Microscopy Flashcards
Describe what a microscope does
A microscope is an instrument which enables you to magnify an object thousands of times. It enables us to see unicellular objects.
Name 4 different types of microscope
- light microscope
- scanning electron microscope
- transmission electron microscope
- laser scanning confocal microscope
Main components of a light microscope (should be able to label one in the exam)
eye piece lens nose piece coarse focusing knob fine focusing knob stage light bulb
SEM stands for
scanning electron microscope
TEM stands for
transmission electron microscope
How does a SEM work?
A beam of electrons (wavelength < 1nm) is sent across the surface of a specimen and the reflected electrons are collected.
- resolution not as good as TEM (3-10nm)
- produces 3D images
How does a TEM work?
A beam of electrons (wavelength < 1nm) is transmitted through a specimen and focused to produce an image
- very high resolution (0.5nm)
- produces 2D images
How does a laser scanning confocal microscope work?
A higher intensity of light is used to illuminate a specimen that has been treated with a fluorescent dye.
A single spot of focused light is moved across a specimen, causing fluorescence from the components labelled with a dye. The light emitted from the specimen is filtered through a pinhole aperture. Only light radiated close to the focal plane is detected (unwanted radiation does not pass through the pinhole and so is not detected).
What is the definition of fluorescence?
The absorption and re-radiation of light
Why is a laser used instead of a light in laser scanning confocal microscopes? (1)
To get higher light intensities which improve the illumination
Explain the purpose of the pinhole aperture in confocal microscopy (3)
- only light radiated very close to the focal plane is detected
- unwanted radiation does not pass through the pinhole so is not detected
- this reduces blurring and improves the resolution of the image
Why can’t confocal microscopy be used for deep tissue imaging? (1)
The light penetration of the sample is limited as it is non invasive
Resolving power of LM, SEM and TEM
LM: 200nm (low!!)
SEM: 3-10nm
TEM: 0.1-0.5nm
What is the maximum useful magnification of LM, SEM and TEM?
LM: x1500
SEM: x500,000 - x2,000,000
TEM: x100,000 - x500,000
In light microscopy specimens are…
living or dead
In electron microscopy specimens are…
dead
Is sample preparation complex and does it lead to distortion in LMs and EMs?
LM: simple, does not usually cause distortion
EM: complex, often distorts material leading to the creation of artefacts
Type of specimen stain used in LMs and EMs
LM: simple stains eg. iodine. Natural colouring is fine.
EM: heavy metal salts
Explain how to use a light microscope to view a specimen at high and low powers (7 steps)
- Rotate nosepiece so that lowest power objective lens is in place
- Place specimen to be viewed onto stage and position so that specimen is in the centre of the field of view. Fasten with stage clips.
- Set up lamp to direct light beam through specimen on stage
- Move the coarse focusing knob so that the lower power objective lens is as close as possible to the stage
- Use eyepiece lens and focussing knobs to bring specimen into clear view by moving stage away from the lenses
- Rotate nosepiece to bring the medium power objective into position and adjust fine focus to view specimen clearly
- Repeat for higher power objective lenses
Describe how to produce a temporary wet mount of living tissue
Place sample on a microscope slide
Add a drop of water
Cover with a coverslip placed on at an angle
Dab away excess water
Explain why slide preparations need to be thin
So that light can pass through it. This means it can be seen by a light microscope
Explain how to use a stage micrometer to work out the distance represented by the small divisions in an eyepiece graticule under 3 different objective lenses
.
Resolution definition
The ability to see two objects (that are close together) as seperate objects
Why are samples stained using a light microscope?
Dyes enable us to distinguish between organelles by increasing the contrast between them. Chemicals can bind to structures to allow them to be seen.
Equation for calculating magnification
Magnification = Image size/ Actual size
Magnification definition
The number of times bigger an object appears compared to its original/ real size
How are samples prepared for electron microscopes?
- in vacuum to ensure electrons travel in straight lines
- fixation using chemicals/freezing
- staining with heavy metals
TEM -> set in resin
SEM -> fractured inside and coated with heavy metals
High resolution allows us to see the … of the cell
ultrastructure
How many micrometres in a millimetre?
1mm = 1000um
diameter of a lysosome (um)
0.5um
length of a flagellum (um)
> 10um
diameter of a centriole (um)
0.2um
length of a cilia (um)
<10um
length of a chloroplast (um)
4-10um
diameter of a nucleus (um)
3-20um
width of a plasma membrane (nm)
5-7nm
width of a plant cell wall (um)
0.1um
length of a mitochondrion (um)
2-5um
Rules for biological drawings
- fill half the space provided
- sharp pencil to make sharp lines
- thin, single, unbroken lines
- no shading or colouring
- label lines drawn in pencil with no arrow heads
- annotate in pencil
- include scale line and magnification
ratio of mm to um and nm
1mm : 1000um : 1,000,000nm
Order of size of organelles
nucleus chloroplast mitochondrion lysosome centriole ribosome
Explain the advantages and disadvantages of using a TEM to study cells (6)
- TEM uses beam of electrons
- which has a short wavelength
- Which allows a greater resolution/ more detail to be seen/ greater useful magnification
- Electrons scattered (by molecules in air)
- vacuum established
Limitations
- Cannot examine living specimens
- Many procedures used in preparing specimens eg. fixing/staining/sectioning
- May result in artifacts
