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Rapid Frequency-Domain FLIM Spinning Disk Confocal Microscope: Lifetime Resolution,Image Improvement and Wavelet Analysis |
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| Authors: | Chittanon Buranachai Daichi Kamiyama Akira Chiba Benjamin D Williams Robert M Clegg |
| Institution: | (1) Center of Biophysics and Computational Biology, University of Illinois, Urbana-Champaign, 1110 W Green St, Loomis Lab, Urbana, IL 61801, USA;(2) Department of Biology, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33124, USA;(3) Cell & Struc Biology, University of Illinois, Urbana-Champaign, 601 S Goodwin, M/C 123, Urbana, IL 61801, USA;(4) Department of Physics, University of Illinois, Urbana-Champaign, 1110 W Green St, Loomis Lab, Urbana, IL 61801, USA |
| Abstract: | A spinning disk confocal attachment is added to a full-field real-time frequency-domain fluorescence lifetime-resolved imaging microscope (FLIM). This provides confocal 3-D imaging while retaining all the characteristics of the normal 2-D FLIM. The spinning disk arrangement allows us to retain the speed of the normal 2-D full field FLIM while gaining true 3-D resolution. We also introduce the use of wavelet image transformations into the FLIM analysis. Wavelets prove useful for selecting objects according to their morphology, denoising and background subtraction. The performance of the instrument and the analysis routines are tested with quantitative physical samples and examples are presented with complex biological samples. |
| Keywords: | FLIM FLI Lifetime imaging Spinning Disk Microscope Polar plot Wavelet Morphology Background subtraction Denoising |
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