Tunable Bessel beam two-photon fluorescence microscopy for high-speed volumetric imaging of brain dynamics
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eLife Assessment
This important study substantially advances the imaging toolbox available to neuroscientists by presenting a tunable Bessel (tBessel-TPFM) platform that enables high-speed volumetric two-photon imaging. The evidence supporting the novel methodology is convincing, with rigorous benchmarking and demonstrations of a wide range of neuroimaging applications covering vascular dynamics, neurovascular coupling, optogenetic perturbation, and microglial responses. The work will be of broad interest to neuroscientists and imaging system tool developers.
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Abstract
High-speed volumetric imaging of the brain is essential for linking diverse cellular events to tissue-level functions. However, the brain’s structural and dynamic heterogeneity—spanning microns to millimeters and milliseconds to hours—requires 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—from vascular physiology and functional connectivity to neuroimmune interactions.
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eLife Assessment
This important study substantially advances the imaging toolbox available to neuroscientists by presenting a tunable Bessel (tBessel-TPFM) platform that enables high-speed volumetric two-photon imaging. The evidence supporting the novel methodology is convincing, with rigorous benchmarking and demonstrations of a wide range of neuroimaging applications covering vascular dynamics, neurovascular coupling, optogenetic perturbation, and microglial responses. The work will be of broad interest to neuroscientists and imaging system tool developers.
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Reviewer #1 (Public review):
Summary:
This manuscript presents a tunable Bessel-beam two-photon fluorescence microscopy (tBessel-TPFM) platform that enables high-speed volumetric imaging with stable axial focus. The work is technically strong and broadly significant, as it substantially improves the flexibility and practicality of Bessel-beam-based two-photon microscopy. The demonstrations are generally strong and bridge a wide range of neuroimaging applications, namely vascular dynamics, neurovascular coupling, optogenetic perturbation, and microglial responses. These convincingly show that the approach enables biological measurements that are difficult or impractical with existing methods.
The evidence supporting the technical and biological claims is generally strong. The optical design is carefully motivated, clearly described, and …
Reviewer #1 (Public review):
Summary:
This manuscript presents a tunable Bessel-beam two-photon fluorescence microscopy (tBessel-TPFM) platform that enables high-speed volumetric imaging with stable axial focus. The work is technically strong and broadly significant, as it substantially improves the flexibility and practicality of Bessel-beam-based two-photon microscopy. The demonstrations are generally strong and bridge a wide range of neuroimaging applications, namely vascular dynamics, neurovascular coupling, optogenetic perturbation, and microglial responses. These convincingly show that the approach enables biological measurements that are difficult or impractical with existing methods.
The evidence supporting the technical and biological claims is generally strong. The optical design is carefully motivated, clearly described, and validated through a combination of simulations and experimental characterization. The biological applications are diverse and well chosen to highlight the strengths of the proposed method, and the data are of high quality, with appropriate controls and comparative measurements where relevant.
Strengths:
(1) The optical innovation addresses a well-recognized limitation of existing Bessel-TPFM implementations, namely axial focus drift during tuning, and does so using a relatively simple, light-efficient, and cost-effective design.
(2) The manuscript provides convincing experimental evidence for this being a versatile platform to map flow dynamics across diverse vessel sizes and orientations in both healthy and pathological states.
(3) Biological demonstrations are comprehensive and span multiple domains such as hemodynamics, neurovascular coupling, and neuroimmune responses.
(4) Quantitative analyses of blood flow across vessel sizes and orientations, including kilohertz line scanning, are particularly compelling and clearly beyond the reach of standard Gaussian TPFM.
(5) Particular advantages are that higher blood slow speeds become measurable up to 23mm/sec (20x more than conventional frame scanning), and that simultaneous (Bessel-)imaging and (Gaussian-)perturbation are possible because of the stable axial focus.
Weaknesses:
(1) At present, the paper does not properly position the new Bessel-beam method against previous work, and fails to compare it to alternative fast volumetric imaging methods without Bessel beams.
(2) The cost-effectiveness of the proposed method is not well described or supported by evidence; it would be useful to include more detail or remove this claim.
(3) Some biological conclusions, e.g., regarding novel features of microglial dynamics (i.e., the observed two-wave responses and coordinated extension-retraction), are based on relatively limited sample size and would benefit from clearer discussion of variability across animals and fields of view.
(4) The use of neural network-based denoising for microglial imaging is reasonable but introduces potential concerns about trustworthiness; additional clarification of validation or failure modes would strengthen confidence in these results.
To conclude, most of the authors' claims are well supported by the data. The central conclusion, namely that tBessel-TPFM provides tunable volumetric imaging enabling experiments not feasible with existing two-photon approaches, is justified. Some biological interpretations would benefit from a more cautious framing, but they do not undermine the main technical and methodological contributions of the study. This is a strong and technically rigorous manuscript that makes a substantial methodological advance with clear relevance to neuroscience and intravital imaging. Minor clarifications and a slightly more measured discussion of certain biological findings are recommended.
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Reviewer #2 (Public review):
Summary:
The authors describe a tunable Bessel beam two-photon microscope (tBessel-TPFM) designed to overcome a common limitation of Bessel-based volumetric imaging: axial shifts of the effective focus during Bessel beam parameter tuning. Their optical design allows independent control of axial beam length and resolution while keeping the axial center fixed. This is extensively validated through simulations and experiments.
Strengths:
A major strength of the work is the breadth of validation combined with the level of technical detail provided. The authors carefully characterize the optical performance of the system and clearly explain the design choices and underlying derivations, which will make it easier for others to understand and implement. The authors demonstrate the utility of the method across …
Reviewer #2 (Public review):
Summary:
The authors describe a tunable Bessel beam two-photon microscope (tBessel-TPFM) designed to overcome a common limitation of Bessel-based volumetric imaging: axial shifts of the effective focus during Bessel beam parameter tuning. Their optical design allows independent control of axial beam length and resolution while keeping the axial center fixed. This is extensively validated through simulations and experiments.
Strengths:
A major strength of the work is the breadth of validation combined with the level of technical detail provided. The authors carefully characterize the optical performance of the system and clearly explain the design choices and underlying derivations, which will make it easier for others to understand and implement. The authors demonstrate the utility of the method across several in vivo applications, including neurovascular imaging, blood flow measurements, optogenetic stimulation, and microglial dynamics.
Weaknesses:
In the in vivo demonstrations, the authors employ different Bessel beam configurations across experiments, but the beam parameters are not dynamically tuned during live imaging. A video example showing continuous or interactive tuning of the Bessel beam within a single in vivo imaging sequence would further highlight the practical advantages of this platform and strengthen the case for its potential applications. In addition, while excitation powers are reported, the manuscript does not place these values in the broader context of known photodamage thresholds for two-photon microscopy, which would be helpful to the readers. Denoising/image restoration are applied in one of the in vivo examples, but it is unclear why this step was used specifically for this dataset and whether it was necessary to achieve adequate SNR or primarily included as an additional demonstration.
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Reviewer #3 (Public review):
Summary:
The manuscript presents an elegant and cost-effective approach for generating a tunable Bessel beam on a conventional two-photon microscope. The authors assemble a compact optical module comprising three axicons and a series of lenses that permits rapid adjustment of both lateral resolution and axial extent without modifying the focal plane. This flexibility enables the system to be readily adapted to a variety of biological preparations. As a proof of concept, the authors employ the device to record blood flow velocities in cortical microcapillaries, arterioles, and venules, thereby directly visualizing vasodilatation and vasoconstriction dynamics and permitting quantitative analysis of neurovascular coupling across cortical layers in awake mice.
The authors demonstrate that the tunability of the …
Reviewer #3 (Public review):
Summary:
The manuscript presents an elegant and cost-effective approach for generating a tunable Bessel beam on a conventional two-photon microscope. The authors assemble a compact optical module comprising three axicons and a series of lenses that permits rapid adjustment of both lateral resolution and axial extent without modifying the focal plane. This flexibility enables the system to be readily adapted to a variety of biological preparations. As a proof of concept, the authors employ the device to record blood flow velocities in cortical microcapillaries, arterioles, and venules, thereby directly visualizing vasodilatation and vasoconstriction dynamics and permitting quantitative analysis of neurovascular coupling across cortical layers in awake mice.
The authors demonstrate that the tunability of the Bessel beam can be exploited to match the numerical aperture to the vessel type: a high NA configuration, albeit slower scan, is optimal for resolving flow in capillaries, whereas a low NA setting provides faster acquisition suitable for arterioles and venules. By implementing a one-dimensional line scan with the Bessel beam, they achieve an imaging speed that is twentyfold faster than conventional frame-by-frame scanning, which proves sufficient to capture hemodynamic transients before and after an induced ischemic stroke.
In addition to pure observation, the authors integrate a co-propagating Gaussian line to the system, allowing simultaneous imaging and photostimulation within the same focal plane. This capability addresses a common limitation of other Bessel beam implementations, in which the observation and perturbation planes often become misaligned when the Bessel beam is altered. The manuscript also emphasizes the advantage of Bessel beam excitation for calcium imaging after a perturbation, because it captures neuronal activity in planes both above and below the nominal focal plane, signals that would be missed with a standard Gaussian focus. Finally, the authors apply the technique to investigate the neuroimmune response following targeted microglial ablation; they report that adjacent microglia extend processes toward the injury site while retracting processes in the opposite direction.
Overall, the work offers a technically straightforward yet powerful extension to existing two-photon platforms, providing high-speed, volumetric imaging and stimulation capabilities that are well-suited to a broad range of neurovascular and neuroimmune studies. The experimental validation is quite thorough, and the presented data convincingly illustrates the benefits of the approach.
Strengths:
The authors present a truly clever and inexpensive optical module that can be integrated into almost any two-photon microscope, providing a tunable Bessel beam with a minimal modification of the existing system. The experimental data and accompanying quantitative analysis convincingly demonstrate that the system can reveal physiological events, such as capillary flow, calcium transients across multiple axial planes, and microglial process dynamics, that are difficult or impossible to capture with a conventional Gaussian beam. The breadth of experiments chosen for the manuscript illustrates the practical utility of the device and supports the authors' conclusions that it extends the functional repertoire of standard two-photon microscopy.
Weaknesses:
The manuscript would benefit from a more detailed contextualisation of the claimed speed advantage. Although the authors mention other techniques in the introduction, they do not provide any direct comparison with other state-of-the-art high-speed two-photon approaches such as light beads microscopy (Demas et al., Nat. Methods 2021), temporal multiplexing schemes (Weisenburger et al., Cell 2019), or random access microscopy (Villette et al., Cell 2019). A brief comparison of imaging speed, spatial resolution, and instrumental complexity would enable readers to assess the relative merits of the present method.
A second limitation that warrants discussion is the inherent trade off between volumetric coverage and image specificity. Because the Bessel beam excites fluorescence throughout an extended axial range, the detector inevitably integrates signal from a three dimensional volume into a two dimensional image. In densely labelled tissue, this can lead to significant signal crosstalk, reducing contrast and complicating quantitative interpretation. A brief analysis of how labeling density affects the fidelity of flow or calcium measurements, or suggestions for mitigating crosstalk (e.g., computational deconvolution, adaptive excitation shaping, or combinatorial sparse labeling), would broaden the applicability of the technique.
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