Microscopes:
- Magnification: number of times larger an image appears, compared with the actual size
of the object.
- Magnification = magnificent power of the objective lens x magnifying power of eyepiece
lens
- Resolution: smallest distance between two objects, the clarity of the image.
Optical microscopes:
- Used in schools, colleges, hospitals and research labs because they are:
- cheap, easy, portable,
- able to use to study whole living organisms.
- Allow magnification up to x1500-2000
- Limited resolution
- Use visible light, wavelength between 400-700.
- Resolution of 200 nanometers.
1M = 1000MM
1MM = 1000MicroM
1MicroM = 1000nM
1nM = 1000pM
Laser scanning microscopes:
- Use laser light to scan an object, by computer, create a pixel image.
- High resolution
- Depth and can focus on structures - clearly observe whole living specimens.
- Used in medicine and biological research.
Electron microscopes
- Use a beam of electrons
- Higher resolutions that optical microscopes give highly magnified images
- Electrons fired from cathode focused by magnets.
Transmission electron
- Specimen is dehydrated and stained with metal salts
- Beam of electrons passes through, some pass through and are focused on the screen.
- Form a 2d black and white image - electron micrograph.
- Magnification of 2 million times.
Scanning electron
- Electrons do not pass through cause secondary electrons to bounce off.
- Gives a 3D image with magnification up to x200 000.
- Black and white image
- Specimen has to be placed in vacuum coated with fine film of metal.
, Both types of electron microscope:
- Large and expensive
- Need a great deal of skill and training to use.
- Have to be dead, as they are in a vacuum and stained by harmful metallic salts.
Use a logarithmic scale - goes up in steps (10 fold increase)
Making slides
- You can use an optical microscope to view live specimens like paramecium, smear
preparations of human blood, thin sections of animal, plant tissue.
Staining:
- Coloured chemicals that bind to molecules in or on the specimen, making the specimen
easy to see.
- Methylene blue is an all purpose stain - stains everything.
- Differential staining is when you use specific stains to bind to specific cell structures and
they can be easily identified.
Specific stains:
- Acetic orcein binds to DNA and stain chromosomes dark red
- Eosin stains cytoplasm, sudan red stains lipids
- Iodine in potassium iodide solution stains the cellulose in plant cell walls yellow and
starch granules blue black.
Preparing specimens:
Dehydrate, embedded in wax to prevent distortion during slice, make thin sections, stain and
mount.
Calculations:
In photomicrographs you get given the magnification
so you can then find the actual size.
Measure the photo in mm.
Convert that measurement to micrometers by
multiplying by 1000.
Divide by the magnification.
If you are told actual size you can calculate magnification.
Measure image size in mm, x 1000 to get to micrometers.
Divide by actual size.
Using graticules
- A micropiece eye piece can be fitted with a graticule.
- Its transparent with a small ruller in it.
- The eyepiece graticule is the large one on the specimen.
- The eyepiece scale has to be calibrated for each different objective lens.
- Magnification: number of times larger an image appears, compared with the actual size
of the object.
- Magnification = magnificent power of the objective lens x magnifying power of eyepiece
lens
- Resolution: smallest distance between two objects, the clarity of the image.
Optical microscopes:
- Used in schools, colleges, hospitals and research labs because they are:
- cheap, easy, portable,
- able to use to study whole living organisms.
- Allow magnification up to x1500-2000
- Limited resolution
- Use visible light, wavelength between 400-700.
- Resolution of 200 nanometers.
1M = 1000MM
1MM = 1000MicroM
1MicroM = 1000nM
1nM = 1000pM
Laser scanning microscopes:
- Use laser light to scan an object, by computer, create a pixel image.
- High resolution
- Depth and can focus on structures - clearly observe whole living specimens.
- Used in medicine and biological research.
Electron microscopes
- Use a beam of electrons
- Higher resolutions that optical microscopes give highly magnified images
- Electrons fired from cathode focused by magnets.
Transmission electron
- Specimen is dehydrated and stained with metal salts
- Beam of electrons passes through, some pass through and are focused on the screen.
- Form a 2d black and white image - electron micrograph.
- Magnification of 2 million times.
Scanning electron
- Electrons do not pass through cause secondary electrons to bounce off.
- Gives a 3D image with magnification up to x200 000.
- Black and white image
- Specimen has to be placed in vacuum coated with fine film of metal.
, Both types of electron microscope:
- Large and expensive
- Need a great deal of skill and training to use.
- Have to be dead, as they are in a vacuum and stained by harmful metallic salts.
Use a logarithmic scale - goes up in steps (10 fold increase)
Making slides
- You can use an optical microscope to view live specimens like paramecium, smear
preparations of human blood, thin sections of animal, plant tissue.
Staining:
- Coloured chemicals that bind to molecules in or on the specimen, making the specimen
easy to see.
- Methylene blue is an all purpose stain - stains everything.
- Differential staining is when you use specific stains to bind to specific cell structures and
they can be easily identified.
Specific stains:
- Acetic orcein binds to DNA and stain chromosomes dark red
- Eosin stains cytoplasm, sudan red stains lipids
- Iodine in potassium iodide solution stains the cellulose in plant cell walls yellow and
starch granules blue black.
Preparing specimens:
Dehydrate, embedded in wax to prevent distortion during slice, make thin sections, stain and
mount.
Calculations:
In photomicrographs you get given the magnification
so you can then find the actual size.
Measure the photo in mm.
Convert that measurement to micrometers by
multiplying by 1000.
Divide by the magnification.
If you are told actual size you can calculate magnification.
Measure image size in mm, x 1000 to get to micrometers.
Divide by actual size.
Using graticules
- A micropiece eye piece can be fitted with a graticule.
- Its transparent with a small ruller in it.
- The eyepiece graticule is the large one on the specimen.
- The eyepiece scale has to be calibrated for each different objective lens.
