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Possible cause: auxiliary filtration issues or detector mis-placement
o It is possible that the fluorescence emission is being blocked by the filter placed in the
removable filter cube, or that the detectors are placed in the wrong positions to suit the
filters installed.
o Try imaging without the filter cube in the main optics block. The red-sensitive detector is
usually placed at the rear of the detection system, and should give a strong signal if the
fluorophore is red (such as AlexaFluor 594), but a blue-green sensitive detector placed
at the rear would give a much weaker signal.
o Confirm that the dichroic mirror in the filter cube is transmitting and reflecting
wavelengths in a manner appropriate to the placement of the detectors: the usual
situation is for the dichroic mirror to reflect shorter (more blue) wavelengths to the side
and to transmit longer (more red) wavelengths towards the rear of the system. Any
auxiliary band-pass filters must be placed in appropriate locations within the cube or no
light will pass through either exit port.
Possible cause: use of a narrow-field objective for imaging in a highly-scattering sample.
o It may be that the multiphoton fluorescence is being generated as expected but is then
being scattered laterally by the sample until it escapes the field of view of the objective.
Try using a smaller scan area or changing to a wider-field objective.
Shading at extremes of field, shading on one side or other, shading at top or bottom
Probable cause: scanner occlusion, poor imaging from scanner mirrors to objective aperture.
o Check the alignment of the imaging system using an appropriate laser-beam viewing
card (wear eye protection during these adjustments!). It may help to set the scanner for
a slow but full-field scan. It may help to adjust the focus of the system to image a
slightly different plane onto the objective’s rear aperture.
o If the problem appears to be new, it may be that the objective lens position has been
adjusted significantly in the vertical direction (perhaps the experiment height has
changed significantly?). In this case re-focusing the beam delivery system should help.
Loss of signal with depth
This is to be expected for two reasons: firstly the laser intensity contributing to the multiphoton
fluorescence is reduced at depth as the sample tissue distorts and scatters the incoming beam.
Secondly, the visible fluorescence is strongly scattered by most tissues and does not reach the
collecting objective so efficiently.
o Consider using substage detection instead of or together with above-sample detection.
Limited field of view (FoV) in multiphoton images
Possible cause: scanning too wide a field or imaging too deeply
o As for the previous problems, visible fluorescence can be scattered laterally by a
significant distance – at least equal to the imaging depth. If the corners of an image lie
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