Biology 200

How to evaluate Microscopy

● scale, features you can see, is it black/white or is it in color● critically evaluate ~ don't just use the checklist
Scale of Things● whole cells are usually in the 10-100µm range● organelles are in the 1-10µm range● macromolecules and their assemblies are in the nm range
Features of Cells● Magnification and resolution do NOT mean the same thing Magnification: expanding something in apparent size → the ration of the size of an image to the size of the object. → the magnification of a microscopy image Resolution: the closest spacing of 2 points which can be distinguished as separate entities → dependent on the λ of the beam used to detect the image. → the ability of the objective lens to gather light.

Light Microscopy

Light Microscopy:● live cells, color, whole tissues● resolution limit: 0.2 µm (typically)
Transmitted Light LM:● brightfield - light passes through specimen and viewed● uses white light, collects all scattered light● tissues must be cut into thin sections in order to see cells in them● ability to identify nucleus, chloroplasts, cell membrane
Emitted Light LM:● colored light (or lasers) is used to excite photons that are then emitted by sample and viewed
Fluorescence:● uses light of one λ to excite fluorochrome● collects light emitted by that fluorochrome● entire specimen is illuminated● fluorescence detected above and below the plane of focus tends to blur the image ● purpose is to localize molecules of structures in cells ● molecules of structures are fluorescently labeled ● epifluorescence microscopes are used to illuminate the whole sample with a light source and the emitted light from the fluorescent label is detected.
Direct Fluorescent Labeling:● some common direct dyes can label the membrane, nucleus (DNA), mitochondria, cytoskeleton
GFP-Fusion:● Green Fluorescent Protein-isolated from bioluminescent jellyfish● proteins fused with GFP can be tracked moving inside cells● GFP gene can be molecularly fused to your gene of interest and transferred inside cells● GFP and RFP can be engineered to produce a broad range of colors, which allows multiple proteins to be tracked simultaneously

Immunofluorescence

● localizes proteins of interest in a cell using primary antibodies● secondary antibodies - covalently linked to a fluorescence molecule, recognize the primary molecule, provides signal amplification
Confocal: ● Incoming light is focused on a single plane● Out-of-focus fluorescence from the specimen is excluded (increases resolution)● Improves topical resolution and contrast● allows optical sectioning

Fluorescent Staining Techniques

Direct staining● fluorochrome is specifically combined with a special type of macromolecule
Indirect immunofluorescence staining● a fluorochrome is covalently linked to the antibodies→ Process:1. Primary antibody is applied, which is made in one type of mammal and is specific to the thing/antigen you want to see.2. A secondary antibody is then applied, which is conjugated to a fluorochrome and made in a different mammal than the primary.
Green Fluorescent protein● GFP is a small luminescent protein that was originally discovered in jellyfish● scientists can genetically engineer their protein of interest with the GFP attached, resulting in a GFP fusion protein that behaves in similar ways to the original protein● using this technique, proteins of interest can be tracked in live cells using fluorescence microscopy

Electron Microscopy

● the electron gun produces a stream of electrons at known λ that are accelerated by a potential difference of many thousands of volts● beyond the gun are electromagnetic lenses that the operator uses to control magnification and focus● electromagnetic lenses induce magnetic fields parallel to the direction of the electron beam, causing it to spiral inwards and converge at a focal point so that the sample can be viewed
→ Transmission Electron Microscopy:Resolution: electrons have shorter wavelengths than light PROS: → good size resolution at size range important for cell studies. → size of organelles to macromolecules CONS: → samples must withstand electron bombardment and vacuum, so elaborate specimen preparation is required. → heavy metal stains must be used to increase contrast. → hard to reconstruct 3D structures from 2D slices, unless even more complex microscopy and digital reconstruction is used.
→ Electron Microscopy Tomography:● allows the study of internal structures of very thinly sliced (50-100nm) specimens● unaffected and diffracted primary electrons which pass through the specimen produce an image on a fluorescent plate● the electron scattering induced by atoms of heavy metals produces the dark parts of the image● TEM cannot be used with living material, as the sample is sliced, embedded in resin, stained with heavy metals, and then placed in the vacuum of the microscope to view it● negative staining is a different type of staining technique
→ Scanning Electron Microscopy:● specimen is usually dried and coated with thin layer of heavy metal● electron beam is scanned across the surface of the specimen in a series of lines● as the bean of the electron hits the specimen, secondary electrons are ejected from the surface of the specimen● these are then collected by a secondary electron detector that electronically builds an image based on electron intensity● produces images of surfaces ONLY PROS: → great for surfaces, 3D images, → can also be for internal images, if the sample has been frozen first and then cut open. CONS: → requires a vacuum, so most specimens have to be fixed and dried.

Determining The Type of Microscopy Used

Questions to ask yourself!● magnification/scale of image - how big/zoomed in is it?● does it look 3D or is there depth?● is there a black background? does it look like the sample is emitting the particles used for imaging?● what types of structure are visible?● is it colored/is the color computer generated?● is it moving/does it look like the sample is alive?