Biomedical Engineering Reference
In-Depth Information
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Photon-counting regime
. On the opposite, if the pulse rate is much lower than the inverse of the
pulse width, the pulses come out well separated in time and in this case the number of pulses
is proportional to the intensity of the detected light (Figure 2.16b). The detection is obtained by
counting the number of pulses with a digital counter. The photon-counting regime is most likely
used in low light level and requires a fast detector with high gain.
As a general rule, proportional regime is most likely used in applications where the signals to be
detected are high and a fast scanning speed is required; photon-counting regime is instead preferable
in applications, where the signals to be detected are low and the required scanning speed is not high.
2.4 optical Scheme of a typical SHG Microscope
In this section, we conclude this chapter presenting the whole optical scheme of a typical SHG micro-
scope. In detail, three representative optical configurations will be considered: the forward detec-
tion geometry (Figure 2.17) and backward detection geometry in both non-de-scan (Figure 2.18) and
de-scan mode (Figure 2.19).
2.4.1 Forward Detection
This optical configuration is the most commonly used in SHG microscopy. This optical configuration is
generally used in SHG imaging of nonmassive samples, such as cultured cells, thin tissue slices, and so
on. Referring to Figure 2.17, the experimental setup is composed by:
L4
BF
DM
PMT
BP
Specimen
RP
λ
/4
POL
Acquisition
TL
λ
/2
PBS
S
Control
L3
G
y
G
x
L1
L2
FIgurE 2.17
Optical scheme of a typical SHG microscope with forward detection geometry. Ti:sapphire:
femtosecond laser source; λ/2: rotating half-wave plate; PBS, polarizing beam splitter cube; S, shutter;
G
x
, galvo-
scanner
x
; L1, relay lens; L2, relay lens;
G
y
, galvo-scanner
y
; L3, scanning lens; TL, tube lens; POL, polarizer; λ/4,
quarter-wave-plate; RP, rotating polarizer; Exc, excitation objective lens; Coll, collection objective lens; DM,
dichroic mirror; BF, laser-blocking filter; L4, collection lens; BP, narrow band-pass filter; PMT, photomultiplier
detector.
