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Imaging in Cell Biology.docx

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Biology 2382B
Jessica Kelly

Lecture 2 – Imaging in Cell Biology Chapter 9: p. 380-391, 400-402 Anton van Leeuwenhoek (1632-1723) • Built many simple, single lens microscopes • First to observe living protozoa and bacteria which he called “animalcules” • Went on to visualize human red blood cells and sperm • With great skill at grinding lenses, naturally acute eyesight and lots of patience he was able to achieve a magnification of 200X Features of a Modern Compound Microscope Bright-field Microscopy • Light source • Condenser lens to focus light on specimen • Objective lens to collect light after it has passed through specimen • Ocular or eyepiece lens to focus image onto eye • Typical light microscope magnification is 100 to 1000X • Only structures with a high refractive index (ability to bend light) are observable • Compound prefix refers to multiple lenses that are used to magnify the specimen • Light source is used to view the specimen 1 • Refraction or interference with the light is what results in the image we see • Condenser lens function to focus the light on the specimen • Objective lens collects the magnified light that has passed through the specimen • Virtual image viewed as light passes through objective lens through the eyepiece • Magnification starts with the objective lenses  actual magnification is increased further with the ocular eyepiece 1000x is usually the upper limit magnification of light microscopy… Resolving Power of Microscopes Resolution (D) - the ability to distinguish between two very closely positioned objects as separate entities  A conventional microscope can never resolve objects that are less than ~0.2 mM apart λ - wavelength of light Nsinα - numerical aperture of lens N - refractive index of medium between the specimen and the objective lens α - 1/2 angle of light entering objective The limit of resolution is 0.2 mm=200 nm Imaging is optimized by microscopy materials that result in adjusting the values in this equation to generating as small a number as possible thus providing greater resolution… • Green light (human eye is not very sensitive to blue light and very sensitive to green light) is used to minimize wavelength • Immersion oil can be used to reduce the refractive index of the medium  thus increasing the numerical aperture • Increasing the angle of light entering objective is dependent on the lens (i.e. how its ground) 2 Obtaining Contrast: Exploiting Changes in the Phase of Light • Objects or materials that refract (slow down) light will change the phase of light Combined wavelengths that are out of phase  interference between phases result in dimmer light  object appears dark Phase Contrast Microscopy Phase Contrast Microscopy - generates an image in which the degree of darkness or brightness of a region of the sample depends on the refractive index of that region.  Used to examine live “unstained” cells  Small differences in refractive index & thickness within the cell are further exploited and converted into contrast visible to the eye 3 • Annular diaphragm focuses the light and the specimen is illuminated with a finer cone of light • Phase differences of light that passes through the specimen as a result of cellular organelles (i.e. nucleus) causes interference and results in dimer light • Phase plate  further acts to cause the interference of out of phase light waves = dim objects (extentuates details in the cell) Differential Interference Contrast Microscopy (DIC microscopy)  Used to examine live “unstained” cells  Small differences in refractive index & thickness within the cell are converted into contrast visible to the eye Uses polarized light  DIC microscope is equipped with polarizers  Interference between polarized light generates contrast • Shadow effect gives an almost 3D image of the object  gives topographical information about the cell Left to right  Bright field, DIC, Phase Contrast… 4 Fluorescence Microscopy  Uses a property of certain molecules to fluoresce (i.e. to emit visible light when they absorb light at a specific wavelength (e.g. invisible UV light) Can visualize more than one protein or cell structure (i.e. with different dyes  tubulin = green, mitochondrion = red, Nuclei = blue Location of fluorescent dyes or fluorescent protein molecules can be imaged - Dyes or “FLUOROPHORES”  absorb energy kicking electrons (e ) into a higher orbital (unstable)  instability causes electrons to drop back into its normal orbital releasing energy as visible light “FLUORESCENCE” • Longer lower energy light photon is emitted after electron is excited • Depending on the molecule there is a wavelength of maximum absorption  this is the wavelength used to allow for optimal fluorescence How Are Fluorescence Images Obtained? 5 Parts of a Fluorescent Microscope Mercury lamp  can produce Emission filter  helps to eliminate the autofluoresence  so we only get the wavelengths of light from UV to Infared fluorescence that is desired Dichroic mirror  does not allow light through but reflects it down towards the Lenses  act to magnify image specimen Filter cube  pre-prepared filters for imaging Excitation filter  acts to select a specific wavelength of light The Many Colours of Fluorophores  Signals are bright on a black background  A variety of fluorophores exist  different excitation and emission wavelengths that allow labeling of more than one protein or organelle at the same time  Fluorescent dyes are available to stain fixed cells and cell organelles (e.g. DAPI to stain nuclei blue, Mitotracker Red to stain mitochondria red)  A dye can be conjugated with antibodies  to localize any molecules of your interest in cells (immunofluorescent staining) 6 Monoclonal Antibodies HAT medium (a selection medium) - is toxic for myeloma cells, which have mutation of specific gene (i.e. HGPRT-) Hybrid cells  survive in HAT medium because they obtain a missing gene product from spleen cells Hybrid cells are immortal (like myeloma cells) and produce desired antibody Hybridomas survive in the HAT medium because the genomes of myeloma and lymphocytes complement each other  allows the purine biosynthesis salvage pathways to function… How is it done? • Difficult to stain very specific proteins  Purified protein can be produce via RDT • The Protein (the antigen) can be injected into mouse  mouse’s (or other experimental animal like a goat) immune system used to produce antibodies for the antigen (protein)  these cells (spleen cells) can be harvested and mixed with mutant (cancer) myeloma mouse cells • Hybrid cells  produced with mutant cells and an immortal cell line fusion  they constantly produce the desired antibodies • Selective media (HAT) can be used to select for only the immortal hybrid cells Hybrid cells can be propagated and used to produce the desired antibody in large amounts for a variety of uses in cell biology experiments… 7 Immunofluorescence Microscopy  Antibodies are made to specific proteins (i.e. microtubules, actin)  Primary Antibodies (i.e. rabbit antibodys) are added to fixed cells which bind only to the specific protein they were designed to recognize  Seco
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