BIO 224 Intro to Molecular and Cell Biology

Microscopes are tools frequently used in cell biology Type of microscope used depends on the specimen being viewed and magnification desired Many types of microscopes are available Light microscopes remain the basic tools of cell biologists

Early microscopes used by Hooke and others were simple light microscopes Earliest microscopes were capable of up to 300 X magnification Early scientists viewed and documented an impressive array of specimens Bacteria Human cells Paramecia

Modern microscopes are capable of 1000X magnification (mag) Most cells range from 10um to 100um diameter, easily seen with light scope Some large organelles can be visualized as well Fine structural details can’t be viewed Requires higher resolution Ability of a microscope to distinguish items separated by small distances More important than magnification

Limit of light microscopes about 0.2 um based on equation Determined by two factors Wavelength of visible light: λ Numerical aperture of the lens: NA Calculated by an equation: (0.61 λ) ÷ NA Wavelength fixed at 0.5 for light microscope NA is found by equation NA=η sinα η is refractive index of the medium which light travels through between the specimen and the lens (1.0 for air, 1.4 with immersion oil) Angle α is half the width of cone of light collected by the lens (maximum value is 90) Highest possible value of NA is 1.4 Maximum theoretical resolution (also called resolving power) had been achieved by late 1800s, no increases expected

Several types used routinely Bright-field microscopy is simplest Light passes directly through the cell Ability to see parts depends on absorption of visible light by cell components Usually requires use of dyes Tissues usually fixed (preserved) prior to staining Requires cells be killed Histology labs routinely examine fixed and stained tissues

Phase-contrast and differential interference- contrast microscopy commonly used for living cells Achieve contrast due to variations in thickness in cell parts Speed of light drops as it passes through intracellular structures, altering its phase compared to light in cytoplasm Changes in phase converted to differences in contrast Allows for improved images of live, unstained cells

Video and cameras can be added to enhance features of microscopes Has allowed visualization of movement of organelles along microtubules Location of certain molecules can be seen using labels and dyes Fluorescence microscopy is used for studying intracellular distribution of molecules

Molecules of interest labeled with fluorescent dye Used in living or fixed cells Fluorescent dye absorbs light at one wavelength and emits it at a second Filters detect the wavelength of light the dye emits Fluorescent labeled antibodies often used to detect specific proteins

Green fluorescent protein (GFP) of jellyfish used to see proteins inside living cells Protein can be expressed in cells and viewed with microscope Other related proteins with blue, yellow, or red emissions also available Other methods developed to follow interactions of GFP- labeled proteins within living cells Fluorescence recovery after photo-bleaching (FRAP) Region of interest bleached by exposure to high-intensity light Unbleached molecules move into bleached region Allows detection of rate of movement of protein within cell

Fluorescence resonance energy transfer (FRET) Two proteins coupled to fluorescent dyes, like two GFP variants that emit different wavelengths of light Light emitted by one GFP variant excites the second If proteins labeled by the two GFP variants interact within cells, the fluorescent molecules will be near each other and light emission will occur

Conventional microscopy produces blurred and out-of- focus images Can be improved by deconvolution: a computer analyzes images obtained by different depths of focus and generates a sharper image Confocal microscopy can be used to allow images of increased contrast and detail Obtained from fluorescence from a single point in specimen Light produced by laser focused on specimen at a certain depth Fluorescence emitted passes through pinhole aperture before hitting detector Allows only light emitted from plane of focus to reach detector Results in sharper image from scanning across image Series of images may be used for 3D image of sample

Multi-photon excitation microscopy Alternative to confocal microscopy Can be applied to living cells Specimen illuminated with wavelength needing absorption of two or more photons to excite fluorescent dye Photons only excite fluorescent dye at point in the specimen where input beam is focused Fluorescence only emitted from plane of focus Allows for 3D resolution without need of pinhole aperture Minimal damage, allowing 3D image of living cells

More powerful than light microscopy Developed in 1930s, first used on biological specimens in 1940s and 1950s Higher resolution due to wavelength of electrons Practical limit of resolution for biological specimens is 1 to 2nm Over 100X improvement on light scopes

Transmission electron microscopy (TEM) Similar to observation of stained cells with bright field scope Specimens fixed and stained with heavy metal salts Scatter electrons to provide contrast Electron beam passed through specimen, focused to form image on fluorescent screen Electrons encountering heavy metal are deflected, causing stained areas to be dark Can use positive or negative staining 3D views can be obtained using electron tomography Uses computer to generate 3D image from 2D scans over range of directions

Metal shadowing is technique used to see surface of structures or molecules Evaporated metal sprayed on specimen from angle so surfaces facing source are more heavily coated Creates shadow effect, allowing 3D look Freeze fracture used in studies of membrane structure, usually interior faces Specimens frozen in liquid N2 and fractured by knife blade Followed by shadowing with Pt and dissolving biological material with acid Produces metal replica of sample surface Freeze etching is variation the allowing visualization of external surface of membranes along with interior

Scanning electron microscopy Used to provide 3D image of cells Surface of specimen coated with heavy metal Beam of electrons used to scan across specimen Electrons scattered or emitted from specimen surface collected by detector Resolution only about 10nm, restricted to whole cells rather than macromolecules or organelles

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