Research progress of intravital microscopy of the pulmonary immune environment_Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Authors：

 CHENG Yufei ¹ , ZHANG Longhao ²

1. Core Facilities, West China Hospital of Sichuan University, Chengdu, 610041, P. R. China;
2. Department of Medical Discipline Construction, West China Hospital of Sichuan University, Chengdu, 610041, P. R. China;

Corresponding author：

CHENG Yufei, Email: cyfandrewtemple@outlook.com

Keywords：

Intravital microscopy; confocal microscope; two-photon microscope; review

DOI：

10.7507/1007-4848.202306065

Video：

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Abstract Full text Figures/Tables Video References Cited by

The development of intravital microscopy (IVM) has enabled researchers to perform in-situ, real-time observations of pulmonary micro-circulation at the cellular level, and has become instrumental for researching the immune micro-environment of pulmonary diseases. This article introduces the developments in constructing the pulmonary imaging window and summarizes the current light microscopy techniques used for lung IVM with regard to its relevant functional and application features, which includes wide field fluorescence microscopy, confocal microscopy, as well as two-photon microscopy. It then provides examples of IVM of pulmonary immune response in inflammation and infection in murine models, and finally specifies the technological limitations to provide reference for researchers to systematically learn and understand the technology.

Citation： CHENG Yufei, ZHANG Longhao. Research progress of intravital microscopy of the pulmonary immune environment. Chinese Journal of Clinical Thoracic and Cardiovascular Surgery, 2023, 30(8): 1204-1209. doi: 10.7507/1007-4848.202306065 Copy

1.	Faust N, Varas F, Kelly LM, et al. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood, 2000, 96(2): 719-726.
2.	Orthgiess J, Gericke M, Immig, K, et al. Neurons exhibit Lyz2 promoter activity in vivo: Implications for using LysM-Cre mice in myeloid cell research. Eur J Immunol, 2016, 46(6): 1529-1532.
3.	Lee PY, Wang JX, Parisini E, et al. Ly6 family proteins in neutrophil biology. J Leukoc Biol, 2013, 94(4): 585-594.
4.	Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature, 2013, 496(7446): 445-455.
5.	Looney MR, Headley MB. Live imaging of the pulmonary immune environment. Cell Immunol, 2020, 350: 103862.
6.	Headley MB, Bins A, Nip A, et al. Visualization of immediate immune responses to pioneer metastatic cells in the lung. Nature, 2016, 531(7595): 513-517.
7.	Alizadeh-Tabrizi N, Hall S, Lehmann C. Intravital imaging of pulmonary immune response in inflammation and infection. Front Cell Dev Biol, 2021, 8: 620471.
8.	Fiole D, Tournier JN, Intravital microscopy of the lung: Minimizing invasiveness. J Biophotonics, 2016, 9(9): 868-878.
9.	Ericsson AC, Crim MJ, Franklin CL. A brief history of animal modeling. Mo Med, 2013, 110(3): 201-205.
10.	Zhao YH, Qu H, Wang YH, et al. Small rodent models of atherosclerosis. Biomed Pharmacother, 2020, 129: 110426.
11.	Vandamme TF. Use of rodents as models of human diseases. J Pharm Bioallied Sci, 2014, 6(1): 2-9.
12.	Coste A, Oktay MH, Condeelis JS, et al. Intravital imaging techniques for biomedical and clinical research. Cytometry A, 2020, 97(5): 448-457.
13.	Frevert U, Nacer A, Cabrera M, et al. Imaging plasmodium immunobiology in the liver, brain, and lung. Parasitol Int, 2014, 63(1): 171-186.
14.	李娟, 关苑君, 梁翠莎, 等. 小鼠双光子激光扫描显微镜活体成像窗口构建技术. 中国医学物理学杂志, 2022, 39(2): 229-235.
15.	Olkon DM, Joannides M, Capillaroscopic appearance of the pulmonary alveoli in the living dog. The Anatomical Record, 1930, 45: 121-127.
16.	Macgregor RG, Examination of the pulmonary circulation with the microscope. J Physiol, 1933, 80: 65-77.
17.	Hall HL. A study of the pulmonary circulation by the trans-illumination method. Am J Physiol, 1925, 72: 446-457.
18.	Hasegawa A, Hayashi K, Kishimoto H, et al. Color-coded real-time cellular imaging of lung T-lymphocyte accumulation and focus formation in a mouse asthma model. J Allergy Clin Immunol, 2010, 125(2): 461-468.
19.	Mitsuoka H, Sakurai T, Unno N, et al. Intravital laser confocal microscopy of pulmonary edema resulting from intestinal ischemia-reperfusion injury in the rat. Crit Care Med, 1999, 27(9): 1862-1868.
20.	Mitsuoka H, Unno N, Sakurai T, et al. Pathophysiological role of endothelins in pulmonary microcirculatory disorders due to intestinal ischemia and reperfusion. J Surg Res, 1999, 87(2): 143-151.
21.	Veith NT, Tschernig T, Gutbier B, et al. Surfactant protein A mediates pulmonary clearance of Staphylococcus aureus. Respir Res, 2014, 15(1): 85.
22.	Entenberg D, Voiculescu S, Guo P, et al. A permanent window for the murine lung enables high-resolution imaging of cancer metastasis. Nat Methods, 2018, 15(1): 73-80.
23.	Rodriguez-Tirado C, Kitamura T, Kato Y, et al. Long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window. J Vis Exp, 2016, 116: e54603.
24.	Tabuchi A, Mertens M, Kuppe H, et al. Intravital microscopy of the murine pulmonary microcirculation. J Appl Physiol, 2008, 104(2): 338-346.
25.	杨桢, 苏波, 钟康颖, 等. 小鼠肺部微循环活体显微成像. 华西医学, 2019, 34(1): 43-49.
26.	林曼娜. 荧光显微镜的成像原理及其在生物医学中的应用. 电子显微镜学报, 2021, 40(1): 90-93.
27.	Podstawka J, Sinha S, Hiroki CH, et al. Marginating transitional B cells modulate neutrophils in the lung during inflammation and pneumonia. J Exp Med, 2021, 218(9): e20210409.
28.	Kim SJ, Carestia A, McDonald B, et al. Platelet-mediated NET release amplifies coagulopathy and drives lung pathology during severe influenza infection. Front Immunol, 2021, 12: 772859.
29.	Barkaway A, Rolas L, Joulia R, et al. Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage. Immunity, 2021, 54(7): 1494-1510.
30.	Park I, Kim M, Choe K, et al. Neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury. Eur Respir J, 2019, 53(3): 1800786.
31.	Lefrancais E, Ortiz-Munoz G, Caudrillier A, et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature, 2017, 544(7648): 105-109.
32.	Kreisel D, Nava RG, Li W, et al. In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation. Proc Natl Acad Sci USA, 2010, 107(42): 18073-18078.
33.	Ueki H, Wang IH, Zhao DM, et al. Multicolor two-photon imaging of in vivo cellular pathophysiology upon influenza virus infection using the two-photon IMPRESS. Nat Protoc, 2020, 15(3): 1041-1065.
34.	Cleary SJ, Hobbs C, Amison RT, et al. LPS-induced lung platelet recruitment occurs independently from neutrophils, PSGL-1, and P-selectin. Am J Respir Cell Mol Biol, 2019, 61(2): 232-243.
35.	Ichise H, Tsukamato S, Hirashimo T, et al. Functional visualization of NK cell-mediated killing of metastatic single tumor cells. Elife, 2022, 11: e76269.
36.	Jeong S, Park SA, Park I, et al. PM2.5 exposure in the respiratory system induces distinct inflammatory signaling in the lung and the liver of mice. J Immunol Res, 2019: 3486841.
37.	Castanheira FVS, Nguyen R, Willson M, et al. Intravital imaging of three different microvascular beds in SARS-CoV-2 infected mice. Blood Adv, 2023.doi: 10.1182/bloodadvances.2022009430.
38.	Ling LQ, Zhang J, Li YS, et al. Platelets play a dual role in the pathophysiology of transfusion-related acute lung injury. Respir Physiol Neurobiol, 2023, 309: 104004.
39.	Kim HK, Missiakas D, Schneewind O. Mouse models for infectious diseases caused by Staphylococcus aureus. J Immunol Methods, 2014, 410: 88-99.
40.	Lefrancais E, Mallavia B, Zhuo H, et al. Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury. JCI Insight, 2018, 3(3): e98178.
41.	Neupane AS, Willson M, Chojnacki AK, et al. Patrolling alveolar macrophages conceal bacteria from the immune system to maintain homeostasis. Cell, 2020, 183(1): 110-125.
42.	Naumenko V, Van S, Dastidar H, et al. Visualizing oncolytic virus-host interactions in live mice using intravital microscopy. Mol Ther Oncolytics, 2018, 10: 14-27.
43.	Brown MB, Hunt WR, Noe JE, et al. Loss of cystic fibrosis transmembrane conductance regulator impairs lung endothelial cell barrier function and increases susceptibility to microvascular damage from cigarette smoke. Pulm Circ, 2014, 4(2): 260-268.
44.	张宇, 卢笑晖, 连新宝. 脓毒症急性肺损伤的发生机制及治疗研究进展. 解放军医学杂志, 2021, 46(11): 1159-1164.
45.	Yipp BG, Kim JH, Lima R, et al. The lung is a host defense niche for immediate neutrophil-mediated vascular protection. Sci Immunol, 2017, 2(10): eaam8929.
46.	Masterson CH, Curley GF, Laffey JG. Modulating the distribution and fate of exogenously delivered MSCs to enhance therapeutic potential: Knowns and unknowns. Intensive Care Med Exp, 2019, 7: 41.

1. Faust N, Varas F, Kelly LM, et al. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood, 2000, 96(2): 719-726.
2. Orthgiess J, Gericke M, Immig, K, et al. Neurons exhibit Lyz2 promoter activity in vivo: Implications for using LysM-Cre mice in myeloid cell research. Eur J Immunol, 2016, 46(6): 1529-1532.
3. Lee PY, Wang JX, Parisini E, et al. Ly6 family proteins in neutrophil biology. J Leukoc Biol, 2013, 94(4): 585-594.
4. Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature, 2013, 496(7446): 445-455.
5. Looney MR, Headley MB. Live imaging of the pulmonary immune environment. Cell Immunol, 2020, 350: 103862.
6. Headley MB, Bins A, Nip A, et al. Visualization of immediate immune responses to pioneer metastatic cells in the lung. Nature, 2016, 531(7595): 513-517.
7. Alizadeh-Tabrizi N, Hall S, Lehmann C. Intravital imaging of pulmonary immune response in inflammation and infection. Front Cell Dev Biol, 2021, 8: 620471.
8. Fiole D, Tournier JN, Intravital microscopy of the lung: Minimizing invasiveness. J Biophotonics, 2016, 9(9): 868-878.
9. Ericsson AC, Crim MJ, Franklin CL. A brief history of animal modeling. Mo Med, 2013, 110(3): 201-205.
10. Zhao YH, Qu H, Wang YH, et al. Small rodent models of atherosclerosis. Biomed Pharmacother, 2020, 129: 110426.
11. Vandamme TF. Use of rodents as models of human diseases. J Pharm Bioallied Sci, 2014, 6(1): 2-9.
12. Coste A, Oktay MH, Condeelis JS, et al. Intravital imaging techniques for biomedical and clinical research. Cytometry A, 2020, 97(5): 448-457.
13. Frevert U, Nacer A, Cabrera M, et al. Imaging plasmodium immunobiology in the liver, brain, and lung. Parasitol Int, 2014, 63(1): 171-186.
14. 李娟, 关苑君, 梁翠莎, 等. 小鼠双光子激光扫描显微镜活体成像窗口构建技术. 中国医学物理学杂志, 2022, 39(2): 229-235.
15. Olkon DM, Joannides M, Capillaroscopic appearance of the pulmonary alveoli in the living dog. The Anatomical Record, 1930, 45: 121-127.
16. Macgregor RG, Examination of the pulmonary circulation with the microscope. J Physiol, 1933, 80: 65-77.
17. Hall HL. A study of the pulmonary circulation by the trans-illumination method. Am J Physiol, 1925, 72: 446-457.
18. Hasegawa A, Hayashi K, Kishimoto H, et al. Color-coded real-time cellular imaging of lung T-lymphocyte accumulation and focus formation in a mouse asthma model. J Allergy Clin Immunol, 2010, 125(2): 461-468.
19. Mitsuoka H, Sakurai T, Unno N, et al. Intravital laser confocal microscopy of pulmonary edema resulting from intestinal ischemia-reperfusion injury in the rat. Crit Care Med, 1999, 27(9): 1862-1868.
20. Mitsuoka H, Unno N, Sakurai T, et al. Pathophysiological role of endothelins in pulmonary microcirculatory disorders due to intestinal ischemia and reperfusion. J Surg Res, 1999, 87(2): 143-151.
21. Veith NT, Tschernig T, Gutbier B, et al. Surfactant protein A mediates pulmonary clearance of Staphylococcus aureus. Respir Res, 2014, 15(1): 85.
22. Entenberg D, Voiculescu S, Guo P, et al. A permanent window for the murine lung enables high-resolution imaging of cancer metastasis. Nat Methods, 2018, 15(1): 73-80.
23. Rodriguez-Tirado C, Kitamura T, Kato Y, et al. Long-term high-resolution intravital microscopy in the lung with a vacuum stabilized imaging window. J Vis Exp, 2016, 116: e54603.
24. Tabuchi A, Mertens M, Kuppe H, et al. Intravital microscopy of the murine pulmonary microcirculation. J Appl Physiol, 2008, 104(2): 338-346.
25. 杨桢, 苏波, 钟康颖, 等. 小鼠肺部微循环活体显微成像. 华西医学, 2019, 34(1): 43-49.
26. 林曼娜. 荧光显微镜的成像原理及其在生物医学中的应用. 电子显微镜学报, 2021, 40(1): 90-93.
27. Podstawka J, Sinha S, Hiroki CH, et al. Marginating transitional B cells modulate neutrophils in the lung during inflammation and pneumonia. J Exp Med, 2021, 218(9): e20210409.
28. Kim SJ, Carestia A, McDonald B, et al. Platelet-mediated NET release amplifies coagulopathy and drives lung pathology during severe influenza infection. Front Immunol, 2021, 12: 772859.
29. Barkaway A, Rolas L, Joulia R, et al. Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage. Immunity, 2021, 54(7): 1494-1510.
30. Park I, Kim M, Choe K, et al. Neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury. Eur Respir J, 2019, 53(3): 1800786.
31. Lefrancais E, Ortiz-Munoz G, Caudrillier A, et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature, 2017, 544(7648): 105-109.
32. Kreisel D, Nava RG, Li W, et al. In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation. Proc Natl Acad Sci USA, 2010, 107(42): 18073-18078.
33. Ueki H, Wang IH, Zhao DM, et al. Multicolor two-photon imaging of in vivo cellular pathophysiology upon influenza virus infection using the two-photon IMPRESS. Nat Protoc, 2020, 15(3): 1041-1065.
34. Cleary SJ, Hobbs C, Amison RT, et al. LPS-induced lung platelet recruitment occurs independently from neutrophils, PSGL-1, and P-selectin. Am J Respir Cell Mol Biol, 2019, 61(2): 232-243.
35. Ichise H, Tsukamato S, Hirashimo T, et al. Functional visualization of NK cell-mediated killing of metastatic single tumor cells. Elife, 2022, 11: e76269.
36. Jeong S, Park SA, Park I, et al. PM2.5 exposure in the respiratory system induces distinct inflammatory signaling in the lung and the liver of mice. J Immunol Res, 2019: 3486841.
37. Castanheira FVS, Nguyen R, Willson M, et al. Intravital imaging of three different microvascular beds in SARS-CoV-2 infected mice. Blood Adv, 2023.doi: 10.1182/bloodadvances.2022009430.
38. Ling LQ, Zhang J, Li YS, et al. Platelets play a dual role in the pathophysiology of transfusion-related acute lung injury. Respir Physiol Neurobiol, 2023, 309: 104004.
39. Kim HK, Missiakas D, Schneewind O. Mouse models for infectious diseases caused by Staphylococcus aureus. J Immunol Methods, 2014, 410: 88-99.
40. Lefrancais E, Mallavia B, Zhuo H, et al. Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury. JCI Insight, 2018, 3(3): e98178.
41. Neupane AS, Willson M, Chojnacki AK, et al. Patrolling alveolar macrophages conceal bacteria from the immune system to maintain homeostasis. Cell, 2020, 183(1): 110-125.
42. Naumenko V, Van S, Dastidar H, et al. Visualizing oncolytic virus-host interactions in live mice using intravital microscopy. Mol Ther Oncolytics, 2018, 10: 14-27.
43. Brown MB, Hunt WR, Noe JE, et al. Loss of cystic fibrosis transmembrane conductance regulator impairs lung endothelial cell barrier function and increases susceptibility to microvascular damage from cigarette smoke. Pulm Circ, 2014, 4(2): 260-268.
44. 张宇, 卢笑晖, 连新宝. 脓毒症急性肺损伤的发生机制及治疗研究进展. 解放军医学杂志, 2021, 46(11): 1159-1164.
45. Yipp BG, Kim JH, Lima R, et al. The lung is a host defense niche for immediate neutrophil-mediated vascular protection. Sci Immunol, 2017, 2(10): eaam8929.
46. Masterson CH, Curley GF, Laffey JG. Modulating the distribution and fate of exogenously delivered MSCs to enhance therapeutic potential: Knowns and unknowns. Intensive Care Med Exp, 2019, 7: 41.

Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Research progress of intravital microscopy of the pulmonary immune environment

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