BIO 224

Intro to Molecular and Cell Biology

Microscopy

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 availableLight microscopes remain the basic tools of

cell biologists

Microscopy

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 specimensBacteriaHuman cellsParamecia

Light Microscopy

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 viewedRequires higher resolution

Ability of a microscope to distinguish items separated by small distances

More important than magnification

Light Microscopy

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

Resolution

Several types used routinelyBright-field microscopy is simplest

Light passes directly through the cellAbility to see parts depends on absorption of

visible light by cell componentsUsually requires use of dyesTissues usually fixed (preserved) prior to

stainingRequires cells be killedHistology labs routinely examine fixed and

stained tissues

Light Microscopy

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

Light Microscopy

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

Light Microscopy

Molecules of interest labeled with fluorescent dye

Used in living or fixed cellsFluorescent dye absorbs light at one

wavelength and emits it at a secondFilters detect the wavelength of light the dye

emitsFluorescent labeled antibodies often used to

detect specific proteins

Fluorescence Microscopy

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 Microscopy

1.28 Fluorescence recovery after photobleaching (FRAP)

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

Fluorescence Microscopy

1.29 Fluorescence resonance energy transfer (FRET)

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

Fluorescence Microscopy

1.30 Confocal microscopy

1.31 Confocal micrograph of human cells

Multi-photon excitation microscopyAlternative to confocal microscopyCan be applied to living cellsSpecimen 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 focusAllows for 3D resolution without need of

pinhole apertureMinimal damage, allowing 3D image of living

cells

Fluorescence Microscopy

1.32 Two-photon excitation microscopy

More powerful than light microscopyDeveloped in 1930s, first used on biological

specimens in 1940s and 1950s Higher resolution due to wavelength of

electronsPractical limit of resolution for biological

specimens is 1 to 2nmOver 100X improvement on light scopes

Electron Microscopy

Transmission electron microscopy (TEM)Similar to observation of stained cells with bright

field scopeSpecimens fixed and stained with heavy metal salts

Scatter electrons to provide contrastElectron beam passed through specimen, focused to

form image on fluorescent screenElectrons encountering heavy metal are deflected,

causing stained areas to be darkCan use positive or negative staining3D views can be obtained using electron

tomographyUses computer to generate 3D image from 2D scans over

range of directions

Electron Microscopy

1.33 Positive staining

1.34 Negative staining

Metal shadowing is technique used to see surface of structures or moleculesEvaporated metal sprayed on specimen from angle so

surfaces facing source are more heavily coatedCreates shadow effect, allowing 3D look

Freeze fracture used in studies of membrane structure, usually interior facesSpecimens frozen in liquid N2 and fractured by knife

bladeFollowed by shadowing with Pt and dissolving

biological material with acidProduces metal replica of sample surface

Freeze etching is variation the allowing visualization of external surface of membranes along with interior

Transmission Electron Microscopy

1.35 Metal shadowing

1.36 Freeze fracture

Scanning electron microscopyUsed to provide 3D image of cellsSurface of specimen coated with heavy metalBeam of electrons used to scan across

specimenElectrons scattered or emitted from specimen

surface collected by detectorResolution only about 10nm, restricted to

whole cells rather than macromolecules or organelles

Electron Microscopy

1.37 Scanning electron microscopy

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