U Ottawa HSS 2305 Molecular Mech. of Disease Lecture Notes PDF

Course HSS 2305 A Molecular Mechanism of Disease Lecture-23 Techniques in Cell and Mol. Biology, Tools Ajoy Basak, Ph. D. Adjunct and Part-time Professor, Pathology and Laboratory Medicine, Faculty of Medicine, U Ottawa,...

Course HSS 2305 A Molecular Mechanism of Disease Lecture-23 Techniques in Cell and Mol. Biology, Tools Ajoy Basak, Ph. D. Adjunct and Part-time Professor, Pathology and Laboratory Medicine, Faculty of Medicine, U Ottawa, Roger Guindon Building 451 Smyth Road Ottawa, ON K1H 8M5 Tel 613-878-7043 (Cell) E-mail: [email protected] Alternate: [email protected] Affiliate Investigator, Chronic Disease Program, Ottawa Hospital Research Institute Web: https://med.uottawa.ca/pathology/people/basak-ajoy 1 2  Introduction Research in cell biology requires complex instrumentation and techniques. Cell and molecular biology is more dependent on the development of new instruments and technologies than any other branch of biology. Understanding the technology helps in understanding the cell. It is important to understand these techniques and the types of information that can be learned by using these techniques. 3  (18.1) The Light Microscope Paths taken by light rays that form the image of the specimen and those that form the background light of the field. Sectional diagram through a compound light microscope that has both an objective and an ocular lens The light microscope uses the refraction of light rays to magnify an object. – A condenser directs light toward the specimen. – The objective lens collects light from the specimen. – The ocular lens forms an enlarged, virtual image. 4  The Light Microscope Resolution Magnification versus resolution: (a) to (b) has Relationship between the stimulation of individual increased magnification and resolution, but (b) to (c) photoreceptors (left) and the resulting scene one would only increased magnification perceive (right). Resolution – Resolution is the ability to see two nearby points as distinct images. The numerical aperture is a measure of the light-gathering qualities of a lens. The limit of resolution depends on the wavelength of light. Optical flaws, or aberrations, affect resolving power. 5  The Light Microscope Visibility Visibility – Visibility deals with factors that allow an object to be observed. It requires that the specimen and the background have different refractive indexes. Translucent specimens are stained with dyes. In bright-field microscope, a light that illuminates the specimen is seen as a bright background; it is suited for Feulgen stain: specific for DNA, showing the chromosomes of an onion root tip cell specimens of high contrast such in metaphase of mitosis at the time it was as stained sections of tissues. fixed. 6  The Light Microscope Visibility Preparation of Specimens for Bright-Field Light Microscopy – A whole mount is an intact object, either living of dead. – A section is a very thin slice of an object. To prepare a section, cells are immersed in a chemical called a fixative. Feulgen stain: specific for DNA, showing the chromosomes of an onion root tip cell in The rest of the procedures metaphase of mitosis at the time it was fixed. minimize alteration from the living state. 7  The Light Microscope Phase-contrast Phase-Contrast Microscopy – The phase-contrast A comparison of cells seen with different microscope makes highly types of light transparent objects more microscopes: a ciliated visible by converting protist under bright-field (top), phase-contrast differences in the refractive (middle), and differential index of some parts of the interference contrast specimen into differences (bottom). in light intensity. – Differential interference contrast (DIC) optics gives a three-dimensional quality to the image. 8  The Light Microscope Fluorescence microscopy Fluorescence Microscopy (and Related Fluorescence-Based Techniques) – Fluorescence microscopy has made possible advances in live-cell imaging. – Fluorochromes are compounds that release visible light upon absorption of UV rays. – Fluorochrome stains cause cell components to glow, a phenomenon called fluorescence. – Fluorochrome-conjugated antibodies are used to locate specific cellular structures (immunofluorescence). – The gene for green fluorescent protein (GFP) from jellyfish can be recombined with genes of interest in model organisms. GFP is expressed with the host gene of interest. GFP is used to follow a gene of interest. 9  The Light Microscope Fluorescence microscopy Use of GFP variants to follow the dynamic interactions between neurons and target cells in vivo: portion of a transgenic mouse brain with two differently colored fluorescent neurons. Fluorescence Microscopy – The gene for green fluorescent protein (GFP) from jellyfish can be recombined with genes of interest in model organisms. GFP is expressed with the host gene of interest. GFP is used to follow a gene of interest. 10  The Light Microscope FRET Fluorescence microscopy – Fluorescence Resonance Energy Transfer (FRET) and a GFP variant which uses fluorochromes to measure changes in distance between labeled cellular components. – Proteins or individual domains can be fused with different GFP variants. – Energy is transferred from Fluorescence resonance energy transfer (FRET). one GFP variant to the This diagram shows an example of the use of FRET other GFP variant. technology to follow the change in conformation of a protein (PKG) following cGMP binding. 11  The Light Microscope Video microscopy Video Microscopy and Image Processing – Video microscopy is used to observe living cells. – Video cameras offer several advantages for viewing specimens. Special types of video cameras (called charge-coupled device, or CCD cameras) are constructed to be very sensitive to light, which allows them to image specimens at very low illumination. They can detect and amplify very small differences in contrast. Images produced by video cameras can be converted to digital electronic images and processed by a computer. – In one technique, the distracting out-of-focus background in a visual field is stored by the computer and then subtracted from the image containing the specimen, greatly increasing the clarity of the image. 12  The Light Microscope Laser scanning confocal microscopy Confocal fluorescence micrographs of three The light paths in separate optical sections, each 0.3 μm thick, of a a confocal yeast nucleus stained with two different fluorescence fluorescently labeled antibodies (DNA in red and a microscope and telomere-binding protein in green). the focal plane Laser Scanning Confocal Microscopy – A laser scanning confocal microscope produces an image of a thin plane located within a much thicker specimen. – A laser beam is used to examine planes at different depths in a specimen. 13  The Light Microscope STORM Breaking the light microscope’s limit of resolution: conventional fluorescence micrograph (left) and STORM micrographs. Shown are microtubules (green) and clathrin-coated pits (red). Super-Resolution Fluorescence Microscopy – STORM (stochastic optical reconstruction microscopy) allows the localization of a single fluorescent molecule within a resolution of