Biol 266 – Cell Biology UNIT 2 PDF

Biol 266 – Cell Biology UNIT 2 “How cells are studied - I” The compound microscope: most common microscope in use today contains several lenses that magnify the image of a specimen The optical pathway in a modern compound optical microscope ocular lens (eyepiece) eye...

Biol 266 – Cell Biology UNIT 2 “How cells are studied - I” The compound microscope: most common microscope in use today contains several lenses that magnify the image of a specimen The optical pathway in a modern compound optical microscope ocular lens (eyepiece) eye reflecting prism objective lens specimen condenser lens iris diaphragm light source The optical pathway in a modern compound optical microscope ocular lens (eyepiece) eye Picks up the light transmitted reflecting prism by the specimen and focus it on the focal plane of the objective lens, creating a magnified image objective lens specimen Focuses the light condenser lens onto the specimen – no magnification iris diaphragm Restricts the amount of light light source entering the lens The optical pathway in a modern compound optical microscope ocular lens (eyepiece) The image on the objective eye focal plane is further magnified by the ocular lens, reflecting prism or eyepiece, and projects it onto the human eye objective lens specimen condenser lens iris diaphragm light source The total magnification is the product of the magnification of the individual lenses. If the objective lens magnifies 100-fold (a 100X lens) and the ocular lens magnifies 10-fold (a 10X lens), the final magnification will be 100 x 10 = 1000-fold However, the most important property of the microscope is not its magnification but its resolving power (resolution). Magnification versus resolution: (a) to (b) has increased magnification and resolution, but (b) to (c) only increased magnification  Resolution (D) is the ability to see two nearby points as distinct images. The smaller the value of D, the better the resolution. D1 = 1 µm D2 = 5 µm Resolution 1 is better than resolution 2 Resolution is determined by the objective lens and its ability to gather the “cone of light” coming from the specimen. The light comes into the objective lens as a cone due to diffraction by the specimen.  objective lens cone of light specimen a represents half the angle of the cone of light 0.61 l D= n sina l is the wavelength of incident light in nm n is the refractive index of the medium between the specimen and the objective  0.61 l D= n sina The lower the wavelength, the better the resolution. The shortest wavelength visible light is 450 nm (blue). In contrast, an electron in an electron microscope with an accelerating voltage of 100,000 V has a wavelength of 0.004 nm. In theory, the resolution of such an electron microscope is ~100,000 times greater than that of the light microscope. A fundamental limitation on all microscopes: a given type of radiation cannot be used to probe details smaller than its own wavelength (l). We can partially circumvent this limitation by increasing a, which will decrease D. The best objectives have an a value of 70° (e.g. sin90 = 1, sin70 = 0.94, sin45 = 0.71). Thus as a increases, the denominator increases, lowering the value of D). 0.61 l D= n sina A fundamental limitation on all microscopes: a given type of radiation cannot be used to probe details smaller than its own wavelength (l). Another way is to increase the refractive index of the medium between the specimen and the objective lens (n). (e.g. n = 1.0 for air, n = 1.5 for oil). Thus, using oil increases resolution by 50%. 0.61 l D= n sina The limit of resolution of a light microscope: With the visible light of shortest wavelength (blue, l = 450 nm), an immersion oil objective (n = 1.5) and the best objective lens (a = 70°, sin α = 0.94): 0.61 x 450 nm D= = 194 nm (~0.2 µm) 1.5 x 0.94 No matter how many times the image is magnified, the light microscope can never resolve objects that are less than ~ 0.2 µm in size  Three common types of light microscopy A. Brightfield – no contrast other than natural is provided, image is projected on a background of the cone of light that enters the objective lens. B. Phase-contrast – as light passes through a sample it is slowed in a medium of higher refractive index. The refracted and unrefracted light are recombined to form the image. If light is out of phase it will be less bright, in phase it will be more bright. Requires a phase plate to be inserted into the microscope after light passes through the objective lens. C. Differential interference contrast (DIC) or Nomarski interference – based on interference between polarized light and the medium (refractive index) through which it travels. Method of choice for thicker samples.  Transmission electron microscope (TEM) Transmission electron microscopes (TEMs) use electrons instead of light to form images. Comparison of skeletal muscle images from a light (top) and electron (bottom) microscope at a comparable magnification of 4500 times actual size.  Comparison between TEM and light microscope vacuum 1. TEM is larger (>2m) compared to a light microscope (~30cm) 2. TEM uses electrons instead of light. 3. Beam of electrons is projected downwards while light is projected upwards 4. TEM uses electromagnetic coils to focus the beam of electrons and to magnify the image while light microscopy uses glass lenses 5. The TEM is maintained in a vacuum while the light microscope operates in air electromagnetic coil  The optical path in a transmission electron microscope cathode anode Electrons are emitted by a cathode when it is electrically heated. The electric potential of the cathode is kept at 50,000 – 100,000 volts electromagnetic The electric potential of the anode condenser lens is zero. The drop in voltage causes the electrons to accelerate as they specimen move toward the anode. A beam of electrons is focused onto the specimen plane by the electromagnetic condenser. Like the light microscope, the condenser does not create a magnified image of the specimen. Specimen is extremely thin (50 – 100 nm), cut with a special instrument called an ultramicrotome.  The optical path in a transmission electron microscope The electromagnetic objective lens picks up the electrons that have passed through the specimen and magnifies the image in the focal plane of the objective lens. The electromagnetic projector lens (equivalent to the ocular lens in the electromagnetic light microscope) picks up the objective lens electrons from the focal plane of the objective lens and both focuses and magnifies them onto the specimen detector. electromagnetic projector lens detector  0.61 l D= n sina For a TEM: l of electron is ~0.004 nm N.A. = 0.01 So, D = 0.2 nm For a light microscope, D = 200 nm. Therefore, a TEM has 1000-fold more resolution compared to a light microscope  The limits of resolution, the sizes of cells and of their component parts, and the units in which they are measured Organelles (0.1 µm – 2 µm) Cells (1 µm – 20 µm) Molecules (0.0002 µm – 0.02 µm) 0.2 mm 0.2 µm 20 µm 2 µm 20 nm 2 nm 0.2 nm (200 µm) (200 nm) Minimum resolvable Minimum resolvable Minimum resolvable by unaided eye by light microscope by electron microscope  Fluorescence microscopy Fluorescent molecules absorb light at one wavelength (the excitation wavelength) and emit light (fluoresce) at another, longer wavelength (the emission wavelength) Excitation at Emission at l = 470 nm l = 540 nm If such a compound is illuminated at its excitation wavelength and viewed through a filter that allows only light of the emitted wavelength to pass, it is seen to glow against a dark background.  E – the energy of a photon of light hc E= [photon is the basic component (unit) of l light] h – Planck’s constant c-speed of light l – wavelength of a photon The energy (E) of a photon of light is inversely proportional to its wavelength  Fluorescence is a three-stage process hc Eex= lex hc Eem= lem Excitation: A photon of energy is supplied by an external source (e.g. a laser) and absorbed by a fluorescent molecule creating an excited state (S1’) Energy dissipation: The energy of S1’ is partially dissipated, yielding a relaxed state (S1) Fluorescence emission: A photon of energy is emitted, returning the fluorescent molecule to its ground state (S0) Fluorescein emits green light when activated by light of the appropriate wavelength Tetramethylrhodamine emits red light when activated by light of the appropriate wavelength  The optical path in a fluorescence microscope 1. First barrier filter: lets through only blue light with a wavelength between 450 and eyepiece 490 nm 2, 3. The fluorescent dye, second barrier fluorescein, in the specimen filter 5 is excited (2) to emit light (fluoresce) at a specific and longer wavelength (3) 4 beam-splitting 1 mirror 2 3 first barrier filter objective lens specimen  The optical path in a fluorescence microscope 4. Beam-splitting mirror reflects light below 510 nm but transmits light above 510 eyepiece nm 5. Second barrier filter cuts second barrier out unwanted fluorescent filter 5 signals, allowing through the green fluorescein emission between 520 and 560 nm 4 beam-splitting 1 mirror 2 3 first barrier filter objective lens specimen Confocal microscopy improves the image of a conventional fluorescence microscope Conventional fluorescence microscopy generates a blurry image because light from above and below the plane of focus are also collected. Confocal microscopy focuses a single point of light at a specific depth in the specimen.  The optical path in a confocal fluorescence microscope 3 4 1 2 1. Light from a 2. A dichroic 3. Emitted light 4. Emitted light laser passes mirror and an from the from elsewhere through a pinhole. objective lens specimen is in the specimen is then focus the focused at a largely excluded light at a specific second (confocal) from the second depth in the pinhole and pinhole. specimen. reaches the detector. Image of a mitotic, fertilized sea urchin egg stained for tubulin  Revealing specific proteins in fixed cells - Immunofluorescence microscopy Primary antibody: rabbit IgG Secondary antibody Secondary antibody (donkey): anti-rabbit IgG (donkey): anti-rabbit IgG fluorescein fluorescein fluoresces fluoresces The use of secondary antibodies results in the amplification of the fluorescent signal, thereby antigen increasing the sensitivity of immunofluorescence microscopy Fixation of a sample “locks” the proteins in place while preserving cell architecture. There are two main types of fixation: 1. Cross-linking reagents such as paraformaldehyde or glutaraldehyde. Glutaraldehyde autofluoresces (green emission) and may interfere with fluorescence signal. 2. Precipitation using a cold organic solutions such as acetone or methanol. Dehydrates the sample and can result in changes to cell architecture. Once the sample is fixed, the membranes will need to be permeabilized to allow for entry of the antibodies. This is accomplished by organic solutions (precipitation fixation accomplishes this simultaneously) or by treatment with detergent such as Triton X-100. Fluorescence micrograph showing the distribution of long actin fibers in a cultured fibroblast cell: Primary antibody: rabbit anti-actin IgG Secondary antibody: fluorescein-conjugated donkey anti-rabbit IgG Excitation/emission spectra of common fluorescent probes * * *proteins * * Different fluorescent probes can be visualized in the same cell A mitotic cell stained for three cellular components. Three different filter sets were used to acquire three separate images. The images were then overlayed to give the composite image. Microtubules – revealed with a green fluorescent antibody Centromeres – revealed with a red fluorescent antibody DNA – revealed with a stain called DAPI DNA CENP-E tubulin merge Considerations when using multiple primary antibodies for immunofluorescence microscopy 1. Make sure the host species (species in which the antibodies are raised – rabbit, mouse, human) for the primary antibodies are different. Otherwise, the secondary antibodies will bind to both primary antibodies and you will not be able to distinguish your proteins of interest. 2. Make sure the fluorescent molecules or proteins on the secondary antibodies emit at a wavelength that is sufficiently different so that you can distinguish the signals.  Revealing specific proteins in fixed cells - Immunogold electron microscopy Attach antibodies directed against a specific protein, catalase, to electron-dense colloidal gold particles (5-20 nm in diameter) These antibodies interact only with their specific antigen (e.g. catalase) antigen (e.g. catalase) primary antibody Gold particle Treat thin sections of glutaraldehyde-fixed cells or tissues with these gold-labeled anti-catalase antibodies Determine the subcellular location of catalase in the electron microscope  Peroxisomes The gold particles (black dots), indicating the presence of catalase, are located exclusively in the peroxisome. 0.5 µm The subcellular location of catalase in a rat liver cell  Revealing specific proteins in living cells Green fluorescent protein (GFP) A naturally fluorescent protein from jellyfish. Emits a green fluorescence when exposed to light of the exciting wavelength. When “linked” to a protein of interest, GFP (or other fluorescent proteins) can be used to follow the localization and movement of the protein of interest in live cells. gene X GFP-encoding gene expression protein X GFP LIVING CELL targeting protein X GFP ORGANELLE fluorescence microscopy There are now many fluorescent proteins, spanning numerous different wavelengths. This allows researchers to investigate several proteins in the same cell. GFP dsRED  Tissue culture Cells can be isolated from tissues 1. Disrupt cell-cell contacts with a protease such as trypsin or collagenase, or with EDTA which chelates Ca2+ (required for cell-cell adhesion) 2. Plate the cells in a plastic dish. Some cells require a layer of collagen to adhere to the plates, others can adhere to the plastic (adherent cells) while others are will not adhere to the plastic or to an extracellular component (non-adherent cells). 3. If there is a mixed population of cells, fluorescence- activated cell sorting (FACS) can be used to separate the population. Sorting cell types by FACS An antibody recognizing a protein facing the outside surface of the cell is coupled to a fluorescent dye and suspended in a fluid. Droplets with single cells pass by a laser to excite the fluorescent dye. If the detector registers fluorescence, the droplet is immediately negatively charged. Otherwise, the drops are not charged. The droplets are then deflected in an electric field and collected. They are then put into culture on plastic dishes. As the cells grow, they occupy more space on the dish. Once they reach 100% confluency (a measure of the surface area occupied by the cells), they must be passaged. They are removed from the dish with trypsin or EDTA and plated onto a new dish. Cells that are derived from a tissue are called primary cells. These cells can only be passaged 25-40 times and then they stop dividing (they become senescent). They do not express telomerase. Primary cells can be immortalized by adding DNA that expresses telomerase. The cells are referred to as a cell line. Some cancer cells have the ability to grow indefinitely and are referred to as transformed cell line. Common cell lines: HeLa (human epithelial), 293 (human kidney), CHO (hamster ovary), MDCK (dog epithelial), COS (monkey kidney) The growth media for cultured cells contains: 9 essential amino acids (Phe, Val, Thr, Trp, Ile, Met, Leu, Lys, His) Glutamine – non-essential amino acid but used as a nitrogen source Vitamins Fatty acids Glucose Serum (non-cellular portion of clotted blood) Ø Hormones (e.g. insulin) Ø Transferrin (iron transport) Ø Growth factors