Microscopes Flashcards
What is a microscope?
An instrument employing lenses to produce a magnified image and fine detail of objects too small to see with the naked eye
What is magnification?
How many times the size of an image is larger than the object itself (it is indicated by a number)
What is resolution?
The degree to which it is possible to distinguish between two separate objects which are close to each other (it is indicated by a length)
What are the two categories of microscopes?
Optical and electron microscopes
What are the key points about the resolution of a optical microscope?
- Resolution of an object/feature in a microscope is limited to half the wavelength of the wave employed
- Optical microscopes can resolve objects up to 200nm or 0.2µm
What is the range of wavelengths used by optical microscopes?
450-600nm
What is the limit of magnification of an optical microscope?
x1500-2000
How does an optical microscope work?
- Objective lens produces a magnified image which is magnified again by the eyepiece lens => object is magnified twice
- Light is shon through the specimen/object to be viewed
How does an electron microscope work?
- A beam of electrons with a wavelength of less than 1nm is used to illuminate the specimen/object
- The lenses are magnets
How do electrons in electron microscopes produce an image?
Electrons produce an image when focused on a fluorescent screen
What is the limit of magnification and resolution in an electron microscope?
- The limit of magnification is x500,000
- The limit of resolution is 0.2nm
What are the two types of electron microscopes?
- Transmission electron microscopes (TEM)
- Scanning electron microscopes (SEM)
How does a TEM work?
- Tissue/cell is inflitrated with plastic resin (=> very hard) and is cut into very thin slices using a diamon knife
- A beam of electrons is passed through exceedingly thin slices of the specimen/material and produces an image on a flourescent screen or photographic film
- This allows for the interior of the cells to be seen
How does a SEM work?
- The specimen is shadowed with a layer of heavy metal and covered with carbon
- It is scanned by a fine electron beam which is scattered from the surface of a specimen and the transmitted to a detector
- This allows the surface of specimens to be seen
- Except when freeze-fracture (the cell is frozen in liquid nitrogen at -70 degrees celcius) is being used where the inside of the cell is exposed
What is the range of magnification and resolution of a TEM?
Magnification: x500,000 - 2,000,000
Resolution: 0.2 - 0.5nm
What is the range of magnification and resolution of a SEM?
Magnification: x100,000 - 500,000
Resolution: 3 - 10nm
What is the difference between a TEM and SEM?
TEM:
- Higher resolution than SEM
- Higher magnifcation than SEM
- Colour of image is black and white
- Can view interior of cells
- 2D image formed
SEM:
- Lower resolution than TEM
- Lower magnifcation than TEM
- Colour of image is black and white
- Can view exterior of cells (=> can only view interior through freeze fracturing)
- 3D image formed
What are the advantages of a light microscope?
- Small and portable
- Natural colour of the material can be observed
- Cheap to purchase
- Material rarely disorted by preparation
- Living material may be viewed
- Preparation of material is relatively quick and simple, requires only a little expertise
- Unaffected by magnetic fields
- Doesn’t require a vacuum
- Cheap to operate
What are the disadvantages of a light microscope?
- The depth of field is restricted
- Magnifies objects up to 1500x
- Can resolve objects 0.2µm apart
What are the advantages of the electron microscope?
- Possible to see a greater depth of field (SEM 3D image)
- Magnifies objects up to 500,000x
- Can resolve objects up to 0.001µm apart
What are the disadvantages of the electron microscope?
- Very large and must be operated in special rooms
- All images in black and white
- Expensive to purchase
- Preparation of material may distort it
- A high vacuum is required
- Living material can’t be viewed
- Preparation of material is length, requires a considerable expertise ad complex equipment
- Affected by magnetic fields
- Expensive to operate: requires up to 100,000V to produce an electron beam
- Can produce artifacts
What are the different ways that samples and specimens can be prepared?
- Dry mount
- Wet mount
- Squash slides
- Smear slides
How is a dry mount prepared?
- Solid specimens are viewed whole or cut into very thin slices with a sharp blade, this is called sectioning
- The specimen is placed on the centre of the slide and a cover slip is placed over the sample
- A cover slip is needed to keep the specimen flat and to maintain the same focal length throughout
How is a wet mount prepared?
- Specimen is placed on the slide
- Drops of a stain/water is put on specimen
- A cover slip is lowered at an angle using a mounted needle to minimise trapping air bubbles
How is a smear slide prepared?
- A spreader slide is pulled backwards across another slide and used to smear the sample
- The spreader slide is pushed forwards in one smooth motion with constant downward pressure
- This creates a thin, even coating on the slide
- A cover slip is placed over the sample
How is a squash slide prepared?
- A wet mount is first prepared, then a lens tissue is used to gently press down the cover slip
- Depending on the material, potential damage to a cover slip can be avoided by squashing the sample between two microscope slides
What are the stages of preparing a permanent slide?
Fixing: chemicals like formaldehyde are used to preserve specimens in as near-natural a state as possible with minimum distortion
Dehydration: specimens are dehydrated (water I removed) with alcohols (to kill bacteria and prevent any from growing)
Clearing: removing the dehydration alcohol to leave specimen transparent
Embedding: placing it in a mould with wax or resin to form a hard block
Sectioning: using a microtome to produce thin slices to form a block
Staining: treating the thin slices with chemical agents (stain) to “dye” different structures different colours
Mounting: the specimens are then secured to a microscope slide and a cover slip placed on top
What are the advantages of staining?
- Provides contrast between components of cells e.g. organelles
- Provides contrast between a structure (e.g. cell) and its background
- Allows different components (e.g. organelles) of cells to be identified
Why does a stain give a different colour with different cellular structures?
Cellular structures are composed of different molecules which interact differently with the specific stain molecules e.g. a positively charged stain will interact with a negatively charged molecule such as DNA
What is differential staining?
- This is staining that uses more than one chemical stain
- This makes differences between cells or between different structures within cells more visible
What is the gram stain technique used for?
It is used to separate bacteria into two groups, Gram-positive bacteria and Gram-negative bacteria
What are the steps of the Gram stain technique?
- Crystal violet is first applied to a bacterial specimen on a slide
- Iodine is added to fix the dye
- The slide is then washed with alcohol
- Gram-positive bacteria retains the crystal violet stain and will appear blue or purple under a microscope
- Gram-negative bacteria have a thinner cell wall and so therefore lose the stain
- Gram-negative bacteria is then stained with Safranin dye which is called a counter stain
- These bacteria will appear red
What is an artificat?
A visible structural detail that is due to the processing of the specimen and not a natural feature of the specimen
What are some examples of positively charged dyes?
Crystal violet and methylene blue
What are some examples of negatively charged dyes?
Nigrosin and congo red
