microscopes and microscopy skills

microscopes

are scientific instruments that are used to visualise objects that are too small to see with the naked eye

light microscopes do?

Light microscopes can be used to view living specimens in their natural colours 

• These microscopes use glass lenses to bend light in order to magnify images (extent of magnification is determined by the lenses used)

how is the cellular sub-structures improved with the use of fluorescent labelling

• Synthetic dyes can be used to bind particular cellular compounds in order to resolve specific structures (e.g. DAPI is a fluorescent dye that stains DNA)

• Immunofluorescence staining uses antibodies that are conjugated to fluorescent probes to specifically target a cellular component of choice

llght microscope structure

electron microscope

Electron microscopes can generate images at a much higher magnification and resolution, however cannot view living specimens in natural colour

• These microscopes use electromagnets to focus electrons and produce monochromatic images (to which false colour may be applied)

transmission electron microscopes

pass electrons through a specimen to generate a cross-section image

scanning electron microscopes

scatter electrons over a surface to differentiate depth and map in 3D

a cryogenic electron microscope and what does it do?

involves freezing samples prior to viewing in order to generate images of a comparable standard to X-ray crystallography

• This allows for the determination of molecular structures at near atomic resolution without requiring the crystallisation of the specimen

• If the frozen specimen is cracked along a plane via freeze fracturing, then internal cellular structures can be studied

• Freeze fracturing was used to demonstrate the presence of integral membrane proteins within the plasma membrane

electron microscope overview

what kind of lense does a light microscope use

Most light microscopes include both an ocular lens (~10×) and objective lens (~10×; 40×; 100×)

what conventions should be followed when using a light microscope

• The image should initially be resolved at the lowest magnification using the coarse focus mechanism

• Higher magnifications are then obtained by changing the objective lens (via the revolving nosepiece) and making fine focus adjustments

• The total magnification of the image is calculated by multiplying the magnification of both lenses (ocular and objective) together

• If the eyepiece has a measurement scale on its surface (eyepiece graticule), this can be used to determine sizes of biological structures

what are the components of a light microscope

1. eyepiece

2. focus knobs

3. nosepiece

4. stage

what is the formula for magnification

• Magnification = Image size (with ruler) ÷ Actual size (according to scale bar)

metric conversion table