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Smearing or “trails” behind narrow, bright objects along scan lines, narrow features appear too wide
(horizontally). Problem mainly evident in high speed scans.
Probable cause: excessive scan speed for the detector bandwidth.
o Ensure that the sampling rate of the data acquisition system is set to at least twice
(preferably three times) the specified bandwidth of the preamplifier.
o Try reducing the scanning speed (either decrease the scanned field or increase the line
time, or both); if this reduces or removes the problem then it is probable that the
preamplifier in the detector cannot cope with the scan speed (it has insufficient
bandwidth). If this is a problem for your experiments contact Scientifica regarding
customised preamplifier bandwidths.
o If your calculations suggest that the scan speed and resolution in use should not be
causing a problem, it is possible that the pre-amplifier filter is faulty. Contact Scientifica
for assistance.
Excess noise in digitized signals (especially compared to the oscilloscope trace)
Probable cause: under-sampling by the data acquisition system.
o It is important that the sampling rate be set at least twice (preferably three times) as fast
as the rated bandwidth of the detectors and preamplifiers; for example, a detector /
preamp with a 500 kHz bandwidth should be sampled at a rate of at least 1 MHz, and
preferably 1.5 MHz regardless of the scan speed in use. This precaution ensures that
the noise floor is as low as possible, and that high-frequency noise is not “aliased” (or
mixed) down to lower frequencies where most of the signal energy resides. Sampling at
half the recommended rate may increase the apparent noise floor in the digitised data
by 40% or more.If you know that the scan will be run more slowly, and that the signal will
not occupy the whole preamplifier bandwidth, then apply a suitable FIR digital low-pass
filter to the line-scan data coming from the digitiser and then decimate the data stream
to achieve the appropriate sampling density. It is never advisable to reduce the
sampling rate! If this processing requires too much storage or computational resource,
consider using a lower bandwidth detector preamplifier.
Low or no multiphoton signal, or unusually large laser power required to obtain any signal.
Possible cause: insufficient laser spot intensity at the sample (multiphoton fluorescence
intensity varies as a power of the applied laser intensity).
o Check that the appropriate or usual laser power is being delivered into the objective and
that the laser is functioning within specification. Check any beam attenuation system
that may be installed – is it functioning and adjusted correctly?
o Is the laser beam filling the rear aperture of the objective lens? If the beam is too
narrow, then the objective will form a wider focus than it might otherwise, with lower
peak intensity, and multiphoton fluorescence may be reduced. This can occur if you
change objectives for a type that has an increased NA or a reduced magnification at the
same NA.
o Is there some aberration in the beam delivery optics that is blurring the laser spot at the
sample?
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