emitted fluorescent light passes through a pinhole that
rejects out-of-focus light, thereby producing a sharp image.
Because light in focus with the image is collected by the
pinhole, the scanned area is an optical section through the
specimen.
The intensity of light from these in-focus areas is recorded by
a photomultiplier tube, and the image is stored in a computer

7. A mitotic fertilized egg from a sea urchin (Psammechinus) was lysed with a detergent, exposed
to an anti-tubulin antibody, and then exposed to a fluorescein-tagged antibody that binds to the
first antibody. (a) When viewed by conventional fluorescence microscopy, the mitotic spindle is
blurred. This blurring occurs because background fluorescence is detected from tubulin above
and below the focal plane as depicted in the sketch. (b) The confocal microscopic image is
sharp, particularly in the center of the mitotic spindle. In this case, fluorescence is detected only
from molecules in the focal plane, generating a very thin optical
section

8. A macrophage cell was stained with fluorochrome-labeled reagents specific for DNA (blue),
microtubules (green), and actin microfilaments (red). The series of fluorescent images obtained at
consecutive focal planes (optical sections) through the cell were recombined in three dimensions.
(a) In this three-dimensional reconstruction of the raw images, the DNA, microtubules, and actin
appear as diffuse zones in the cell. (b) After application of the deconvolution algorithm to the
images, the fibrillar organization of microtubules and the localization of actin to adhesions become
readily visible in the reconstruction.

9. ADVANTAGES over Fluorescence Microscopy
 Conventional fluorescence microscopy has two major limitations.
 First, the physical process of cutting a section destroys material, and so
in consecutive (serial) sectioning a small part of a cell’s structure is lost.
 Second, the fluorescent light emitted by a sample comes from molecules
above and below the plane of focus; thus the observer sees a blurred
image caused by the superposition of fluorescent images from molecules
at many depths in the cell.
 The blurring effect makes it difficult to determine the actual three-
dimensional molecular arrangement.
 Two powerful refinements of fluorescence microscopy produce much
sharper images by reducing the image-degrading effects of out-of-focus
light.

10. ADVANTAGES
 Ability to serially produce thin (0.5 to 1.5 micrometer) optical sections
- fluorescent specimens (Thickness ranging up to 50 micrometers or
more).
 Analysis x-z and y-z planes can be readily generated by Confocal
software programs.
 3D representation of the specimen with volume rendering
computational techniques – interrelationship in biological
investigations.
 Internal structures of interest at differing levels within the specimen.
 Multidimensional analysis of living cells and tissues.
 Digitization of the sequential analog image data – readily prepared for
print out for publication.

11. DISADVANTAGES
PHOTOBLEACHING: Reaction Involve the interaction of
Fluorophore + Light & Oxygen, destroys Fluorescence and yield
a free radicals, that cause death of live cells and tissues.
Monochromatic LASER beam is harmful.
It is cost effective.

12. APPLICATIONS
Biosciences
Pathology
Pharmaceutical industries
Plant biology
 Veterinary research
 food technology
dairy technology
 microbiology
 cytogenetics
 molecular biology
Industrial fields

13. BIBLIOGRAPHY
1. Class lecture-presentation- Sreenivas sir
2. The Cell 5th edition- Bruce Alberts et al.
3. Molecular Cell Biology 5th edition - Lodish et al.

14. Thank you