Automatic detector synchronization for long-term imaging using confocal light-sheet microscopy.

Light sheet fluorescence microscopy (LSFM) is an important tool in developmental biology. In this microscopy technique confocal line detection is often used to improve image contrast. To this end, the image of the illuminating scanned focused laser beam must be mapped onto a line detector. This is not trivial for long-term observations, since the spatial position of the laser beam and therefore its image on the detector may drift. The problem is aggravated in two-photon excitation LSFM, since pulsed laser light sources exhibit a lower laser beam pointing stability than continuous wave lasers. Here, we present a procedure for automatic synchronization between the excitation laser and detector, which does not require any additional hardware components and can therefore easily be integrated into existing systems. Since the recorded images are affected by noise, a specific, noise-tolerant focus metric was developed for calculating the relative displacement, which also allows for autofocusing in the detection direction. Furthermore, we developed an image analysis approach to determine a possible tilt of the excitation laser, which is executed in parallel to the autofocusing and enables the measurement of three solid angles. This allows to automatically correct for the tilting during a measurement. We demonstrated our approach by the observation of the migration of oligodendrocyte precursor cells in two-day-old fluorescent Tg(olig2:eGFP) reporter zebrafish larvae over a time span of more than 20 hours.