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However, the brain\u2019s structural and dynamic heterogeneity\u2014spanning microns to millimeters and milliseconds to hours\u2014requires imaging techniques with tunable spatiotemporal resolution, flexible 3D sampling, and compatibility with targeted perturbations. Here, we present tunable Bessel beam two-photon fluorescence microscopy (tBessel-TPFM), a compact, low-cost, and versatile platform for intravital brain imaging across millimeter scale with subcellular resolution. tBessel-TPFM transforms slow 3D volume scans into fast 2D frame scans via an axially elongated Bessel focus, achieving acquisition rates ~100 fold faster and reduced motion artifacts compared with conventional TPFM. Exploiting its full tunability of the Bessel focus, we applied tBessel-TPFM for quantitative mapping of cerebral blood flow and neurovascular coupling in normal and ischemic stroke mice. Unlike existing Bessel focus generation methods, the axial center of tBessel-TPFM remains fixed at the objective focal plane during profile tuning. Leveraging this advantage, we integrated tBessel-TPFM with simultaneous 3D targeted optogenetic stimulation for volumetric neuronal connectivity mapping. We also tracked microglial process dynamics following single-cell laser ablation, revealing diverse neuroimmune responses across spatial and temporal scales. By combining high speed, deep penetration, tunable sampling, and multimodal perturbation, tBessel-TPFM empowers a broad spectrum of neurobiological investigations\u2014from vascular physiology and functional connectivity to neuroimmune interactions.<\/jats:p>","DOI":"10.7554\/elife.110228","type":"journal-article","created":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T14:30:29Z","timestamp":1770906629000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.110228","source":"Crossref","is-referenced-by-count":0,"title":["Tunable Bessel beam two-photon fluorescence microscopy for high-speed volumetric imaging of brain dynamics"],"prefix":"10.7554","volume":"15","author":[{"given":"Mengyang Jacky","family":"Li","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00hx57361","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Electrical and Computer Engineering, Princeton University","place":["Princeton, United 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