Confocal Laser Scanning Microscopy

 The principle of confocal microscopy was originally

patented by Marvin Minsky in 1957

Marvin Minsky
(MIT, USA)
 In 1978, Thomas and Christoph
Cremer designed a laser scanning
process, which scans the three
dimensional surface of an object point-
by-point by a laser beam

 Creates the over-all picture by

Thomas Cremer Christoph Cremer electronic imaging process
(Germany) (Germany)
Optical sectioning and 3D reconstruction
Basic Concept
 Conventional fluorescence microscope:

 The entire specimen is illuminated by light

 All parts of the specimen are excited and the resulting fluorescence is
detected by photodetector including a large unfocused background part.

 Confocal microscope:

 Uses point illumination

 Eliminates out-of-focus signal

 Ability to collect serial optical sections from thick specimens

 Creates 2D and 3D views

 Fixed or living cells and tissues that are labeled with fluorescent probes
can be imaged
 An image stack

 The layer of cells are optically

sectioned by a confocal microscope.

 The stack is collected in 0.25

micron steps.

It makes it easy to see that

different events are occurring at
different levels within the cell layer.
 Confocal Laser Scanning microscope

Basic components of a confocal laser scanning microscope (CLSM)

Confocal Microscopy

Inverted Scope Upright Scope

Optical pathway in a confocal scan head

 Laser

 Dichroic mirror

 Scan control

 Objective lens
Laser
 Barrier filter : allows emitted light

 Pinhole : blocks the out of focus light

 PMT :
Confocal principle in fluorescence laser scanning microscopy

pinhole

Laser

mirror

Objective lens
 The scan-control mechanism in a CLSM

Delivery of the excitatory laser

beam to the specimen by one of two
galvanometer-driven mirrors

 mirrors vibrate in mutually

perpendicular axes within the confocal
scan head

One mirror controls scanning

along the x-axis, the other along the y-axis

 Both motions are coordinated to

generate a pattern of a raster on the
Specimen.

 Raster scanning mechanism create image by scanning by point by

point over sample and recording intensely at each point
159
 Confocal microscope image showing
insect immune cells (green) containing
E.coli bacteria (red).

http://thewere42.wordpress.com/2009/07/page/4/
 Applications of Confocal Microscopy

1. FRAP : Fluorescence Recovery After Photo-bleaching

2. FRET : Fluorescence Resonance Energy Transfer

FRAP
3. FLIM: Fluorescence Lifetime Imaging
FRET
FLIM
4. FLIP: Fluorescence
FLIP loss in photobleaching
FLAP
FISH
5. FLAP: Fluorescence localization after photobleaching

6. FISH: Fluorescence in situ hybridization

 Fluorescence resonance energy transfer

 Mechanism describing energy transfer between two chromophores

 A donor chromophore, initially in its electronic excited state, may transfer

energy to an acceptor chromophore (in proximity, typically less than 10 nm)
through nonradiative dipole–dipole coupling without emission of a
photon.
 FRET is an important technique for investigating a variety of biological
phenomena that produce changes in molecular proximity

 When FRET is used as a contrast mechanism, colocalization of proteins

and other molecules can be imaged with spatial resolution beyond the
limits of conventional optical microscopy

 Primary Conditions for FRET

 Donor and acceptor molecules must be in close proximity (typically 10–

100 Å).

 The absorption spectrum of the acceptor must overlap the fluorescence

emission spectrum of the donor (Figure).

 Donor and acceptor transition dipole orientations must be approximately

parallel.
 Fluorescence in situ hybridization (FISH)

 FISH involves the preparation of short sequences of cDNA complementary

to the DNA sequence of interest.

 These cDNA probes are labeled with fluorescent tags, and once the probes
hybridize to the complementary DNA, the area of DNA fluoresces.

 FISH can be performed on nondividing cells as well as actively dividing

cells.

 Used in medical diagnostics to assess chromosomal integrity

Used to measure and localize mRNAs and other transcripts within tissue
sections or whole mounts.

 Used to study microbial diversity ( using 16S rRNA fluorescence in situ

hybridization)