Fluorescence lifetime imaging microscopy is a valuable tool for probing biological phenomena independent of luminescence intensity and fluorophore concentrations. Here, I demonstrate an adaptation of a laser scanning confocal microscope (LSCM) for time-resolved lifetime imaging without any add-on equipment. I have named this technique luminescence lifetime imaging microscopy by confocal pinhole shifting (Acronym: LLIM-CPS). I used LLIM-CPS to image europium (Eu 3+) microspheres, a red emitting long lifetime luminescent probe, simultaneously with short life time green-fluorescing microspheres and/or fluorescein and rhodamine in solution. With a one Airy unit pinhole diameter, short lifetime luminescence disappeared rapidly as the pinhole was repositioned in the lagging direction with complete disappearance at one Airy unit distance displacement, whereas long life time luminescence of Eu3+ was retained. In contrast, repositioning the pinhole in the leading or orthogonal directions to the rasting laser spot caused equal loss of short and long lifetime luminescence. These results show the ability of pinhole in the lagging direction to selectively image long lifetime luminescence. By making measurements at 1, 2 and 3 Airy unit lag pinhole positions, lifetime for Eu3+ was estimated to be 270 μs. The effect of pinhole diameter and laser dwell times on LLIM-CPS were studied. Pinhole diameters of 3 and 5 Airy units caused streaking of long lifetime europium microspheres with a one Airy unit pinhole diameter resolving the europium to its true diameter. Dwell times of 51 and 102 μs were required to image the europium microspheres compared to the shorter 3 μs dwell time that could not image the europium microspheres.
LLIM-CPS was used to quantify oxygen-dependent changes in intensity and lifetime of Tris-4, 7 diphenyl 1, 10-phenanthroline ruthenium an oxygen sensing long lifetime luminophore. LLIM-CPS images of heart cells in the presence of the oxygen-sensing phosphorescent luminophore, PtTBP-AG2-PEG, visualized oxygen surrounding the respiring cells. Thus, in this dissertation I have demonstrated an adaptation of a LSCM to perform quantitative long lifetime luminescence imaging and presented a biological application of oxygen sensing with this technique.
|Advisor:||Lemasters, John J.|
|Commitee:||Costello, Joseph M., Gallippi, Caterina M., Knisley, Stephen B., Lalush, David S.|
|School:||The University of North Carolina at Chapel Hill|
|Department:||Biomedical Engineering Joint Program|
|School Location:||United States -- North Carolina|
|Source:||DAI-B 68/11, Dissertation Abstracts International|
|Keywords:||Confocal pinhole shifting, Luminescence lifetime imaging microscopy|
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