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Two-Photon Microscopy for Biomedical Studies Yassel Acosta University of Texas at El Paso, [email protected]

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Two-Photon Microscopy for Biomedical Studies Yassel Acosta University of Texas at El Paso, Yassel.Acosta@Gmail.Com University of Texas at El Paso DigitalCommons@UTEP Open Access Theses & Dissertations 2014-01-01 Two-Photon Microscopy For Biomedical Studies Yassel Acosta University of Texas at El Paso, [email protected] Follow this and additional works at: https://digitalcommons.utep.edu/open_etd Part of the Biology Commons, and the Physics Commons Recommended Citation Acosta, Yassel, "Two-Photon Microscopy For Biomedical Studies" (2014). Open Access Theses & Dissertations. 1185. https://digitalcommons.utep.edu/open_etd/1185 This is brought to you for free and open access by DigitalCommons@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of DigitalCommons@UTEP. For more information, please contact [email protected]. TWO-PHOTON MICROSCOPY FOR BIOMEDICAL STUDIES YASSEL ACOSTA Department of Physics APPROVED: Chunqiang Li, Ph.D., Chair Vivian Incera, Ph.D. Kyung-An Han, Ph.D. Bess Sirmon-Taylor, Ph.D. Interim Dean of the Graduate School Copyright © by Yassel Acosta 2014 TWO PHOTON MICROSCOPY FOR BIOMEDICAL STUDIES by YASSEL ACOSTA, B.S. Electronic Engineering THESIS Presented to the Faculty of the Graduate School of The University of Texas at El Paso in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Department of Physics THE UNIVERSITY OF TEXAS AT EL PASO August 2014 Table of Contents Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii Chapter 1: Two-Photon Microscopy ................................................................................................1 1.1 Theory of Two-Photon Absorption .............................................................................1 1.2 Two-Photon Fluorescence Intensity ...........................................................................8 1.3 Axial Resolution in Two-Photon Microscopy ............................................................9 1.4 Scattering and Absorption.........................................................................................11 1.5 Two-Photon Laser-Scanning Fluorescence Microscope ..........................................13 Chapter 2: Fluorescence Resonance Energy Transfer ...................................................................22 2.1 The Rate of Energy Transfer ...............................................................................22 2.2 The efficiency of Energy Transfer ........................................................................24 2.3 Semi-classical Derivation of FRET ..........................................................................25 2.4 Orientation Factor ................................................................................................31 2.5 The Fӧster distance and The Overlap Integral ..........................................................33 2.6 Measuring FRET .......................................................................................................35 Chapter 3: Two-Photon FRET Microscopy to Quantify Mycobacterium Phagosomal Rupture in Macrophages .....................................................................................................................38 3.1 Background Knowledge............................................................................................38 3.2 Mechanism for Tracking Phagosomal Rupture ........................................................39 3.3 Experimental Procedure ............................................................................................40 3.4 Results .......................................................................................................................43 3.5 Conclusions ...............................................................................................................46 Chapter 4: Functional Imaging of Drosophila Brain Using Two-Photon Microscopy ..................47 4.1 Visualization of dopaminergic neurons in the protocerebrum ..................................47 4.2 Functional imaging using the fluorescent calcium reporter GCaMP5G ...................49 4.3 Imaging Drosophila cerebral trachea with two-photon fluorescence microscopy....52 4.4 Conclusions ................................................................................................................57 iv Bibliography ............................................................................................................................58 Vita…. ......................................................................................................................................59 v List of Tables Table 1.1: Transverse and axial resolution calculated based on the PSF ..................................... 10 Table 1.2: Lines per image corresponding to each frame rate. ..................................................... 16 Table 1.3: Configuration of the control inputs A, B, C, D, E and F for each frame rate. ............. 18 Table 4.1 Comparison of tracheal volume for the structures shown in figure 4.8 (c and d). ....... 57 vi List of Figures Figure 1.1: Process of one-photon absorption described by Eq. 1.13............................................. 4 Figure 1.2: Convention followed for labeling the various levels for two-photon absorption......... 5 Figure 1.3: Approximation of from Eq. (1.20) as a Dirac delta function. ................................. 6 Figure 1.4: Fluorescence intensity generated at each z-plane as a function of the distance from the focal plane. .............................................................................................................................. 11 Figure 1.5: Schematic of the Two-Photon Laser Scanning Fluorescence Microscope developed in the Biophotonics Laboratory of the Physics Department at UTEP. ............................................. 13 Figure 1.6: Schematic of the synchronization electronic circuit................................................... 17 Figure 2.1: Diagram describing the decay process in terms of the rates. ..................................... 23 Figure 2.2: Diagram describing FRET and showing decaying and transfer rates. ....................... 24 Figure 2.3: Plot of equation (2.9) .................................................................................................. 25 Figure 2.4: Dipole moments in a Cartesian coordinate system. ................................................... 29 Figure 2.5: Four possible relative orientations between D and A................................................. 32 Figure 2.6: Representation of the overlap integral . .................................................................. 34 Figure 3.1: Stages of Mtb infection. ............................................................................................. 39 Figure 3.2: Fluorescence Emission Spectra of CCF4 (two-photon excitation at 700 ) ........... 41 Figure 3.3: Mechanism for tracking phagosomal rupture. ............................................................ 41 Figure 3.4: Images acquired after 48 and 96 hours of infection. .................................................. 44 Figure 3.5: Comparison at 48 and 96 hours. ................................................................................. 45 Figure 3.6: Comparison with Ms after 96 hours. .......................................................................... 45 Figure 4.1: TH-GAL4 expressing neurons in the Drosophila brain. ............................................ 48 Figure 4.2: Maximum intensity projections of two Drosophila brains generated with Imajej. .... 49 Figure 4.3: GCaMP5G fluorescent spectra. .................................................................................. 50 Figure 4.4: Three-dimensional reconstructions of two adult brains expressing calcium sensor GFP. .............................................................................................................................................. 52 Figure 4.5: Three-dimensional reconstructions of one adult brain (a, b, c) and one third instar larvae brain (d,e,f) autofluorescence upon excitation with 710 . ............................................ 53 Figure 4.6: Maximum intensity projections of tracheal branches in two different brains showing correlation between GFP expression and autofluorescence.......................................................... 54 Figure 4.7: Treshholded images using Imagej. ............................................................................. 55 Figure 4.8: Three-dimensional reconstruction of the ROI. ........................................................... 56 vii Chapter 1: Two-Photon Microscopy 1.1 THEORY OF TWO-PHOTON ABSORPTION The simultaneous absorption of two photons by an atom was first predicted by Maria Gӧppert-Mayer in 1931. The probability for such quantum event is proportional to the square of the excitation intensity and to the molecular cross section. In order to calculate this probability the laws of quantum mechanics must be taken into consideration, more specifically, time- dependent perturbation theory should be applied to the interaction of atomic electrons with a classical radiation field. To begin we write the Hamiltonian ̂ of the system
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