Keller, P. J. | bioRxiv Science

bioRxiv2018

BigStitcher: Reconstructing high-resolution image datasets of cleared and expanded samples

New methods for clearing and expansion of biological objects create large, transparent samples that can be rapidly imaged using light-sheet microscopy. Resulting image acquisitions are terabytes in size and consist of many large, unaligned image tiles that suffer from optical distortions. We developed the BigStitcher software that efficiently handles and reconstructs large multi-tile, multi-view acquisitions compensating all major optical effects, thereby making single-cell resolved whole-organ datasets amenable to biological studies.

bioinformatics↗

bioRxiv2017

A general method to fine-tune fluorophores for live-cell and in vivo imaging

AO_SCPCAPBSTRACTC_SCPCAPPushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. Here, we refine and extend this strategy, showing that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties with unprecedented precision. This strategy yields a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red with utility in live cells, tissue, and animals.

cell biology↗

bioRxiv2017

Reconstruction of cell lineages and behaviors underlying arthropod limb outgrowth with multi-view light-sheet imaging and tracking

During development coordinated cell behaviors orchestrate tissue and organ morphogenesis to suit the lifestyle of the organism. We have used here the crustacean Parhyale hawaiensis to study the cellular basis of limb development. Transgenic Parhyale embryos with fluorescently labeled nuclei were imaged at high spatiotemporal resolution with multi-view light-sheet fluorescence microscopy over several days of embryogenesis spanning appendage morphogenesis from early specification up to late differentiation stages. Cell tracking with a new tool called Massive Multi-view Tracker (MaMuT) enabled the reconstruction of the complete cell lineage of an outgrowing thoracic limb with single-cell resolution. In silico clonal analyses suggested that the limb primordium becomes subdivided from an early stage first into anterior-posterior and then into dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb bud formation is associated with the spatial modulation of cell proliferation, while limb elongation is also driven by the preferential orientation of division of epidermal cells along the proximal-distal axis of growth. Cellular reconstructions were predictive of the expression patterns of limb development genes including the Decapentaplegic (Dpp) morphogen.\n\nHIGHLIGHTSO_LIMulti-view light-sheet microscopy of crustacean embryos from species Parhyale hawaiensis are ideal for cellular-level analysis of organ morphogenesis.\nC_LIO_LILineages of 3-dimensional organs were reconstructed at single-cell resolution with the Fiji/ImageJ plugin Massive Multi-view Tracker.\nC_LIO_LIThe Parhyale limb primordium undergoes early lineage restrictions associated with particular cell behaviors and patterns of gene expression.\nC_LIO_LIDifferential rates of cell proliferation and oriented cell divisions guide appendage proximal-distal outgrowth.\nC_LI

developmental biology↗