Biomedical Engineering Reference
In-Depth Information
• The capability to estimate the mean orientation and/or the spatial distribution of the emitters
contained in the focal volume with respect to the optical axis by measuring the ratio between
forward and backward scattered light (LaComb et al., 2008, Nadiarnykh et al., 2010). In fact, as
reported in Figure 2.13, the front-to-back emission ratio (F/B) depends on the angle between the
emitter and the optical axis with a monotonic behavior.
2.3.3 optical Filtering
Optical filtering is a crucial point for an SHG microscope, since the power density of the excitation is
extremely high in comparison to the signals to be detected. Filtering is commonly achieved by using
three optical elements:
• A
dichroic mirror
for diverting from the laser path the SHG light to be detected.
• In principle, the dichroic mirror needs to be chosen with a cutoff wavelength comprised between
the excitation wavelength and the SHG wavelength. As a general rule, the dichroic mirror has
to be placed as close as possible to the collection objective. This serves to minimize the distance
between the detector and the objective, therefore maximizing the detected signal (Oheim et al.,
2001, Svoboda and Yasuda, 2006). Furthermore, in backward detection, dichroic filters with a
thin substrate are recommended to minimize the wave front distortions in the excitation beam.
• A
short-pass filter
for blocking spurious contamination of the excitation light in the detection path.
• Such filter has to be placed in the detection path independently if the microscope works with
backward or forward detection. Its function is to remove spurious laser light from the detection
path. The optical density at the laser emission wavelength should be at least 6-7, in order to avoid
detection of undesired NIR photons. On the other hand, the filter transmission at the SHG spec-
tral range should be as high as possible (generally more than 90%). The most common laser (e.g.,
Ti:sapphire laser) blocking filters for SHG microscopy have a cutoff wavelength between 650 and
700 nm, in order to block the laser light (λ > 700 nm) and transmit both SHG and fluorescence
light. In fact, while in principle a shorter cutoff wavelength can be chosen for an SHG microscope,
it could be of interest for the researcher to be able to detect also two-photon fluorescence signals
with the same instrument. Additional filtering for removing fluorescence and selectively detect
SHG is performed by using another filter, as described below.
• A
narrow band-pass filter
selectively detects only photons involved in the SHG process.
• This filter is placed in the detection path to selectively detect only photons involved in the SHG
process. Considering that the emitted SHG light has a wavelength of exactly one-half the excita-
tion wavelength, the central wavelength of the bandpass is chosen at simply half the excitation
wavelength. On the other hand, the optimal filter width depends on the spectral broadening of
excitations laser pulses. For a Ti:sapphire laser, a dielectric bandpass filter with 20 nm FWHM is
a good choice. Narrower filters, based on interference, can be used as well.
2.3.4 Detectors
This section describes the most common detectors that can be used in an SHG microscope and the
features to be taken into account when choosing the detector. In particular, the detection modality, the
spectral sensitivity, and the dimension of the sensitive area will be considered.
2.3.4.1 Detector technology
The main technologies used for building detectors suitable for SHG microscopy are summarized below.
•
Photomultiplier tubes (PMTs):
The most used detector in SHG microscopy is the PMT. A conven-
tional PMT is a vacuum device which contains a photocathode, a number of dynodes (amplifying
stages) and an anode which delivers the output signal (Figure 2.15a). An electrical field accelerates
