Microscopy, Magnification and Cell Fractionation

Magnification is image size divided by actual object size, using the same length units. Resolution is the ability to distinguish two close points as separate. Enlarging a blurred image increases magnification without improving the underlying resolution; a visible outline is not proof that every internal feature can be resolved.

Calibrate an eyepiece graticule using a stage micrometer at the objective setting being used. The graticule divisions do not have a fixed physical length independent of magnification. A scale bar remains useful when an image is resized together with its bar; a printed magnification value may no longer be valid after resizing.

Optical microscopy can observe suitable living material and offers lower resolution than electron microscopy. Stains increase contrast but can alter or kill cells. A transmission electron microscope uses electrons passing through a thin specimen to reveal internal ultrastructure in a two-dimensional section.

A scanning electron microscope builds information from a specimen’s surface and can produce a three-dimensional-looking surface image. Conventional electron microscopy requires extensive preparation and cannot show living cells under normal operating conditions. Preparation can introduce artefacts: apparent structures should be interpreted with knowledge of method and corroborating evidence.

For cell fractionation, homogenise tissue to break cells, filter out large debris, then centrifuge the homogenate. A cold, buffered, appropriately isotonic medium helps limit enzyme damage, maintain pH and prevent organelles bursting or shrinking osmotically. These conditions preserve the structures being studied; they are not arbitrary recipe details.

In differential centrifugation, a low-speed spin pellets relatively large components such as nuclei. Transfer the supernatant and spin faster to pellet smaller components in later steps. Separation depends on properties including size and density. Fractions are enriched rather than automatically pure, so marker enzymes or microscopy can help evaluate contamination.

Try each question before opening the worked response. Use precise biological terms, connect each cause to its effect, and check units and assumptions when interpreting data.