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Microscopy and Cell Fractionation in General Biology

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  • What is magnification in microscopy?

    Magnification is the ratio of an object’s image size to its real size, showing how much larger the image appears compared to the actual specimen.

  • Define resolution in microscopy.

    Resolution is the measure of image clarity, specifically the minimum distance two points can be separated and still be distinguished as separate points.

  • What limits the resolution of a standard light microscope?

    The light microscope cannot resolve details finer than about \(0.2\ \mu m\) (200 nm) due to the wavelength of visible light.

  • What is contrast in microscopy and how is it enhanced?

    Contrast is the difference in brightness between light and dark areas of an image, enhanced by staining or labeling cell components.

  • Who first observed cell walls and when?

    Robert Hooke first observed cell walls in 1665 using a microscope to look at dead cells from oak tree bark.

  • What was Antoni van Leeuwenhoek's contribution to microscopy?

    He crafted lenses that allowed visualization of living cells and microorganisms, called "very little animalcules," in 1674.

  • How does a light microscope (LM) work?

    Visible light passes through the specimen and glass lenses, which refract the light to magnify the image projected to the eye or camera.

  • What is the size range of most cells?

    Most cells range between 1 and 100 micrometers (\(\mu m\)) in diameter.

  • What is the principle behind electron microscopy (EM)?

    EM uses a beam of electrons instead of light to achieve much higher resolution due to electrons’ shorter wavelengths.

  • What is the resolution limit of modern electron microscopes?

    Electron microscopes can theoretically resolve structures as small as about \(0.002\ nm\), but practically about 2 nm.

  • What is the main use of scanning electron microscopy (SEM)?

    SEM provides detailed 3D images of the surface topography of specimens by scanning with an electron beam.

  • How does transmission electron microscopy (TEM) differ from SEM?

    TEM transmits electrons through a thin specimen section to study internal cell structures, while SEM scans surfaces.

  • Why are specimens stained with heavy metals in TEM?

    Heavy metals increase electron density in certain structures, enhancing contrast by scattering electrons more in denser regions.

  • What is a disadvantage of electron microscopy compared to light microscopy?

    Electron microscopy requires specimen preparation that kills cells and may introduce artifacts, unlike light microscopy which can study living cells.

  • What advances have revitalized light microscopy recently?

    Fluorescent markers, confocal microscopy, and super-resolution techniques have improved detail and resolution beyond traditional limits.

  • What is cryo-electron microscopy (cryo-EM)?

    Cryo-EM preserves specimens at very low temperatures without preservatives, allowing visualization of structures in their natural cellular environment.

  • What is cell fractionation?

    Cell fractionation is a technique where broken cells are centrifuged to separate components by size and density into pellets for study.

  • How does centrifugation separate cell components?

    Spinning broken cells at increasing speeds causes larger components to pellet first, followed by smaller components in subsequent spins.

  • How did cell fractionation help identify mitochondria function?

    Biochemical tests on fractions with mitochondria showed enzymes for cellular respiration, linking mitochondria to energy production.

  • Why is microscopy important in cytology?

    Microscopy allows visualization of cell structures, essential for understanding cell function and integrating with biochemical data.