Applications of Total Internal Reflection Fluorescence Microscopy for Studies of Chemical Phenomena at the Substrate-Liquid Interface

Persistent Link:
http://hdl.handle.net/10150/194335
Title:
Applications of Total Internal Reflection Fluorescence Microscopy for Studies of Chemical Phenomena at the Substrate-Liquid Interface
Author:
Phimphivong, Samrane
Issue Date:
2008
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Applications of TIRFM for quantitative measurements of cells are limited due to high background fluorescence which can result in a low S/N ratio and therefore contribute to inaccuracy of measurements. Time-resolved total internal reflection fluorescence microscopy (TR-TIRFM) was developed by temporally gating a CCD camera using a liquid crystal shutter to optically filtering the short-live fluorescence and recording only the long-live emission. This technique was then applied to examine the extent of cell-substrate contacts. Tb+3 chelates such as DTPA-PhenylAS-Tb+3 was synthesized and applied as a membrane staining agent but was observed to internalized into the cell nucleus. A modified chelate molecule was therefore synthesized using DOPE as a carrier molecule. DOPE-DTPA-pAS-Tb+3 has a similar emission lifetime (1.5 msec) and appeared to stain only the cell membrane. TR-TIRF was applied to examine adherent cells on polystyrene-coated substrate. TR-TIRF images showed cellular autofluorescence and polystyrene emissions were optically filtered out, while the long-lived emission intensity of Tb+3 chelate was recorded. These results conclude that TR-TIRFM, with the use of long-live emission label (Tb+3 and Eu+3 chelates), is suitable as an analytical tool for probing a large number of cellular and molecular events occurring in the cell membrane and on the cell surface where background fluorescence would usually be problematic. Detection of K+ transported across a cell membrane is a prerequisite in the development of devices for screening drugs targeting K+ ion channels. K+ sensing film was fabricated by encapsulating a squaraine dye (aza-crown-SQR) in a sol-gel matrix for detection of K+. Sol-gel films were prepared by the hydrolysis and condensation reactions of TEOS or TMOS, APTS and GOPS mixtures. Formation of a DPhPC bilayer on sol-gel films was achieved by the vesicle fusion method and had diffusion coefficients of 2.3 and 2.1x10-8 cm2s-1 as measured by FRAP on TEOS-APTS-GOPS and TMOS-APTS-GOPS film, respectively. The time-based fluorescence intensity data from the H+ blocking experiments showed the sol-gel-supported DPhPC bilayers are impermeable to H+, and the K+ blocking experiments showed K+ was passively transported across a DPhPC bilayer by valinomycin.
Type:
text; Electronic Dissertation
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Chemistry; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Saavedra, S. Scott
Committee Chair:
Saavedra, S. Scott

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleApplications of Total Internal Reflection Fluorescence Microscopy for Studies of Chemical Phenomena at the Substrate-Liquid Interfaceen_US
dc.creatorPhimphivong, Samraneen_US
dc.contributor.authorPhimphivong, Samraneen_US
dc.date.issued2008en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractApplications of TIRFM for quantitative measurements of cells are limited due to high background fluorescence which can result in a low S/N ratio and therefore contribute to inaccuracy of measurements. Time-resolved total internal reflection fluorescence microscopy (TR-TIRFM) was developed by temporally gating a CCD camera using a liquid crystal shutter to optically filtering the short-live fluorescence and recording only the long-live emission. This technique was then applied to examine the extent of cell-substrate contacts. Tb+3 chelates such as DTPA-PhenylAS-Tb+3 was synthesized and applied as a membrane staining agent but was observed to internalized into the cell nucleus. A modified chelate molecule was therefore synthesized using DOPE as a carrier molecule. DOPE-DTPA-pAS-Tb+3 has a similar emission lifetime (1.5 msec) and appeared to stain only the cell membrane. TR-TIRF was applied to examine adherent cells on polystyrene-coated substrate. TR-TIRF images showed cellular autofluorescence and polystyrene emissions were optically filtered out, while the long-lived emission intensity of Tb+3 chelate was recorded. These results conclude that TR-TIRFM, with the use of long-live emission label (Tb+3 and Eu+3 chelates), is suitable as an analytical tool for probing a large number of cellular and molecular events occurring in the cell membrane and on the cell surface where background fluorescence would usually be problematic. Detection of K+ transported across a cell membrane is a prerequisite in the development of devices for screening drugs targeting K+ ion channels. K+ sensing film was fabricated by encapsulating a squaraine dye (aza-crown-SQR) in a sol-gel matrix for detection of K+. Sol-gel films were prepared by the hydrolysis and condensation reactions of TEOS or TMOS, APTS and GOPS mixtures. Formation of a DPhPC bilayer on sol-gel films was achieved by the vesicle fusion method and had diffusion coefficients of 2.3 and 2.1x10-8 cm2s-1 as measured by FRAP on TEOS-APTS-GOPS and TMOS-APTS-GOPS film, respectively. The time-based fluorescence intensity data from the H+ blocking experiments showed the sol-gel-supported DPhPC bilayers are impermeable to H+, and the K+ blocking experiments showed K+ was passively transported across a DPhPC bilayer by valinomycin.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorSaavedra, S. Scotten_US
dc.contributor.chairSaavedra, S. Scotten_US
dc.contributor.committeememberAspinwall, Craig A.en_US
dc.contributor.committeememberHruby, Victor J.en_US
dc.contributor.committeememberPolt, Robinen_US
dc.contributor.committeememberEvans, Dennis H.en_US
dc.identifier.proquest2915en_US
dc.identifier.oclc659749566en_US
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