Microscopy

• Bacteria are typically 0,5 to micrometers 10 in size.
• Magnification is how many times bigger the image of a specimen observed is compared to the actual size of the specimen
• the lenses on a light microscope are the eyepiece lens with often a magnification of x10 and a series of objective lenses with different magnifications each
• Total magnification =eyepiece lens magnification x objective lens magnification
• using more lenses means more magnification
• Amount of detail depends on resolution also known as resolving power of the microscope
• Resolution(resolving) power is the ability to distinguish between two separate points
• resolution(resolving power) of a light microscope is limited by the wavelength of light
• when objects are much smaller than wavelength of light, they are not detected due to being unable to interrupt the waves
• Electron microscopes have a higher resolution and magnification than light microscopes but smaller wavelengths
• The maximum resolution of a light microscope is 200nm
• The smallest wavelength of visible light is 400nm
• Points separated by a distance less than 200nm cannot be resolved by a light microscope therefore won’t be distinguished as separate.
• Specimens viewed by a light microscope can be living or dead
• Light microscopes look at whole cells not organelles due to resolution
• Compound microscopes are microscopes that use several lenses to obtain high magnification
• Electrons from electron microscope can be produced using hot wire, focused using electromagnets and detected using a phosphor screen or photographic film
• Magnification of electron microscope is better than light microscope due to higher frequency therefore shorter wavelength
• Electrons in microscopy can be produced using a hot wire, focused using electromagnets and detected using a phosphor screen or photographic film.
• Electron microscopes have higher magnification and resolution than light microscopes due to higher frequency therefore shorter wavelength
• specimens viewed using electron microscope have extensive fixation in plastic and viewed in a vacuum therefore must be dead
• Electron beam can damage the specimens and they must be stained with an electron dense chemical like osmium, lead or gold
• The two major types of electron microscopy are scanning electron microscopy and transmission electron microscopy
• In Scanning electron microscop, electrons are reflected off the surface of a specimen previously coated in heavy metals focused on a fluorescent screen to make an image.
• Larger, thicker structures can be seen under Scanning Electron Microscopy as electrons do not have to pass through the sample to form the image
• Scanning Electron Microscopy gives excellent 3D images but has a lower resolution than Transmission Electron Microscope
• Transmission Electron Microscope transmits electrons through thin specimen and displayed on a fluorescent screen and is used to obtain detailed internal structures of cells.
• Transmission Electron Microscopy has the best resolution
• Resolution of light microscope is 200nm while resolution of electron microscope is 0,5nm
• Preparation of sample distorts material in electron microscope
• Natural color of sample is maintained in light microscope
• Sample preparation is complex in electron microscopy
• In light microscopy, samples need to be thin to allow light to pass through
• Slide preparation methods include dry mount, wet mount, squamous slides and smear slides
• Dry mount is for solid specimens, coverslip placed on top and uses hair, pollen , dust , muscle tissue and plant tissue
• Wet mount is used for wet specimens suspended in water or immersion oil. Coverslip placed at an angle. Uses aquatic samples and other living organisms
• Squamous slide is used for soft specimens. Wet mount squashed between slide and coverslip. Uses root cells
• Smear slides used for body fluid specimens. Edge of slide used to smear sample creating thin, even coating. Uses blood smears
• The six Stages in preparation of slide samples are fixation, dehydration, clearing, embedding, sectioning, staining and mounting
• Fixation ensures material preserved in life like condition and does not distort the specimen. Dehydration is important for electron microscopy as water particles deflect electron beam thus blurring the image.