title: Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.
abstract: High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas.
authors.affiliation: Department of Mechanical Engineering, Pohang University of Science and Technology, San 31 Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784, Republic of Korea.
authors.forename: Jun Ho
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Mechanical Engineering, Pohang University of Science and Technology, San 31 Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784, Republic of Korea.
authors.forename: Seunghun
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Life Science and Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31 Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784, Republic of Korea.
authors.forename: Yong Song
authors.initials: YS
authors.lastname: Gho
authors.affiliation: Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea.
authors.forename: In Seok
authors.initials: IS
authors.lastname: Song
authors.affiliation: Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea.
authors.forename: Hungwon
authors.initials: H
authors.lastname: Tchah
authors.affiliation: Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea. Electronic address: joon@amc.seoul.kr.
authors.forename: Myoung Joon
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Department of Mechanical Engineering, Pohang University of Science and Technology, San 31 Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784, Republic of Korea; Department of Mechanical Engineering and Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, San 31 Hyoja-dong, Nam-gu, Pohang, Gyeongbuk 790-784, Republic of Korea. Electronic address: kiheankim@postech.edu.
authors.forename: Ki Hean
authors.initials: KH
authors.lastname: Kim
mesh_terms: D000818:Animals; D003094:Collagen / Q000032:analysis; D003315:Cornea / Q000033:anatomy & histology*; D016510:Corneal Neovascularization / Q000473:pathology*; D003319:Corneal Stroma / Q000166:cytology; D004195:Disease Models, Animal; D019573:Epithelium, Corneal / Q000166:cytology; D049452:Green Fluorescent Proteins / Q000032:analysis; D021621:Imaging, Three-Dimensional / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008853:Microscopy; D018613:Microscopy, Confocal / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins; D003094:Collagen
keywords: Animal model; Confocal microscopy; Corneal suture model; High-resolution 3D imaging; In vivo imaging; Two-photon microscopy