Table 1 :

Summary of Approaches to Intravital Lung Imaging

	Approach	Methodology	Pros/Cons	Well-suited for Immune Study	Animal Models Studied	Key References
1	Bronchus Clamping	Bronchus of one lobe is clamped preventing airflow induced motion	Pros: a) Minimal motion artifacting    b) Relatively simple surgical preparation Cons: a) Imaged lobe is subjected to severe hypoxia    b) Only short imaging duration achievable	No	Rabbit, Cat, Dog, Mouse	1) H. L. Hall, Am. J. Physiol. 72, 446–457 (1925). 2) R. G. Macgregor, J. Physiol. 80, 65–77 (1933) 3) A. Hasegawa, K. Hayashi, et al., J. Allergy Clin. Immunol. 125, 461–468.e6 (2010).
2	Prolonged Apnea	Image capture restricted to the expiratory plateau which is prolonged to ~15secs through mechanical ventilation	Pros: a) Minimized hypoxia compared to clamping    b) Relatively minimal surgical trauma Cons: a) Frequency of timelapse and overall Imaging duration very short    b) Image stability is generally poor	No	Rat, Mouse	1) A. Tabuchi, M. Mertens, et al., J. Appl. Physiol. 104, 338–346 (2008). 2) H. Mitsuoka, N. Unno, et al., J. Surg. Res. 87, 143–151 (1999). 3) H. Mitsuoka, T. Sakurai, et al., Crit. Care Med. 27, 1862–1868 (1999). 4) P. Schneider, T. Foitzik, et al., Microvasc. Res. 62, 421–434 (2001). 5) E. P. Schmidt, Y. Yang, et al., Nat. Med. 18, 1217–1223 (2012). 6) L. Preu, M. Bischoff, et al., Inflammation (2015). 7) N. T. Veith, T. Tschernig, et al., Respir. Res. 15, 85 (2014). 8) T. Tschernig, N. T. Veith, et al., Exp. Toxicol. Pathol. 65, 883–886 (2013).
3	Glue-based Stabilization	Lung parenchyma is exposed and glue is utilized to affix a small portion ofthe lung to a coverslip for imaging.	Pros: a) Long duration of highly stable continuous imaging is possible    b) Has been applied to the development of a permanently-placed window enabling longitudinal imaging studies Cons: a) In some studies substantial inflammation has been reported shortly after window implantation which may complicate interpretation	Yes	Mouse	1) D. Kreisel, R. G. Nava, et al., Proc. Natl. Acad. Sci. U.S.A. 107, 18073–18078 (2010). 2) J. H. Spahn, W. Li, et al., J. Immunol. 194, 4039–4048 (2015). 3) D. Entenberg, S. Voiculescu, et al., Nat. Methods. 113. 3073 (2017)
4	Suction-based Stabilization	Lung parenchyma is exposed and vacuum-pressure is utilized to stabilize a small portion ofthe lung beneath a coverslip for imaging.	Pros: a) Long duration of highly stable continuous imaging is possible    b) Minimal early inflammation Cons: a) Imaging sessions limited to ~12 hours    b) Imaging >6 hours may be confounded by method-induced inflammation	Yes	Dog, Rat, Mouse	1) W.W. Wagner, J. Appl. Physiol. 26, 375–377 (1969) 2) D. G. McCormack, S. Mehta, etal, Microvasc. Res. 60, 131–140(2000). 3) N. Funakoshi, M. Onizuka, et al. , Microvasc. Res. 59, 361–367 (2000). 4) H. M. Razavi, L. F.Wang, etal., Am. J. Respir. Crit. Care Med. 170, 227–233 (2004). 5) R. G. Presson, M. B. Brown, etal., Am.J.Pathol.179,75–82 (2011). 6) M. R. Looney, E. E. Thornton, et al., , Nat. Methods 8, 91–96 (2011). 7) E. E. Thornton, M. R. Looney,et al.,,J. Exp. Med.209, 1183–1199 (2012). 8) S. Devi, Y. Wang, etal., , J. Exp. Med. 210, 2321–2336 (2013). 9) G. Ortiz-Muñoz, B. Mallavia, et al., Blood 124, 2625–2634 (2014). 10) M. F. Bennewitz, et al., IntraVital 3, e29748 (2014). 11) A. Caudrillier, B. Mallavia, et al., PLoS One 10, e0133022 (2015). 12) R. N. Hanna, C. Cekic, et al., Science 350, 1–9 (2015). 13) D Entenberg. C. Rodriguez-Tirado, et. al., Intravital 4:31–11 (2015) 14) M.B. Headley, A. Bins, etal.,Nature.531. 513–517.(2016) 15) S.Z. Chong, M. Evrard, et al., J.Exp.Med.213. 2293–2314. (2016) 16) E. Lefrancais, G. Ortiz-Munoz, etal., Nature. 544.105–109. (2017) 16) B.G.Yipp, J.H. Kim, etal., SciImmunol.2 (2017) 17) E. Lefrancais, B. Mallavia, etal.JCI Insight. 3(3) (2018)