Lecture 2 Microscopy 2023.pdf

TA Contact Information For questions, please contact the TA that has been assigned to you: Student Name Abdul --- Benest Bengtsson --- Diallo Diba --- Iqbal Isaak --- Lofaro Loghmani --- Okonowele Onusko --- Stanzel Stiles --- Znamenski Your TA Elena Esina Emily Gallipeau Ryan Lambert Anthea Mavri...

TA Contact Information For questions, please contact the TA that has been assigned to you: Student Name Abdul --- Benest Bengtsson --- Diallo Diba --- Iqbal Isaak --- Lofaro Loghmani --- Okonowele Onusko --- Stanzel Stiles --- Znamenski Your TA Elena Esina Emily Gallipeau Ryan Lambert Anthea Mavridis Mohamed Ramadan Brianna Raven Victor Wiltenburg TA's email [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] 1 Research Techniques I: Microscopy and Cell Imaging Seeing at the cellular and subcellular level 2 Several Types of Microscopy •Light microscope •Conventional (e.g. bright field, phase contrast etc.) •Fluorescence microscope •Confocal microscope •Two-photon microscope •Electron microscope (next lecture) vesicles membrane bilayers mitochondria 3 The Light Microscope (LM) • Utilizes basic light path. • Used for live or fixed cells and tissue. • Tissues: upright microscope. • Isolated cells: inverted microscope. An “upright” microscope 4 Four types of LM 1) Bright Field 1) Bright field • transmitted light. 2) Phase contrast • converts phase differences into changes in brightness. • Frits Zernike, 1953 Nobel Prize. 2) Phase contrast 5 Four types of LM • Wave properties of light can be exploited. • In unstained cells, a phase shift will occur as light travels through the cell. • Phase alignment is related to increased brightness; if not aligned, decreased brightness. • Observable with phase contrast. l phase shift a Figure 9-7B. Molecular Biology of the Cell, 4th Edition. 6 Four types of LM 3) Differential interference contrast • similar principles as with phase contrast. • polarized light is separated and recombined. • more definition. 4) Dark field • lateral light source shows only scattered light. 3) Differential Interference Contrast (DIC) 4) Dark Field 7 Phase and DIC Compared Figure A6, 8. Becker et al. 2006 World of the Cell. 8 Fluorescence Microscopy • Useful for detection of specific molecules or ions. • Works on the principle that some molecules absorb and emit photons of light at specific wavelengths. • Atomic absorption of a photon is followed by emission at a longer wavelength, and a light signal is detected. wavelength shift Figure A10. Becker et al. 2006 World of the Cell. 9 Fluorescence Microscopy • A variety of fluorescent molecules are used in fluorescence microscopy (e.g. DAPI, GFP, FITC). • Note spectral characteristics of each dye/molecule. 10 Fluorescence Microscopy • Fluorescence microscope is optically similar to LM. • High energy lamps (Hg) provide bright source. • Filters reduce light of unwanted wavelengths. • Chromophores excited at specific wavelengths. 11 GFP in Research • • Transgenic mice generated to express GFP and other “FPs”. Permits selective labelling and imaging of cells in live specimens. Hippocampal neurons Dendritic spines Neuromuscular junction (GFP+YFP) Spinal cord: double expression (with YFP) ACh receptors From Feng et al. 2000. Neuron 28:41-51. 12 Jackson et al. (2013) Expression of sall4 in taste buds of zebrafish. Dev Neurobiol. 73:543-58. 13 Tissue Preparation • • • • • To observe cells in tissue, in most cases tissues must be histologically prepared (e.g. not for GFP). Fixation: exposure to chemical reagents (aldehydes, acids, alcohols) to preserve and stabilize. May produce unwanted effects. Embedding: plastic or polyethylene glycol. Sectioning: cutting of thin (1–10 µm) tissue sections on a microtome. Staining: if applicable, involves exposure to dyes, e.g. hematoxylin, eosin, antibodies. 14 Immunofluorescence • Antibodies are produced in host animal and collected. • Fixed tissue is permeabilized and treated with primary antibodies directed against a specific antigen. • Antibodies bind to antigen on or within cell. • Secondary antibodies conjugated with fluorescent marker bind to primary antibodies. • Indirect immunohistochemistry labels cell structures. 15 The Confocal Microscope Advantages: • Technique that provides clear images with reduced “background” signal. • Particularly useful for applications involving thick sections or whole-mount preparations. Disadvantages: • Costly. 16 The Confocal Microscope pinhole • “Confocal” refers to equidistance between light source and object, and object and detector.** • Utilizes fluorescence and high energy lasers (He-Ne and Ar). • Pinhole focuses light at a single point in specimen, producing an optical section with low background “noise”. d2 pinhole d1 d1=d2 object 17 The Confocal Microscope • Only light focused at the pinhole will enter the detector.** • This allows the confocal to provide clear images a few µm into tissue. • In addition to the x and y axes, imaging may occur in the z axis. • 3D reconstructions possible. 18 Comparison of Techniques Fluorescence Glia (red) Neurons (green) Confocal Figure A15. Becker et al. 2006 World of the Cell. Actin (green) in Drosophila embryo 19 Two-Photon Microscopy (non-linear) Advantages: • Non-linear technique that uses higher-order light-matter interactions from multiple photons to generate contrast. • Allows deep tissue imaging (up to 1 mm depth possible). • In this process, absorption occurs in the near IR region, and NIR light penetrates deep into tissue. Disadvantage: • Very costly. 20 Two-Photon Microscopy • Two-photon absorption involves “simultaneous” (~0.5 fs) arrival at excitable molecule. • Excitation and emission occurs, as in fluorescence. (e.g. Confocal) • However, signal is dependent upon photon density, so absorption is spatially confined. • In confocal, single photon absorption occurs throughout exc. light cone. 21 From Helmchen and Denk. 2005. Nature Meth 2:932 Two-Photon Microscopy • Differs from confocal by excitation laser and detection pathway. • Rapid high-energy laser pulses (100 fs; 100 MHz) are emitted. • Signal collected by detector depending on sample thickness. • No pinholes needed, as in confocal. 22 Two-Photon Microscopy • Example of in vivo deep-tissue imaging. • Possible configurations. • Intact neocortex. 23 From Helmchen and Denk. 2005. Nature Meth 2:932 Things to Consider… 1. What are the primary differences between the different types of microscopy discussed so far? 2. Think about appropriate applications in which you would use regular fluorescence, confocal and two-photon microscopy. 24