Wayne M. Becker Lewis J. Kleinsmith Jeff Hardin Gregory Paul Bertoni The World of the Cell Seventh Edition Appendix Visualizing Cells and Molecules Copyright © 2009 Pearson Education, Inc..

 All types of microscopes should have : 1. Source of illumination 2. Specimens 3. System of lenses  Microscope functions: 1.Resolution 2.Magnification 3.Contrast

Types of Microscopes  Compound Microscopes - Phase-Contrast Microscope (PCM) - Differential Interference Contrast (DICM) - Fluorescence Microscopy ( FM) - Confocal Microscope (CM)  The Electron Microscope - Transmission Electron Microscope (TEM) - Scanning Electron Microscope (SEM)

Figure A-1 Optical Systems of LM and EM

Comparison between LM & TEM TEMLM Comparison Phase Electron beamlightSource of illumination Electromagnetic lenses glassLenses Adjust current Adjust distance between objects and lenses Focusing 300,000x1000xmagnifying vacuumAir, oilPath of beam (rays) 0. 1µ m1-3 µ sections Fluorescent coated with ZnS or photographic film Eye, photographic filmImage seen by Salts of heavy metalsStains (dyes)Contrast improved by (60, ,000)v6vVoltage 5 Å 0.2 µ mResolution limit

Important definitions  Interference: The process by which two or more waves combine to reinforce or cancel one another producing a wave equal to the sum of the combining wave  Diffraction: Pattern of either additive or cancelling interference of the waves that went through the lenses  Resolution the minimum distance that can separate two points when viewed by microscope

Focal length : the distance between the midline of the lens and the point at which rays passing through the lens converge to a focus The focal length

Angular aperture is half the angle α of the cone light entering the objective lens of microscope from the specimen

Factors that determine resolution(r) and size of diffraction limited spot r = 0.61 λ N sin α  The quantity of of N sin α is called numerical aperture which reflects the ability of lens to collect light rays 1.The wave length (λ) of light used to illuminate the specimen 2.Angular aperture (α): the best glass lenses have about 70⁰ 3.Refractive index (N) : a measure of the change in the velocity of light as it passes from one medium to another  0.61 represents the degree to which image points can overlap and still be recognized as a separate points by an observer

How resolution is improved?  The resolution improved as r becomes smaller by : - Decreasing the value of numerator as small as possible by using λ shortest as possible (blue light 450 nm) - Increasing the value of denominator as large as possible by α is equal to 70⁰ sin= 0.94 and N= 1 in air so NA=0.94X1=0.94

The Compound Light Microscope (brightfield)

Path of light through a single lens Formation of an image of a single point of light by a lens Formation of an image of a point of light in the presence of two other points Formation of an image of a section of an equally bright tube of light Formation of an image of a brightened section of a tube of light

Phase-Contrast Microscope (PCM)  PCM can detect differences in relative index and thickness  PCM is used in microbiology and tissue culture, cellular organelles in living specimens PCM of epithelial cells in unstained state which is the major advantage of PCM

Differential Interference Contrast (DICM) Four cell sea urchin embryo. Shadow casting effect that makes these cell appear dark at the bottom and light at top  DIC utilizes a spelt light beam to detect phase differences  DIC is used for studying living unstained specimens

Fluorescence Microscopy ( FM) Endothelial cells stained with an anti-β tubulin mouse monoclonal antibody and green secondary antibody green to label microtubules, Texas red phalloidin for labeling F-actin and DAPI for labeling DNA in nuclei

The principle of fluorescence (a) Light of a certain energy is absorbed (the blue light here) the e jumps from its ground state to an excited state (b) The absorption and emission spectra of a typical fluorescent molecule. The blue curve represents the amount of light absorbed as a function of λ, and the green curve shows the amount of emitted light as a function of λ.

Immunostaining using fluorescent antibodies Direct immunofluorescence Indirect immunofluorescence

Fluorescent Probes  Fluorescein (green)  Rhodamine (red)  Phalloidin is mushroom toxin binds to actin filaments  Fura-2 detect ca conc. In cytosol  Green Fluorescent Protein (GFP) from jellyfish A. victoria, by recombinant DNA (fuse DNA gene encoding GFP with a gene coding for a particular cellular protein ) Using GFP to visualize proteins. An image series of a living, one cell worm embryo undergoing mitosis. The embryo expressing β-tubulin that is taged with GFP. Elapsed time from the first frame is shown in minutes : seconds

Laser Scanning Confocal Microscope (LSCM) LSCM. A laser is used to illuminate one spot at a time in the specimen (blue lines). Scanning mirrors move the spot in a given plane of focus through a precise pattern. The fluorescent light being emitted from the specimen (red lines) bounces off the same scanning mirrors and returns along the original path of the illumination beam. The emitted light does not return to the laser but instead is transmitted through the dichroic mirror. A pinhole in the image plane blocks the extraneous rays that are out of focus. The light is detected by photomultiplier tube, whose signal is digitized and stored by a computer

Comparison of confocal fluorescent microscopy (CFM) with traditional fluorescent microscopy (TFM) In the TFM the entire specimen is illuminated so fluorescent material above and below the plane of focus tends to blur the image In CFM the incoming light is focused on a single plane and out of focus fluorescence from the specimen is excluded, the resulting image is much sharper

Sample preparation techniques for LM  fixation with acids and aldehydes e.g acetic acid, picric acid, formaldehyde, and glutaraldehyde  dehydration in alcohol  embedding in paraffin wax nor epoxy plastic resi  sectioning  Mounting  Staining

Transmission Electron Microscope (TEM)  TEM forms an image from electrons that pass through the specimen  Accelerating voltage results from difference between Anode voltage v = 0, and cathode voltage v = kv  Voltage -For CTEM specimens must be extremely thin usually no more than 100nm V= kv - for HVEM for specimens with 1µm thick V= kv  The contrasting light, dark, and intermediate areas of specimen create a final image  Specimen preparation imparts selective electron density to the specimen

Scanning Electron Microscope (SEM)

 SEM reveals the surface architecture of cells and organelles allowing the surface topography of specimens to be studied  A beam of electrons (primary electrons) is focused on the surface of the specimen and sweeps rapidly over it  The beam deflector (located between condenser and lens) attracts or repels the beam by signals from deflector circuitry  The image is generated by secondary electrons emitted by the specimen and captured by scintillator located above the specimen

Sample preparation techniques for EM  Ultrathin sectioning and staining  Radioisotopes and antibodies  Negative staining  Shadowing  Freeze fractioning and freeze etching

Ultrathin sectioning and staining (50-100)nm  Specimen is chemically fixed and stabilized using aldehyde or glutaraldehyde  Staining with 1-2% of OsO4 give electron dense  Dehydration with alcohol  Placing tissue in acetone or propylene oxide  Embedding in liquefied in plastic epoxy resin  Specimen is put into mold and heated to harden the plastic  Specimen is sliced into ultrathin sections by ultramicotome  Mounting of specimen on the arm of the ultramicrotome  section is float from blade of the microtome onto water surface  sections are picked up on a circular copper specimen grid  Stained again with solution containing uranium and lead to give electron density to a specific part of the cell  photograpgy

Radioisotopes and antibodies  In TEM the specimen containing the radioactively labeled compound is simply examined in ultrathin sections on copper specimen grid  The antibodies used in EM is called immunoelectron microscopy  Fluorescent can’t be seen in EM, instead antibodies are visualized by linking them to electron dense substance and visible as opaque dots  when ultrathin tissue sections are stained with gold labeled antibodies directed against various proteins this can reveal the subcellular location of these proteins

Negative staining NS  NS can highlight small objects in relief against darkly stained background  NS study the surface appearance of very small objects e.g viruses, organelles without cutting  The copper specimen grid is overlaid with ultrathin plastic film  Specimen is suspended in a small drop of liquid and specimen is dried in air on the surface of the grid  A drop of stain of e.g uranyl acetate, phosphotungstic acid is applied to the film surface  Filter paper absorbs the excess stain, this draws stain down and around the specimen and its ultrastructure features  Examined by TEM specimen is seen in negative contrast

Negative staining NS of a bacteriophage using TEM

Shadowing Vacuum evaporator Metal electrode Carbon electrode specimen To vacuum system

Shadowing specimen Mica surface 1. The specimen is spread on a mica surface and dried Metal atoms Metal wire Heated filament 2. The specimen is shadowed by coating it with atoms of a heavy metal that are evaporated from a heated filament Metal replica Carbon atoms 4. The replica is floated onto the surface of an acid bath to dissolve away the specimen, leaving a clean metal replica Acid path Specimen dissolving away 3. The specimen is coated with carbon atoms evaporated from an overhead electrode Metal replica Copper grid 5. The replica is washed and picked up on a copper grid for examination on TEM

Shadowing An electron micrograph of tobacco mosaic virus particle visualized by shadowing

Freeze fracturing

Freeze etching  A further step is added to the conventional freeze-fracturing procedure which occurs following the fracture of the specimen but prior to shadowing, the microtome arm is placed directly over the specimen for a short period of time  This cause a small amount of water to evaporate from the surface of specimen to the cold knife this produces etching effect an accentuation of surface detail