Chapter 1 Flashcards
Microscope Slide Preparation
-In order to observe cellular material in more detail, specimens can be prepared for viewing under a light microscope -Samples need to be thin enough to allow light to pass through -The type of preparation that is appropriate is dependent on the cellular material that needs to be viewed
Samples which need to be stained, why and how
as the cytosol and other cell structures may be transparent or difficult to distinguish -To stain a slide the sample needs to be first air-dried and then heated by passing it through a Bunsen burner flame – this will allow the sample to be fixed to the slide and to take up the stain -As with the type of preparation required, the type of stain used is dependent on what type of specimen is being used
Slide preparation methods table
common microscope stains and uses
Magnification Calculations
-Magnification is how many times bigger the image of a specimen observed is in comparison to the actual (real-life) size of the specimen -The magnification (M) of an object can be calculated if both the size of the image (I), and the actual size of the specimen (A), is known actual = image/magnification
converting units of measurements
Eyepiece Graticules & Stage Micrometers
-An eyepiece graticule and stage micrometer are used to measure the size of the object when viewed under a microscope -Each microscope can vary slightly so needs to be calibrated when used -The calibration is done with a stage micrometer, this is a slide with a very accurate scale in micrometres (µm), it is usually in 10 µm divisions, so 1 mm divided into 100 divisions -The eyepiece graticule is a disc placed in the eyepiece with 100 divisions, this has no scale -To know what the divisions equal at each magnification the eyepiece graticule is calibrated to the stage micrometer at each magnification 1 graticule division = number of micrometres ÷ number of graticule division
Magnification
Magnification is how many times bigger the image of a specimen observed is in compared to the actual (real-life) size of the specimen
A light microscope has two types of lens:
-An eyepiece lens, which often has a magnification of x10 -A series of (usually 3) objective lenses, each with a different magnification
To calculate the total magnification
eyepiece lens magnification x objective lens magnification = total magnification
Resolution
-Resolution is the ability to distinguish between two separate points -If two separate points cannot be resolved, they will be observed as one point -The resolution of a light microscope is limited by the wavelength of light -As light passes through the specimen, it will be diffracted -The longer the wavelength of light, the more it is diffracted and the more that this diffraction will overlap as the points get closer together -Electron microscopes have a much higher resolution and magnification than a light microscope as electrons have a much smaller wavelength than visible light ——This means that they can be much closer before the diffracted beams overlap
The concept of resolution
is why the phospholipid bilayer structure of the cell membrane cannot be observed under a light microscope -The width of the phospholipid bilayer is about 10nm -The maximum resolution of a light microscope is 200nm (half the smallest wavelength of visible light, 400nm) -Any points that are separated by a distance less than 200nm (such as the 10nm phospholipid bilayer) cannot be resolved by a light microscope and therefore will not be distinguishable as “separate”
Light microscopes
are used for specimens above 200 nm -Light microscopes shine light through the specimen, this light is then passed through an objective lens (which can be changed) and an eyepiece lens (x10) which magnify the specimen to give an image that can be seen by the naked eye -The specimens can be living (and therefore can be moving), or dead -Light microscopes are useful for looking at whole cells, small plant and animal organisms, tissues within organs such as in leaves or skin
Electron microscopes
both scanning and transmission, are used for specimens above 0.5 nm -Electron microscopes fire a beam of electrons at the specimen either a broad static beam (transmission) or a small beam that moves across the specimen (scanning) -The electrons are picked up by an electromagnetic lens which then shows the image -Due to the higher frequency of electron waves (a much shorter wavelength) compared to visible light, the magnification and resolution of an electron microscope is much better than a light microscope -Electron microscopes are useful for looking at organelles, viruses and DNA as well as looking at whole cells in more detail -Electron microscopy requires the specimen to be dead however this can provide a snapshot in time of what is occurring in a cell eg. DNA can be seen replicating and chromosome position within the stages of mitosis are visible
Light v electron microscope features
