10Identifying the cellular origins and mapping the dendritic and axonal arbors of neurons have 11 been century old quests to understand the heterogeneity among these brain cells. Classical 12 chemical and genetic methods take advantage of light microscopy and sparse labeling to 13 unambiguously, albeit inefficiently, trace a few neuronal lineages or reconstruct their 14 morphologies in each sampled brain. To improve the analysis throughput, we designed Bitbow, 15 a digital format of Brainbow which exponentially expands the color palette to provide tens of 16 thousands of spectrally resolved unique labels. We generated transgenic Bitbow Drosophila 17 lines, established statistical tools, and streamlined sample preparation, image processing and 18 data analysis pipelines to allow conveniently mapping neural lineages, studying neuronal 19 morphology and revealing neural network patterns with an unprecedented speed, scale and 20 resolution. 21 One way to generate more unique labels for lineage tracing is to localize the same FPs to 48 different subcellular compartments. In strategies such as CLoNe and MAGIC, Brainbow 49 cassettes targeted to cytoplasm, cell membrane, nucleus, and/or mitochondria were co-50 electroporated with transposase for genome integration, which allowed the differentiation of 51 neighboring progenies in chick and mouse embryos with fewer color collisions 26,27 . However, 52 the number of expression cassettes being integrated in each cell is random in these 53 experiments, leading to uncertainty in each color's appearance probability which complicates 54 quantitative analysis. The Raeppli strategy solves this problem by generating a transgenic 55 Drosophila which utilizes 4 FPs to create up to 4 x 4 = 16 membrane and nucleus color 56 combinations 16 . In parallel, strategies such as TIE-DYE and MultiColor FlpOut (MCFO) attempt to 57 generate more color combinations by stochastically removing the expression stops from each 58 3 FP module 15,28 . While inserting 3 different modules into 3 genomic loci allows generating up to 59 2 3 -1=7 unique labels, it is difficult to insert more modules to more genomic loci in a single 60 transgenic animal. 61 Here we present Bitbow, a digital format of Brainbow to greatly expand the unique color 62 pool from a single transgenic cassette. Unlike the original Brainbow, whose FP choices are 63 exclusive in one cassette, Bitbow allows each FP to independently express in an ON or OFF state 64 upon recombination. Color coding by each FP's binary status is similar to the information coding 65 by each bit in computer memory, thus leading to the name Bitbow. In a recent study, we 66 implemented the Bitbow1 design to target 5 spectrally distinct FPs to the nucleus for lineage 67 tracing 33 . Here, we present novel Bitbow1 flies which encode up to 32,767 unique "colors" 68 (Bitbow codes) in a single transgenic animal. This allows reliable lineage tracing without 69 complicated statistical tests 33 . To better enable morphology tracing, we generated Bitbow2, 70 which...