Abstract：Two-photon microscopy (TPM) is a new technology with the characteristics of strong penetrability and low photo-toxicity that breaks through the limitations of conventional light microscopy and laser confocal scanning microscopy (LSCM). Based on the two-photon absorption theory and femtosecond pulse technology, TPM provides the special advantages in studying long-term visualization of the microcirculation system in the brain of living animals. It has unique advantages to study the structure of cerebral cortex microcirculation network, blood perfusion and blood oxygen metabolism. In recent years, TPM has been developing iteratively, and its application has been expanding. TPM can be used to detect and photochemical control the blood flow signal in a single sub leptomeningeal microvascular, which opens a valuable microcirculation window for the basic and clinical research of the pathogenesis of ischemic cerebrovascular disease and Alzheimer's disease. Starting with the imaging principle, this review introduced the technological characteristics and progress of TMP, and summarized its application status and prospects from four aspects including quantifying hemodynamic changes, measuring oxygen partial pressure, detecting leukocyte-endothelial cell interaction, and establishing a stroke model, and discussed a few key questions and proposed solutions in the research field as well.
[1] Mostany R, Miquelajauregui A, Shtrahman M, et al. Two-photon excitation microscopy and its applications in neuroscience[M]// Methods Mol Biol. New York: Humana Press, 2015: 25-42. [2] Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluorescence microscopy [J]. Science, 1990, 248(4951): 73-76. [3] Zipfel WR, Williams RM, Webb WW. Nonlinear magic: Multiphoton microscopy in the biosciences [J]. Nat Biotechnol, 2003, 21(11): 1369-1377. [4] Oheim M, Michael DJ, Geisbauer M, et al. Principles of two-photon excitation fluorescence microscopy and other nonlinear imaging approaches [J]. Adv Drug Deliv Rev, 2006, 58(7): 788-808. [5] Dunn KW, Young PA. Principles of multiphoton microscopy [J]. Nephron Exp Nephrol, 2006, 103(2): 33-40. [6] Sofroniew NJ, Flickinger D, King J, et al. A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging [J]. Elife, 2016, 5: e14472 [7] Terada SI, Kobayashi K, Ohkura M, et al. Super-wide-field two-photon imaging with a micro-optical device moving in post-objective space [J]. Nat Commun, 2018, 9(1): 3550-3564. [8] Stirman JN, Smith IT, Kudenov MW, et al. Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain[J]. Nat Biotechnol, 2016, 34(8): 857-862. [9] Yang Weijian, Miller JE, Carrillo-Reid L, et al. Simultaneous multi-plane imaging of neural circuits [J]. Neuron, 2016, 89(2): 269-284. [10] Liang W, Hall G, Messerschmidt B, et al. Nonlinear optical endomicroscopy for label-free functional histology in vivo [J]. Light Sci Appl, 2017, 6(11): e17082 [11] Dana H, Sun Y, Mohar B, et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments [J]. Nat Methods, 2019, 16(7): 649-657. [12] Kannan M, Vasan G, Huang C, et al. Fast, in vivo voltage imaging using a red fluorescent indicator[J]. Nat Methods, 2018, 15(12): 1108-1116. [13] Lu Rongwen, Sun Wenzhi, Liang Yajie, et al. Video-rate volumetric functional imaging of the brain at synaptic resolution [J]. Nat Neurosci, 2017, 20(4): 620-628. [14] Kazemipour A, Novak O, Flickinger D, et al. Kilohertz frame-rate two-photon tomography[J]. Nat Methods, 2019, 16(8): 778-786. [15] Yildirim M, Sugihara H, So P, et al. Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy [J]. Nat Commun, 2019, 10(1): 177-180. [16] Ouzounov DG, Wang T, Wang M, et al. In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain [J]. Nat Methods, 2017, 14(4): 388-390. [17] Zong Weijian, Wu Runlong, Li Mingli, et al. Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice[J]. Nat Methods, 2017, 14(7): 713-719. [18] Kleinfeld D, Mitra PP, Helmchen F, et al. Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex [J]. Proc Natl Acad Sci USA, 1998, 95(26): 15741-15746. [19] Jonkman J, Stelzer E. Resolution and contrast in confocal and two-photon microscopy [M]//Confocal and Multiphoton Microscopy. New York: Wiley, 2002: 101-126. [20] Mostany R, Portera-Cailliau C. A craniotomy surgery procedure for chronic brain imaging [J]. J Vis Exp, 2008, 15(12): e680. [21] Marker DF, Tremblay ME, Lu SM, et al. A thin-skull window technique for chronic two-photon in vivo imaging of murine microglia in models of neuroinflammation [J]. J Vis Exp, 2010, 19(43): e2059. [22] Shih AY, Driscoll JD, Drew PJ, et al. Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain[J]. J Cereb Blood Flow Metab, 2012, 32(7): 1277-1309. [23] Driscoll JD, Shih AY, Drew PJ, et al. Two-photon imaging of blood flow in the rat cortex [J]. Cold Spring Harb Protoc, 2013, 2013(8): 759-767. [24] Zhang Shengxiang, Boyd J, Delaney K, et al. Rapid reversible changes in dendritic spine structure in vivo gated by the degree of ischemia [J]. J Neurosci, 2005, 25(22): 5333-5338. [25] Kobat D, Durst ME, Nishimura N, et al. Deep tissue multiphoton microscopy using longer wavelength excitation [J]. Opt Express, 2009, 17(16): 13354-13364. [26] Hudetz AG, Feher G, Weigle CG, et al. Video microscopy of cerebrocortical capillary flow: Response to hypotension and intracranial hypertension [J]. Am J Physiol, 1995, 268(6): 2202-2210. [27] Tiret P, Chaigneau E, Lecoq J, et al. Two-photon imaging of capillary blood flow in olfactory bulb glomeruli [J]. Methods Mol Biol, 2009, 489: 81-91. [28] Hirase H, Creso J, Buzsaki G. Capillary level imaging of local cerebral blood flow in bicuculline-induced epileptic foci [J]. Neuroscience, 2004, 128(1): 209-216. [29] Huang Jiyun, Li Litao, Wang Huan, et al. In vivo two-photon fluorescence microscopy reveals disturbed cerebral capillary blood flow and increased susceptibility to ischemic insults in diabetic mice [J]. CNS Neurosci Ther, 2014, 20(9): 816-822. [30] Schrandt CJ, Kazmi SM, Jones TA, et al. Chronic monitoring of vascular progression after ischemic stroke using multiexposure speckle imaging and two-photon fluorescence microscopy [J]. J Cereb Blood Flow Metab, 2015, 35(6): 933-942. [31] Hillman EM. Coupling mechanism and significance of the BOLD signal: A status report[J]. Annu Rev Neurosci, 2014, 37: 161-181. [32] Iadecola C. The neurovascular unit coming of age: A Journey through neurovascular coupling in health and disease [J]. Neuron, 2017, 96(1): 17-42. [33] Chow BW, Nunez V, Kaplan L, et al. Caveolae in CNS arterioles mediate neurovascular coupling [J]. Nature, 2020, 579(7797): 106-110. [34] Estrada AD, Ponticorvo A, Ford TN, et al. Microvascular oxygen quantification using two-photon microscopy[J]. Opt Lett, 2008, 33(10): 1038-1040. [35] Torres FI, Intaglietta M. Microvessel PO2 measurements by phosphorescence decay method [J]. Am J Physiol, 1993, 265(4): 1434-1438. [36] Golub AS, Pittman RN. PO2 measurements in the microcirculation using phosphorescence quenching microscopy at high magnification [J]. Am J Physiol Heart Circ Physiol, 2008, 294(6): 2905-2916. [37] Lecoq J, Parpaleix A, Roussakis E, et al. Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels [J]. Nat Med, 2011, 17(7): 893-898. [38] Sakadzic S, Roussakis E, Yaseen MA, et al. Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue[J]. Nat Methods, 2010, 7(9): 755-759. [39] Kazmi SM, Salvaggio AJ, Estrada AD, et al. Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion [J]. Biomed Opt Express, 2013, 4(7): 1061-1073. [40] Xu Kui, Boas DA, Sakadzic S, et al. Brain tissue PO2 measurement during normoxia and hypoxia using two-photon phosphorescence lifetime microscopy [J]. Adv Exp Med Biol, 2017, 977: 149-153. [41] Sullender CT, Mark AE, Clark TA, et al. Imaging of cortical oxygen tension and blood flow following targeted photothrombotic stroke [J]. Neurophotonics, 2018, 5(3): 1-10. [42] Lyons DG, Parpaleix A, Roche M, et al. Mapping oxygen concentration in the awake mouse brain [J]. Elife, 2016, 5: 1-16 [43] Jain RK, Munn LL, Fukumura D. Measuring leukocyte-endothelial interactions in mice [J]. Cold Spring Harb Protoc, 2013, 2013(6): 561-563. [44] Leick M, Azcutia V, Newton G, et al. Leukocyte recruitment in inflammation: Basic concepts and new mechanistic insights based on new models and microscopic imaging technologies [J]. Cell Tissue Res, 2014, 355(3): 647-656. [45] Zenaro E, Pietronigro E, Della BV, et al. Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin [J]. Nat Med, 2015, 21(8): 880-886. [46] Pietronigro E, Zenaro E, Constantin G. Imaging of leukocyte trafficking in Alzheimer′s disease [J]. Front Immunol, 2016, 7: 33-43. [47] Cruz HJ, Bracko O, Kersbergen CJ, et al. Neutrophil adhesion in brain capillaries reduces cortical blood flow and impairs memory function in Alzheimer′s disease mouse models[J]. Nat Neurosci, 2019, 22(3): 413-420. [48] Bracko O, Njiru BN, Swallow M, et al. Increasing cerebral blood flow improves cognition into late stages in Alzheimer's disease mice [J]. J Cereb Blood Flow Metab, 2020, 40(7): 1441-1452. [49] Wang Huan, Hong Lingjuan, Huang Jiyun, et al. P2RX7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy [J]. Cell Res, 2015, 25(6): 674-690. [50] El AM, Gluck C, Binder N, et al. Neutrophils obstructing brain capillaries are a major cause of no-reflow in ischemic stroke[J]. Cell Rep, 2020, 33(2): e108260. [51] Liu Shen, Wei Cong, Kang Ning, et al. Chinese medicine Tongxinluo capsule alleviates cerebral microcirculatory disturbances in ischemic stroke by modulating vascular endothelial function and inhibiting leukocyte-endothelial cell interactions in mice: A two-photon laser scanning microscopy study [J]. Microcirculation, 2018, 25(2): e12437. [52] Langer HF, Chavakis T. Leukocyte-endothelial interactions in inflammation [J]. J Cell Mol Med, 2009, 13(7): 1211-1220. [53] Wahl SM, Feldman GM, Mccarthy JB. Regulation of leukocyte adhesion and signaling in inflammation and disease[J]. J Leukoc Biol, 1996, 59(6): 789-796. [54] Herr N, Mauler M, Witsch T, et al. Acute fluoxetine treatment induces slow rolling of leukocytes on endothelium in mice[J]. PLoS ONE, 2014, 9(2): e88316. [55] Talley WL, Zheng W, Garling RJ, et al. Rose bengal photothrombosis by confocal optical imaging in vivo: A model of single vessel stroke[J]. J Vis Exp, 2015(100): e52794. [56] Shih AY, Nishimura N, Nguyen J, et al. Optically induced occlusion of single blood vessels in rodent neocortex [J]. Cold Spring Harb Protoc, 2013, 2013(12): 1153-1160. [57] Nishimura N, Schaffer CB, Friedman B, et al. Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: Three models of stroke [J]. Nat Methods, 2006, 3(2): 99-108. [58] Zhang S, Boyd J, Delaney K, et al. Rapid reversible changes in dendritic spine structure in vivo gated by the degree of ischemia [J]. J Neurosci, 2005, 25(22): 5333-5338. [59] Schaffer CB, Friedman B, Nishimura N, et al. Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion [J]. PLoS Biol, 2006, 4(2): 258-270. [60] Nimmagadda A, Park HP, Prado R, et al. Albumin therapy improves local vascular dynamics in a rat model of primary microvascular thrombosis: A two-photon laser-scanning microscopy study [J]. Stroke, 2008, 39(1): 198-204. [61] Zhu Haihao, Fan Xiang, Yu Zhanyang, et al. Annexin A2 combined with low-dose tPA improves thrombolytic therapy in a rat model of focal embolic stroke [J]. J Cereb Blood Flow Metab, 2010, 30(6): 1137-1146. [62] Davalos D, Grutzendler J, Yang G, et al. ATP mediates rapid microglial response to local brain injury in vivo [J]. Nat Neurosci, 2005, 8(6): 752-758. [63] Eichhoff G, Brawek B, Garaschuk O. Microglial calcium signal acts as a rapid sensor of single neuron damage in vivo [J]. Biochim Biophys Acta, 2011, 1813(5): 1014-1024. [64] Santisakultarm TP, Kersbergen CJ, Bandy DK, et al. Two-photon imaging of cerebral hemodynamics and neural activity in awake and anesthetized marmosets [J]. J Neurosci Methods, 2016, 271: 55-64.
