Light-based in-vivo brain imaging relies on light transport over large distances of highly scattering tissues. Scattering gradually reduces imaging contrast and resolution, making it difficult to reach structures at greater depths even with the use of multiphoton techniques. To reach deeper, minimally invasive endo-microscopy techniques have been established. These most commonly exploit graded-index rod lenses and enable a variety of modalities in head-fixed and freely moving animals. A recently proposed alternative is the use of holographic control of light transport through multimode optical fibres promising much less traumatic application and superior imaging performance. We present a 110 μm thin laser-scanning endo-microscope based on this prospect, enabling in-vivo volumetric imaging throughout the whole depth of the mouse brain. The instrument is equipped with multi-wavelength detection and three-dimensional random access options, and it performs at lateral resolution below 1 μm. We showcase various modes of its application through the observations of fluorescently labelled neurones, their processes and blood vessels. Finally, we demonstrate how to exploit the instrument to monitor calcium signalling of neurones and to measure blood flow velocity in individual vessels at high speeds.

Institute of Applied Optics Friedrich Schiller University Jena Fröbelstieg 1 07743 Jena Germany

Institute of Biophysics of the Czech Academy of Sciences Královopolská 135 612 65 Brno Czech Republic

Institute of Scientific Instruments of the Czech Academy of Sciences Královopolská 147 612 64 Brno Czech Republic

Leibniz Institute of Photonic Technology Albert Einstein Straße 9 07745 Jena Germany

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Cornejo VH, Ofer N, Yuste R. Voltage compartmentalization in dendritic spines in vivo. Science. 2022;375:82–86. doi: 10.1126/science.abg0501. PubMed DOI PMC

Aime M, et al. Paradoxical somatodendritic decoupling supports cortical plasticity during rem sleep. Science. 2022;376:724–730. doi: 10.1126/science.abk2734. PubMed DOI

Yildirim M, Sugihara H, So PTC, Sur M. Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy. Nat. Commun. 2019;10:177. doi: 10.1038/s41467-018-08179-6. PubMed DOI PMC

Mittmann W, et al. Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo. Nat. Neurosci. 2011;14:1089–1093. doi: 10.1038/nn.2879. PubMed DOI

Kawakami R, et al. In vivo two-photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high-peak power gain-switched laser diode. Biomed. Opt. Express. 2015;6:891–901. doi: 10.1364/BOE.6.000891. PubMed DOI PMC

Ji N, Milkie DE, Betzig E. Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues. Nat. Methods. 2010;7:141–147. doi: 10.1038/nmeth.1411. PubMed DOI

Bocarsly ME, et al. Minimally invasive microendoscopy system for in vivo functional imaging of deep nuclei in the mouse brain. Biomed. Opt. Express. 2015;6:4546–4556. doi: 10.1364/BOE.6.004546. PubMed DOI PMC

Stamatakis AM, et al. Miniature microscopes for manipulating and recording in vivo brain activity. Microscopy. 2021;70:399–414. doi: 10.1093/jmicro/dfab028. PubMed DOI PMC

Campos P, Walker JJ, Mollard P. Diving into the brain: deep-brain imaging techniques in conscious animals. J. Endocrinol. 2020;246:R33–R50. doi: 10.1530/JOE-20-0028. PubMed DOI PMC

Aharoni D, Hoogland TM. Circuit investigations with open-source miniaturized microscopes: past, present and future. Front. Cellular Neurosci. 2019;13:141. doi: 10.3389/fncel.2019.00141. PubMed DOI PMC

Antonini A, et al. Extended field-of-view ultrathin microendoscopes for high-resolution two-photon imaging with minimal invasiveness. eLife. 2020;9:e58882. doi: 10.7554/eLife.58882. PubMed DOI PMC

Conkey DB, Caravaca-Aguirre AM, Piestun R. High-speed scattering medium characterization with application to focusing light through turbid media. Opt. Express. 2012;20:1733–1740. doi: 10.1364/OE.20.001733. PubMed DOI

Bianchi S, Di Leonardo R. A multi-mode fiber probe for holographic micromanipulation and microscopy. Lab Chip. 2012;12:635–639. doi: 10.1039/C1LC20719A. PubMed DOI

Čižmár T, Dholakia K. Exploiting multimode waveguides for pure fibre-based imaging. Nat. Commun. 2012;3:1027. doi: 10.1038/ncomms2024. PubMed DOI PMC

Papadopoulos IN, Farahi S, Moser C, Psaltis D. High-resolution, lensless endoscope based on digital scanning through a multimode optical fiber. Biomed. Opt. Express. 2013;4:260–270. doi: 10.1364/BOE.4.000260. PubMed DOI PMC

Plöschner M, Tyc T, Čižmár T. Seeing through chaos in multimode fibres. Nat. Photonics. 2015;9:529–535. doi: 10.1038/nphoton.2015.112. DOI

Morales-Delgado EE, Psaltis D, Moser C. Two-photon imaging through a multimode fiber. Opt. Express. 2015;23:32158–32170. doi: 10.1364/OE.23.032158. PubMed DOI

Gulino M, Kim D, Pané S, Santos SD, Pêgo AP. Tissue response to neural implants: The use of model systems toward new design solutions of implantable microelectrodes. Front. Neurosci. 2019;13:689. doi: 10.3389/fnins.2019.00689. PubMed DOI PMC

Kozai TDY, Jaquins-Gerstl AS, Vazquez AL, Michael AC, Cui XT. Brain tissue responses to neural implants impact signal sensitivity and intervention strategies. ACS Chem. Neurosci. 2015;6:48–67. doi: 10.1021/cn500256e. PubMed DOI PMC

Leite IT, et al. Three-dimensional holographic optical manipulation through a high-numerical-aperture soft-glass multimode fibre. Nat. Photonics. 2018;12:33–39. doi: 10.1038/s41566-017-0053-8. DOI

Popoff SM, et al. Measuring the transmission matrix in optics: An approach to the study and control of light propagation in disordered media. Phys. Rev. Lett. 2010;104:100601. doi: 10.1103/PhysRevLett.104.100601. PubMed DOI

Čižmár T, Dholakia K. Shaping the light transmission through a multimode optical fibre: Complex transformation analysis and applications in biophotonics. Opt. Express. 2011;19:18871–18884. doi: 10.1364/OE.19.018871. PubMed DOI

Leite IT, Turtaev S, Boonzajer Flaes DE, Čižmár T. Observing distant objects with a multimode fiber-based holographic endoscope. APL Photonics. 2021;6:036112. doi: 10.1063/5.0038367. DOI

Ohayon S, Caravaca-Aguirre A, Piestun R, Dicarlo JJ. Minimally invasive multimode optical fiber microendoscope for deep brain fluorescence imaging. Biomedical Optics Express. 2018;9:1492–1509. doi: 10.1364/BOE.9.001492. PubMed DOI PMC

Vasquez-Lopez SA, et al. Subcellular spatial resolution achieved for deep-brain imaging in vivo using a minimally invasive multimode fiber. Light-Sci. Appl. 2018;7:110. doi: 10.1038/s41377-018-0111-0. PubMed DOI PMC

Turcotte R, Schmidt CC, Emptage NJ, Booth MJ. Focusing light in biological tissue through a multimode optical fiber: refractive index matching. Opt. Lett. 2019;44:2386–2389. doi: 10.1364/OL.44.002386. PubMed DOI PMC

Turtaev S, et al. High-fidelity multimode fibre-based endoscopy for deep brain in vivo imaging. Light-Sci. Appl. 2018;7:92. doi: 10.1038/s41377-018-0094-x. PubMed DOI PMC

Yuste R, Denk W. Dendritic spines as basic functional units of neuronal integration. Nature. 1995;375:682–684. doi: 10.1038/375682a0. PubMed DOI

Turtaev S, et al. Comparison of nematic liquid-crystal and DMD based spatial light modulation in complex photonics. Opt. Express. 2017;25:29874–29884. doi: 10.1364/OE.25.029874. PubMed DOI

Gomes AD, Turtaev S, Du Y, Čižmár T. Near perfect focusing through multimode fibres. Opt. Express. 2022;30:10645–10663. doi: 10.1364/OE.452145. PubMed DOI

Silveira BM, et al. Side-view holographic endomicroscopy via a custom-terminated multimode fibre. Opt. Express. 2021;29:23083–23095. doi: 10.1364/OE.426235. PubMed DOI

Madisen L, et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 2010;13:133–140. doi: 10.1038/nn.2467. PubMed DOI PMC

Katayama H, Yamamoto A, Mizushima N, Yoshimori T, Miyawaki A. GFP-like proteins stably accumulate in lysosomes. Cell Struct. Function. 2008;33:1–12. doi: 10.1247/csf.07011. PubMed DOI

Kleinfeld D, Mitra PP, Helmchen F, Denk W. Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex. Proc. Natl Acad. Sci. 1998;95:15741–15746. doi: 10.1073/pnas.95.26.15741. PubMed DOI PMC

Mateo C, Knutsen PM, Tsai PS, Shih AY, Kleinfeld D. Entrainment of arteriole vasomotor fluctuations by neural activity is a basis of blood-oxygenation-level-dependent “resting-state” connectivity. Neuron. 2017;96:936–948.e3. doi: 10.1016/j.neuron.2017.10.012. PubMed DOI PMC

Fox MD, Raichle ME. Spontaneous fluctuations in brain activity observed with functional magnetic resonance imaging. Nat. Rev. Neurosci. 2007;8:700–711. doi: 10.1038/nrn2201. PubMed DOI

Greicius MD, Krasnow B, Reiss AL, Menon V. Functional connectivity in the resting brain: A network analysis of the default mode hypothesis. Proc. National Acad. Sci. 2003;100:253–258. doi: 10.1073/pnas.0135058100. PubMed DOI PMC

Knöpfel T, Song C. Optical voltage imaging in neurons: moving from technology development to practical tool. Nat. Rev. Neurosci. 2019;20:719–727. doi: 10.1038/s41583-019-0231-4. PubMed DOI

Tzang O, et al. Wavefront shaping in complex media with a 350 khz modulator via a 1d-to-2d transform. Nat. Photonics. 2019;13:788–793. doi: 10.1038/s41566-019-0503-6. DOI

Tao C, Zhang G, Xiong Y, Zhou Y. Functional dissection of synaptic circuits: in vivo patch-clamp recording in neuroscience. Front. Neural Circuits. 2015;9:23. doi: 10.3389/fncir.2015.00023. PubMed DOI PMC

Jin M, Glickfeld LL. Magnitude, time course, and specificity of rapid adaptation across mouse visual areas. J Neurophysiol. 2020;124:245–258. doi: 10.1152/jn.00758.2019. PubMed DOI PMC

Barretto RPJ, et al. Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy. Nat. Med. 2011;17:223–228. doi: 10.1038/nm.2292. PubMed DOI PMC

Aghajan ZM, et al. Impaired spatial selectivity and intact phase precession in two-dimensional virtual reality. Nat. Neurosci. 2015;18:121–128. doi: 10.1038/nn.3884. PubMed DOI

Ravassard P, et al. Multisensory control of hippocampal spatiotemporal selectivity. Science. 2013;340:1342–1346. doi: 10.1126/science.1232655. PubMed DOI PMC

Mikulovic S, et al. Ventral hippocampal olm cells control type 2 theta oscillations and response to predator odor. Nat. Commun. 2018;9:3638. doi: 10.1038/s41467-018-05907-w. PubMed DOI PMC

Farahi S, Ziegler D, Papadopoulos IN, Psaltis D, Moser C. Dynamic bending compensation while focusing through a multimode fiber. Opt. Express. 2013;21:22504–22514. doi: 10.1364/OE.21.022504. PubMed DOI

Gordon GSD, et al. Characterizing optical fiber transmission matrices using metasurface reflector stacks for lensless imaging without distal access. Phys. Rev. X. 2019;9:041050.

Li S, Horsley SAR, Tyc T, Čižmár T, Phillips DB. Memory effect assisted imaging through multimode optical fibres. Nat. Commun. 2021;12:3751. doi: 10.1038/s41467-021-23729-1. PubMed DOI PMC

Loterie D, Psaltis D, Moser C. Bend translation in multimode fiber imaging. Opt. Express. 2017;25:6263–6273. doi: 10.1364/OE.25.006263. PubMed DOI

Boonzajer Flaes DE, et al. Robustness of light-transport processes to bending deformations in graded-index multimode waveguides. Phys. Rev. Lett. 2018;120:233901. doi: 10.1103/PhysRevLett.120.233901. PubMed DOI

Čižmár T, Mazilu M, Dholakia K. In situ wavefront correction and its application to micromanipulation. Nat. Photonics. 2010;4:388–394. doi: 10.1038/nphoton.2010.85. DOI

Rudolf B, Du Y, Turtaev S, Leite IT, Čižmár T. Thermal stability of wavefront shaping using a dmd as a spatial light modulator. Opt. Express. 2021;29:41808–41818. doi: 10.1364/OE.442284. DOI

Goodman, J. W. Introduction to Fourier Optics (McGraw-Hill, 1968).

Bianco M, et al. Comparative study of autofluorescence in flat and tapered optical fibers towards application in depth-resolved fluorescence lifetime photometry in brain tissue. Biomed. Opt. Express. 2021;12:993–1009. doi: 10.1364/BOE.410244. PubMed DOI PMC

Paxinos, G. & Franklin, K. B. Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates (Academic Press, 2019).

Allen Reference Atlas—Mouse Brain—Coronal Sections, available from atlas.brain-map.org.

Advancing the path to in-vivo imaging in freely moving mice via multimode-multicore fiber based holographic endoscopy

"There's plenty of room at the bottom": deep brain imaging with holographic endo-microscopy