Optimal image quality in light-sheet microscopy requires a perfect overlap between the illuminating light sheet and the focal plane of the detection objective. However, mismatches between the light-sheet and detection planes are common owing to the spatiotemporally varying optical properties of living specimens. Here we present the AutoPilot framework, an automated method for spatiotemporally adaptive imaging that integrates (i) a multi-view light-sheet microscope capable of digitally translating and rotating light-sheet and detection planes in three dimensions and (ii) a computational method that continuously optimizes spatial resolution across the specimen volume in real time. We demonstrate long-term adaptive imaging of entire developing zebrafish (Danio rerio) and Drosophila melanogaster embryos and perform adaptive whole-brain functional imaging in larval zebrafish. Our method improves spatial resolution and signal strength two to five-fold, recovers cellular and sub-cellular structures in many regions that are not resolved by non-adaptive imaging, adapts to spatiotemporal dynamics of genetically encoded fluorescent markers and robustly optimizes imaging performance during large-scale morphogenetic changes in living organisms.

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Systematic optimisation with central composite design offers an efficient route for rapid optimisation of resolution with multiple interacting parameters in chiral CE. This is illustrated by separations of amlodipine with a-CD as chiral selector in the running buffer, for which the predicted performance of central composite design is assessed. The utility of response surface methodology for locating optimum ruggedness in CE is also described.o 1995 Wiley-Liss, Inc.KEY WORDS: central composite design, liquid chromatography, capillary electrophoresis, amlodipine, cyclodextrin, ruggedness Systematic methods for optimisation of chiral resolution are well established in liquid chromatography (LC). '7' They have significant potential for rapid method development in capillary electrophoresis (CE). In LC, the use of central composite design (CCD) has already been established by Zoest et aL3 for nitroimidazole separations in a reversed-phase system, involving the optimisation of two variables. More recently Tucker et aL4 showed that CCD is generally applicable for the optimisation ' of three variables in chiral LC separations. The fact that interactions between operational parameters can be assessed by CCD, and that modelling of the response surface can be achieved in four-dimensional space to predict rapidly and reliably the experimental conditions required for optimum response (e.g., resolution), were shown to be key advantages of this approach. Indeed, in chiral LC separations of tioconazole using the experimental conditions predicted by CCD, it was shown that there was excellent correlation between the response (i.e., resolution) observed in practice, with that predicted by SAS.4In the field of CE, however, by comparison with LC limited attention has so far been given to the use of systematic optimisation strategies, despite the acknowledged complexities of the separations involved. In 1991 Atanha et aL5 explored the use of factorial design for the optimisation of two variables in capillary zone electrophoresis. In the same year Vindevogel et aL6 demonstrated the use of a Plackett-Burman design for the first time in the micellar electrokinetic chromatography (MEKC) of steroids. In 1992 Li and co-workers7 also examined systematic optimisation for the separation of flavonoids by MEKC. Altria et aL8 explored the use of univariate optimisation in the chiral CE of P-aminoalcohol enantiomers, using cyclodextrins as buffer additives. In 1994 this group used a Plackett-Burman design for chiral CE in the separation of clenbuterol enantiomers. These authors have recently re-0 1995 Wiley-Liss, Inc.viewed various modes of CE with LC for drug analysis and discussed operational strategies for their optimisation. lo Given the advantages of CCD already identified in LC,4 there is signhcant potential for its use as a novel approach to rapid method development in all modes of CE. This approach has important advantages in high-performance CE systems, particularly for chiral separations, compared with the ad hoc univariate opti...

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