Variations of Confocal

Microscope

Arun Jyothi S.P

Laboratory for Confocal microscopy and Experimental pathology
SCTIMST

 There are more than one way to achieve

confocal effect.
To achieve confocal effect it is not
necessary to use laser and pinhole.
There are three major types of confocal
system available.

1. Laser Scanning Confocal Microscope

(LSCM)
2. Multiphoton Laser Confocal Microscope
(MP)
3. Disk Scanning Confocal Microscope
(Nipkow disk system)

Laser Scanning Confocal

Microscope (LSCM)
The archetype of confocal microscope in
the confocal family.
It utilizes laser and illumination pinhole to
get point-like light source illumination on
the specimen.
Detecting pinhole is used to get rid of outof-focus signal.
PMTs are used as detecting device.

Laser Scanning Confocal

Microscope (LSCM)

Advantages

Good image quality.

Versatile functionality.
Reliability.
Easy to maintain.

Disadvantages
Slow frame rate.
Photo bleaching.
Photo-cytotoxicity due to the indiscriminate
illumination and excitation.
Exciting on the full length of the specimen
(although only focal-plane signal is
collected by PMT).

Multiphoton Laser Confocal

Microscope (MP)
Uses two lasers for a single excitation.
It is based on the phenomena that two
high-energy lasers of longer wavelength
focused on a single point will produce a
shorter excitation at the half of their
original wavelength.
High peak-power, pulsed femto-laser is
used to produce high energy laser of
longer wavelength.

The sample is illuminated with a wavelength (700nm)

around twice the wavelength of the absorption peak
(350nm) of the fluorophore being used.

 Since we are using two laser sources, twophoton events will occur at the single point of
focus.
At this point of focus the photon density is
high that two photons can be absorbed by the
fluorophore simultaneously.
In this way, fluorophore excitation will only
occur at the point of focus thereby eliminating
excitation of out-of-focus fluorophore and
achieving optical sectioning without pinholes.

As shown in figure on the right side, the excitation of fluorophore

occurs only at the focal point where two laser meet, (while in single
photon system, the excitation is on the whole illuminated length,
although only emission from focal point is detected).

A cuvette of fluorescent dye excited by single photon

excitation (right line) and multiphoton excitation (localized
spot of fluorescence at left) illustrating that two photon
excitation is confined to the focus of the excitation beam

Advantages
Low photo-cytotoxicity, since the long wavelength
infra red laser has less toxicity.
Longer wavelengths are scattered to a lesser
degree, which is a benefit to high-resolution
imaging.
It also bleaches less than LSCM because only the
focal point is affected.
MP suits living cell imaging, point bleach
experiment, and other physiological studies.
MP can work with even thicker specimen (1mm)
than single photon confocal.

Disadvantages
Slightly lower resolution with a given fluorophore
when compared to confocal imaging, since there
is no pinhole.
Thermal damage can occur in a specimen if it
contains chromophores that absorb the
excitation wavelengths, such as the pigment
melanin.
Only works with fluorescence imaging.
High price for building-up and maintaining the
system.

Thank You..