Why does Z-step size matter for corneal epithelial imaging?

Setting the Z step to one micrometer enables precise optical sectioning of superficial wing and basal cells in the corneal epithelium, allowing mapping of single cells between layers and visualization of hexagonal cell shapes. This resolution supports quantitative analysis of epithelial dynamics and structural integrity in live tissue.

How does dual excitation wavelength selection enable specific signal detection?

Using 880 nm excitation for SHG EGFP and 940 nm for tdTomato allows spectral separation of second harmonic generation and fluorescent signals, enabling simultaneous label-free stromal imaging and membrane protein visualization. This approach prevents signal bleed-through and ensures accurate attribution of cellular and structural signals.

What does median 3D filtering enable in post-processing?

Applying the Median 3D filter plugin in Fiji removes background noise from Z-stacks while preserving structural details of corneal stroma, nerve fibers, and capillary networks. This step enhances signal-to-noise ratio before contrast optimization and 3D reconstruction.

Why does ocular surface stabilization impact imaging quality?

Stabilizing the eyeball with moderate pressure via the custom holder and forceps prevents movement artifacts during Z-serial imaging across the corneal and conjunctival surface. Insufficient or excessive pressure compromises image quality by inducing tissue deformation or ischemia, directly affecting data reliability.

What spectral filters are required to separate SHG and tdTomato signals?

Using 434-17 nm, 510-84 nm, and 585-40 nm band pass filters spectrally separates SHG EGFP, EGFP, and tdTomato signals, enabling clear detection of collagenous stroma, nuclear, and membrane signals. This filter set is essential for multiplexed imaging without spectral overlap in the detection pathway.

Niestandardowa platforma mikroskopii wielofotonowej do obrazowania na żywo rogówki i spojówki myszy

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Cited by 1

06:53 min

May 17th, 2020

10.3791/60944-v

May 17th, 2020

5.3K views

Yueh-Feng Wu¹ , Rai-Teng Ye¹ , Ming-Kai Pan²^,³ , Sung-Jan Lin^*¹^,³^,⁴^,⁵ , Hsin-Yuan Tan^*⁶^,⁷

¹Department of Biomedical Engineering, National Taiwan University, ²Institute of Pharmacology, College of Medicine, National Taiwan University, ³Molecular Imaging Center, National Taiwan University, ⁴Department of Dermatology, National Taiwan University Hospital, and College of Medicine, ⁵Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, ⁶Department of Ophthalmology, Chang Gung Memorial Hospital, ⁷College of Medicine, Chang Gung University

^* These authors contributed equally

Prezentowana tutaj jest wielofotonowa mikroskopijna platforma do obrazowania powierzchni oka myszy na żywo. Fluorescencyjna mysz transgeniczna umożliwia wizualizację jąder komórkowych, błon komórkowych, włókien nerwowych i naczyń włosowatych na powierzchni oka. Nieliniowe sygnały generujące drugą harmoniczną pochodzące ze struktur kolagenowych zapewniają obrazowanie bez znaczników dla architektur zrębowych.