Biomedical Engineering Reference
In-Depth Information
Excitation
light
Scanning system
Detector
DM
Pinhole
X-Y
Scanner
Non - descanned detector
Detector
DM
Objective
lens
Focal
Plane
Scattering specimen
Fig. 4.6.
Laser sources in use at LAMBS Microscobio research center of the
University of Genoa for TPE
to rapidly modulate the laser intensity: in this way it is possible to define
regions to be imaged with different powers during the very same scan.
As depicted in Fig. 4.6 it is possible to consider two popular approaches
to TPE microscopy, the descanned and non-descanned modes. The first con-
figuration uses the same optical pathway as used in confocal microscopy, and
the light emitted by the sample is deflected back by the scanning mirrors and
brought to the photomultipliers. The pinhole is usually kept open - or removed
- as it is not necessary for TPE optical sectioning. In the non-descanned mode
the microscope architecture is optimized for collecting the maximum TPE
signal: pinholes are removed, the emitted light is collected through a dichroic
mirror and it does not cross the scanning system. [61, 76, 86]
Despite the configuration used, the fluorescent light is brought by the
detection system by a dichroic mirror. Several types of detectors can be used,
