Biomedical Engineering Reference
In-Depth Information
Photomultiplier
Rejection
pin-hole
Detector
pin-hole
Illumination
pin-hole
Confocal
unit
Frame buffer
Dichroic
mirror
Laser
Control
electronics
Excitation
filter
x/y scanning
unit
PC
Conventional
microscope
Objective
Focus
motor
Specimen
Stage
1. 8
Components of a laser scanning confocal microscope (LSCM).
1.4.1 Mechanics of LSCM
In LSCM a beam of laser light is focused into a small point at the focal plane
of the specimen. In this case the specimen may be a human cell containing
a fl uorescent molecular probe. The illuminating laser beam is focused onto
the specimen by fi rst passing it through a light source pin-hole aperture
after it leaves the laser excitation source which is usually an Argon/Krypton
gas laser. The laser light beam is then refl ected downward by a dichromatic
mirror so that it passes through the lens assemblies of the objective and is
highly focused at the focal plane of the specimen. A computer-controlled
scanning mirror can move or scan this beam in the
x
and
y
directions at the
focal plane. Fluorescent emission is created at each point of the scanned
laser beam in the specimen focal plane with considerable fl uorescence
also being created above and below the focal plane. This divergent fl uo-
rescence, if it makes its way to the photon multiplier tube or detector, will
cause blurring of the fi nal image. To get rid of this light a detector pin-hole
aperture is introduced between the detector and the specimen. Light out-
side of the specimen focal plane is largely excluded from hitting the photon
multiplier tube. The geometry of the light in the focal plane is such that it
