Visualization of Intravital Immune Cell Dynamics After Conjunctival Surgery Using Multiphoton Microscopy

Kojima, Sachi; Inoue, Toshihiro; Kikuta, Junichi; Furuya, Masayuki; Koga, Ayaka; Fujimoto, Tomokazu; Ueta, Mayumi; Kinoshita, Shigeru; Ishii, Masaru; Tanihara, Hidenobu

doi:10.1167/iovs.15-18507

Cited by 6 publications

(3 citation statements)

References 24 publications

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“…The setup of Steven et al (54), investigating leukocyte transmigration into lymphatic vessels in the peripheral cornea and limbus, was not temperature controlled, which as we demonstrate, is necessary to visualize central CL kinetics in a noninvasive manner, and furthermore, did not use transgenic mice. More recently, 2 studies have used MPM to investigate endogenously, fluorescently labeled neutrophil kinetics on the ocular surface of mice (69,70). Whereas the setup used by Ueta et al (69) for analyzing the cornea likely maintains the correct ocular surface temperature required for analysis of CLs, it is highly invasive, as the entire eye is dislocated to immobilize the cornea and compensate/ correct for the various image stability issues (e.g., drift and ocular pulsations) and positions the mouse on its back for the entire length of imaging inside a dark, 37°C box.…”

Section: Discussionmentioning

confidence: 99%

Kinetics of corneal leukocytes by intravital multiphoton microscopy

et al. 2018

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Corneal immune privilege is integral in maintaining the clear avascular window to the foreign world. The presence of distinct populations of corneal leukocytes (CLs) in the normal cornea has been firmly established. However, their precise function and kinetics remain, as of yet, unclear. Through intravital multiphoton microscopy (IV-MPM), allowing the means to accumulate critical spatial and temporal cellular information, we provide details for long-term investigation of CL morphology and kinetics under steady state and following inflammation. Significant alterations in size and morphology of corneal CD11c dendritic cells (DCs) were noted following acute sterile inflammation, including cell volume (4364.4 ± 489.6 vs. 1787.6 ± 111.0 μm, P < 0.001) and sphericity (0.82 ± 0.01 vs. 0.42 ± 0.02, P < 0.001) compared with steady state. Furthermore, IV-MPM analyses revealed alterations in both the CD11c DC and major histocompatibility complex class II (MHC)-II mature antigen-presenting cell population kinetics during inflammation, including track displacement length (CD11c: 16.57 ± 1.41 vs. 4.64 ± 0.56 μm, P < 0.001; MHC-II: 9.03 ± 0.37 vs. 4.09 ± 0.39, P < 0.001) and velocity (CD11c: 1.91 ± 0.07 μm/min vs. 1.73 ± 0.1302 μm/min; MHC-II: 2.97 ± 0.07 vs. 1.62 ± 0.08, P < 0.001) compared with steady state. Our results reveal in vivo evidence of sessile CL populations exhibiting dendritic morphology under steady state and increased velocity of spherical leukocytes following inflammation. IV-MPM represents a powerful tool to study leukocytes in corneal diseases in context.-Seyed-Razavi, Y., Lopez, M. J., Mantopoulos, D., Zheng, L., Massberg, S., Sendra, V. G., Harris, D. L., Hamrah, P. Kinetics of corneal leukocytes by intravital multiphoton microscopy.

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Section: Discussionmentioning

confidence: 99%

Kinetics of corneal leukocytes by intravital multiphoton microscopy

et al. 2018

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“…In addition, cell velocities increased significantly until 3 h post-surgery (5.9 ± 3.2 µm/min; p < 0.0001) and the elevated level was sustained until 72 h after injury (5.9 ± 3.3 µm/min). The authors concluded that this type of imaging may be a useful modality for visualizing the molecular processes of wound healing after a variation of ocular trauma or injuries, such as glaucoma surgery [48].…”

Section: Ocular Surface Diseasementioning

confidence: 99%

Two-Photon and Multiphoton Microscopy in Anterior Segment Diseases of the Eye

Hong,

Chong,

Goh

et al. 2024

IJMS

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Two-photon excitation microscopy (TPM) and multiphoton fluorescence microscopy (MPM) are advanced forms of intravital high-resolution functional microscopy techniques that allow for the imaging of dynamic molecular processes and resolve features of the biological tissues of interest. Due to the cornea’s optical properties and the uniquely accessible position of the globe, it is possible to image cells and tissues longitudinally to investigate ocular surface physiology and disease. MPM can also be used for the in vitro investigation of biological processes and drug kinetics in ocular tissues. In corneal immunology, performed via the use of TPM, cells thought to be intraepithelial dendritic cells are found to resemble tissue-resident memory T cells, and reporter mice with labeled plasmacytoid dendritic cells are imaged to understand the protective antiviral defenses of the eye. In mice with limbal progenitor cells labeled by reporters, the kinetics and localization of corneal epithelial replenishment are evaluated to advance stem cell biology. In studies of the conjunctiva and sclera, the use of such imaging together with second harmonic generation allows for the delineation of matrix wound healing, especially following glaucoma surgery. In conclusion, these imaging models play a pivotal role in the progress of ocular surface science and translational research.

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“…MPM can also be directly used to image fresh tissues without any other treatment. Near-infrared laser excitation allows for deeper imaging depths and less photodamage to biological tissues, and SHG does not involve the excitation of molecules, making it immune to phototoxicity and photobleaching, enabling in situ in vivo imaging and providing real-time diagnostic information [17][18][19]. MPM has been used in diagnostic studies of precancerous lesions [20,21].…”

Section: Introductionmentioning

confidence: 99%

Label‐free assessment of pathological changes in pancreatic intraepithelial neoplasia by biomedical multiphoton microscopy

Miao,

Zhang,

Zhang

et al. 2024

Journal of Biophotonics

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Pancreatic intraepithelial neoplasia (PanIN) is the most common precursor lesion that has the potential to progress to invasive pancreatic cancer, and early and rapid detection may offer patients a chance for treatment before the development of invasive carcinoma. Therefore, the identification of PanIN holds significant clinical importance. In this study, we first used multiphoton microscopy (MPM) combining two‐photon excitation fluorescence and second‐harmonic generation imaging to label‐free detect PanIN and attempted to differentiate between normal pancreatic ducts and different grades of PanIN. Then, we also developed an automatic image processing strategy to extract eight morphological features of collagen fibers from MPM images to quantify the changes in collagen fibers surrounding the ducts. Experimental results demonstrate that the combination of MPM and quantitative information can accurately identify normal pancreatic ducts and different grades of PanIN. This study may contribute to the rapid diagnosis of pancreatic diseases and may lay the foundation for further clinical application of MPM.

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Visualization of Intravital Immune Cell Dynamics After Conjunctival Surgery Using Multiphoton Microscopy

Cited by 6 publications

References 24 publications

Kinetics of corneal leukocytes by intravital multiphoton microscopy

Kinetics of corneal leukocytes by intravital multiphoton microscopy

Two-Photon and Multiphoton Microscopy in Anterior Segment Diseases of the Eye

Label‐free assessment of pathological changes in pancreatic intraepithelial neoplasia by biomedical multiphoton microscopy

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