Confocal Laser Scanning

Microscopy (CLSM)

Nethravathi R

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

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.

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

Schematic diagram of CLSM

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

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 thefirst antibody. (a) When viewed by conventional fluorescence microscopy, the mitotic spindle is blurred. This blurring occurs because background fluorescence is detected from tubulin aboveand 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 onlyfrom molecules in the focal plane, generating a very thin opticalsection

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 actinappear as diffuse zones in the cell. (b) After application of the deconvolution algorithm to theimages, the fibrillar organization of microtubules and the localization of actin to adhesions become readily visible in the reconstruction.

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.

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.

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.

APPLICATIONS

Biosciences

Pathology

Pharmaceutical industries

Plant biology

Veterinary research

food technology

dairy technology

microbiology

cytogenetics

molecular biology

Industrial fields

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.

Thank you