Two-photon fluorescence excitation microscopy to assess transscleral diffusional pathways in an isolated perfused bovine eye model

W.K. Kek, Wallace S. Foulds, Gail McConnell, Amanda Wright, John Girkin, Clive Wilson

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract


To assess the feasibility of using two-photon microscopy to study the pattern of diffusion through the sclera of a tracer (tazarotenic acid [TA]).

Polyvinyl alcohol films containing 1% tazarotenic acid (PVA-TA) were applied to the equatorial sclera of isolated perfused bovine eyes. Two-photon microscopy (TPM) was used to determine the lateral spread and depth of penetration of TA in the sclera over time. Protein and collagen binding were determined, and calibration standards were prepared by TPM imaging at 10 μm depth in scleral samples that had been immersed for 24 hours in solutions of TA of 0.7, 7.0, or 70 μg/mL.

TA was weakly bound to collagen and sclera (<55%) but strongly bound to plasma protein (95%). In perfused eyes, 50 minutes after PVA-TA application, peak fluorescence in the sclera was detected at a 210-μm depth. By 85 minutes after application of the PVA-TA film, fluorescence had disappeared from surface layers of the sclera and was at maximum at 250 to 290 μm. Penetration of the tracer followed the track of scleral collagen bundles rather than that of the proteoglycan ground substance between collagen bundles.

TPM can image in real time the progressive diffusion of TA from its source in a PVA-TA film applied to the equatorial sclera of the isolated perfused bovine eye and follow its subsequent penetration deeper into the sclera. The data suggest that lateral spread and deeper penetration of the test compound occurred along the course of scleral collagen bundles. Imaging was possible to a depth of 340 μm, the average thickness of the human equatorial sclera.
LanguageEnglish
Pages5182-5189
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume51
Issue number10
DOIs
Publication statusPublished - Oct 2010

Fingerprint

Sclera
Photons
Fluorescence Microscopy
Collagen
Microscopy
Fluorescence
tazarotenic acid
Polyvinyl Alcohol
Proteoglycans
Protein Binding
Calibration
Blood Proteins

Keywords

  • two-photon microscopy
  • isolated perfused bovine eye
  • trans-scleral pathways
  • polyvinyl alcohol
  • tazarotenic acid

Cite this

@article{b1111eba30be4e898109a3cf6332eb28,
title = "Two-photon fluorescence excitation microscopy to assess transscleral diffusional pathways in an isolated perfused bovine eye model",
abstract = "To assess the feasibility of using two-photon microscopy to study the pattern of diffusion through the sclera of a tracer (tazarotenic acid [TA]).Polyvinyl alcohol films containing 1{\%} tazarotenic acid (PVA-TA) were applied to the equatorial sclera of isolated perfused bovine eyes. Two-photon microscopy (TPM) was used to determine the lateral spread and depth of penetration of TA in the sclera over time. Protein and collagen binding were determined, and calibration standards were prepared by TPM imaging at 10 μm depth in scleral samples that had been immersed for 24 hours in solutions of TA of 0.7, 7.0, or 70 μg/mL.TA was weakly bound to collagen and sclera (<55{\%}) but strongly bound to plasma protein (95{\%}). In perfused eyes, 50 minutes after PVA-TA application, peak fluorescence in the sclera was detected at a 210-μm depth. By 85 minutes after application of the PVA-TA film, fluorescence had disappeared from surface layers of the sclera and was at maximum at 250 to 290 μm. Penetration of the tracer followed the track of scleral collagen bundles rather than that of the proteoglycan ground substance between collagen bundles.TPM can image in real time the progressive diffusion of TA from its source in a PVA-TA film applied to the equatorial sclera of the isolated perfused bovine eye and follow its subsequent penetration deeper into the sclera. The data suggest that lateral spread and deeper penetration of the test compound occurred along the course of scleral collagen bundles. Imaging was possible to a depth of 340 μm, the average thickness of the human equatorial sclera.",
keywords = "two-photon microscopy, isolated perfused bovine eye, trans-scleral pathways, polyvinyl alcohol, tazarotenic acid",
author = "W.K. Kek and Foulds, {Wallace S.} and Gail McConnell and Amanda Wright and John Girkin and Clive Wilson",
year = "2010",
month = "10",
doi = "10.1167/iovs.09-3854",
language = "English",
volume = "51",
pages = "5182--5189",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
number = "10",

}

Two-photon fluorescence excitation microscopy to assess transscleral diffusional pathways in an isolated perfused bovine eye model. / Kek, W.K.; Foulds, Wallace S.; McConnell, Gail; Wright, Amanda; Girkin, John; Wilson, Clive.

In: Investigative Ophthalmology and Visual Science, Vol. 51, No. 10, 10.2010, p. 5182-5189.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Two-photon fluorescence excitation microscopy to assess transscleral diffusional pathways in an isolated perfused bovine eye model

AU - Kek, W.K.

AU - Foulds, Wallace S.

AU - McConnell, Gail

AU - Wright, Amanda

AU - Girkin, John

AU - Wilson, Clive

PY - 2010/10

Y1 - 2010/10

N2 - To assess the feasibility of using two-photon microscopy to study the pattern of diffusion through the sclera of a tracer (tazarotenic acid [TA]).Polyvinyl alcohol films containing 1% tazarotenic acid (PVA-TA) were applied to the equatorial sclera of isolated perfused bovine eyes. Two-photon microscopy (TPM) was used to determine the lateral spread and depth of penetration of TA in the sclera over time. Protein and collagen binding were determined, and calibration standards were prepared by TPM imaging at 10 μm depth in scleral samples that had been immersed for 24 hours in solutions of TA of 0.7, 7.0, or 70 μg/mL.TA was weakly bound to collagen and sclera (<55%) but strongly bound to plasma protein (95%). In perfused eyes, 50 minutes after PVA-TA application, peak fluorescence in the sclera was detected at a 210-μm depth. By 85 minutes after application of the PVA-TA film, fluorescence had disappeared from surface layers of the sclera and was at maximum at 250 to 290 μm. Penetration of the tracer followed the track of scleral collagen bundles rather than that of the proteoglycan ground substance between collagen bundles.TPM can image in real time the progressive diffusion of TA from its source in a PVA-TA film applied to the equatorial sclera of the isolated perfused bovine eye and follow its subsequent penetration deeper into the sclera. The data suggest that lateral spread and deeper penetration of the test compound occurred along the course of scleral collagen bundles. Imaging was possible to a depth of 340 μm, the average thickness of the human equatorial sclera.

AB - To assess the feasibility of using two-photon microscopy to study the pattern of diffusion through the sclera of a tracer (tazarotenic acid [TA]).Polyvinyl alcohol films containing 1% tazarotenic acid (PVA-TA) were applied to the equatorial sclera of isolated perfused bovine eyes. Two-photon microscopy (TPM) was used to determine the lateral spread and depth of penetration of TA in the sclera over time. Protein and collagen binding were determined, and calibration standards were prepared by TPM imaging at 10 μm depth in scleral samples that had been immersed for 24 hours in solutions of TA of 0.7, 7.0, or 70 μg/mL.TA was weakly bound to collagen and sclera (<55%) but strongly bound to plasma protein (95%). In perfused eyes, 50 minutes after PVA-TA application, peak fluorescence in the sclera was detected at a 210-μm depth. By 85 minutes after application of the PVA-TA film, fluorescence had disappeared from surface layers of the sclera and was at maximum at 250 to 290 μm. Penetration of the tracer followed the track of scleral collagen bundles rather than that of the proteoglycan ground substance between collagen bundles.TPM can image in real time the progressive diffusion of TA from its source in a PVA-TA film applied to the equatorial sclera of the isolated perfused bovine eye and follow its subsequent penetration deeper into the sclera. The data suggest that lateral spread and deeper penetration of the test compound occurred along the course of scleral collagen bundles. Imaging was possible to a depth of 340 μm, the average thickness of the human equatorial sclera.

KW - two-photon microscopy

KW - isolated perfused bovine eye

KW - trans-scleral pathways

KW - polyvinyl alcohol

KW - tazarotenic acid

UR - http://www.iovs.org/cgi/content/abstract/iovs.09-3854v1

U2 - 10.1167/iovs.09-3854

DO - 10.1167/iovs.09-3854

M3 - Article

VL - 51

SP - 5182

EP - 5189

JO - Investigative Ophthalmology and Visual Science

T2 - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 10

ER -