Xlpln25xsvmp2

A half-waveplate and polarizing beam splitter set the excitation power sent to the microscope following pulse gating. The beam was expanded to fill the microscope objective back aperture, which was either a 25× objective (Olympus XLPLN25XSVMP2, 1.0 NA) for in vivo imaging or a 10× objective (Nikon CFIPlan10×, 0.25 NA) for cuvette experimentation. The excitation beam was scanned using a pair of galvanometer mirrors (Thorlabs, QS7XY-AG) conjugated to the objective back aperture using a scan lens (Thorlabs, SL50-2P2, $f=50\mathrm{mm}$ ) and tube lens (Thorlabs, two AC508-400-C lenses in Plössl configuration, $f=200\mathrm{mm}$ ) combination. The backscattered fluorescent signal was directed to a photomultiplier tube (PMT) (Hamamatsu, H10770PB-50) with a 775 nm cutoff dichroic filter (Semrock, FF775-Di01) and was further filtered with a 609/181 bandpass filter (Semrock, FF01-609/181-25), which immediately preceded the PMT photocathode. Imaging was controlled using a custom LabVIEW software. Although it may be necessary to synchronize EOM gating with the data acquisition sampling in some situations, this was not done for this work given our low sampling rate (160 kHz) relative to the gating frequency (1 MHz).