Microscopy Flashcards
What is the equation used with microscopes?
image size = magnification x actual size
What is magnification?
Magnification is the ratio of an object’s size when viewed through a microscope to its actual size
It’s a way to make small objects appear larger
What is resolution?
The minimum distance between two objects where they can still be seen as two separate objects
What is a photomicrograph?
A photograph of a microscopic object, taken with the aid of a microscope
What are artifacts?
A result that doesn’t represent the true biological material or function
Light Microscope
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What are light microscopes?
The most commonly used microscope, that used light rays to magnify the specimen
How do light microscopes work?
- Lenses focus rays of light and magnify the view of the thin slice of the specimen
- Different structures absorb different amounts and wavelengths of light
- Reflected light is transmitted to the observer via the objective lens and eyepiece
What are the lenses used within a light microscope?
- Objective lens
- Eyepiece lens
Advantages of light microscopes
- Relatively cheap
- Easy to use
- Magnify living organisms
- Colour image
Disadvantages of light microscopes
- Low magnification
- Low resolution
- 2D image
Electron Microscope
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When was the electron microscope invented?
1930’s
How do electron microscopes work?
A beam of electrons focused by an electromagnet that magnifies the specimen
Advantages of an electron microscope
- High magnification
- High resolution
- Can see smaller details
- 3D images
Disadvantages of an electron microscope
- Cant use live specimens
- No colour
- Expensive
Why cant electron microscopes have live specimens?
An air vacuum is required as air particles would interfere with the beam of electrons
Differences between light and electron microscopes
- EM more expensive
- EM higher resolution and magnification
- LM colour results
- LM live specimens
What are the different types of electron microscopes?
- Transmission Electron Microscope (TEM)
- Scanning Electron Microscope (SEM)
How does the TEM work?
- Pass a high energy beam of electrons through a thin slice if the specimen
- More dense structures appear darker since they absorb more energy
- Less dense structure appear lighter since the electrons pass through the area
- Focus image onto fluorescent screen or photogenic plate using magnetic lenses
What is the TEM usually used to study?
Good for the inside of the cells - how components are structured
What is the resolving power of the TEM?
0.1nm
How can we build a 3D image using a TEM?
- When using a TEM, the specimen has to be really thin to allow electrons to penetrate it therefore a 2d image is produced
- To obtain a 3D image you would have to take a series of photomicrographs and then combine these together
What are the limitations of TEM’s?
- Requires a vacuum - can not show living organisms
- Heavily stained areas wont show - stain required
- No colour images
- Extensive preparation may introduce artefacts - time consuming
- 2D image
- Specimen must be very thin
What are the advantages of TEM’s?
- Electrons have a shorter wavelength than light - high resolution, so ultrastructure visible
- High magnification (x 500000)
How does the SEM work?
- Focuses a beam onto the specimen’s surface using electromagnetic lenses
- Reflected electrons (off the surface of structures) hit a collecting device and are amplified to produce an image on a photographic plate
What is the SEM usually used to study?
Used to study the surface of cells
What is the resolving power of the SEM?
20nm
How can we build a 3D image using a SEM?
A 3D image can be built on a computer when the pattern of the scattered electrons is analysed and secondary electrons are produced
What are the limitation of SEM’s?
- Requires a vacuum - can’t show living structures
- No colour images
- Only shows outer surface
- Has a lower resolution that TEM
*Specimen has to be coated in metal - more risk of artifacts - Can distort the sample
What are the advantages of SEM’s
- 3D image
- Specimen doesn’t need to be sliced as thin
- Good for showing outer surface
What is an eye piece graticule?
- It is a glass disc that is placed in the eyepiece of a microscope
- Has a scale etched into it
- Scale is typically 10mm long and divided into 100 subdivisions
- The scale is visible when looking down the eyepiece
- The scale is not used to directly measure the object size since the objective lenses will magnify it to different degrees
- The eyepiece graticule remains constant no matter the magnification cells are viewed at
What is a stage micrometer?
- A microscope slide that has a scale etched into it
- Used to calibrate the eye piece graticule for a particular objective lens
- Scale is typically 2mm long, smallest sub-divisions are 0.1mm (10um)
How do you calibrate the eye piece graticule and stage micrometer together?
The eyepiece graticule has regular divisions that need to be calibrated for each magnification
You place the stage micrometer on the microscope stage, focus on it clearly, then align the divisions on the eyepiece graticule with the divisions on the stage micrometer, counting how many eyepiece divisions correspond to a known distance on the stage micrometer to determine the actual size represented by each eyepiece division
How do you use an eyepiece graticule and stage micrometer to measure the size of a structure?
- Place micrometer on stage to calibrate eyepiece graticule
- Line up scales on graticule and micrometer. Count how many graticule divisions are in 100um on the micrometer
- Length of 1 eyepiece division = 100um/number of divisions
- Use calibrated values to calculate length of structures
What id the equation to find out 1 division?
length on stage micrometer/number of lines on eyepiece graticule
What is a cover slip?
A square of clear glass or plastic that protects the specimen
What is a temporary mount?
Where the specimen is suspended in a drop liquid on the slide
How do you set up a microscope?
- Place the microscope on a stable surface
- Select the lowest power objective lens
- Position your prepared slide on the stage, adjust the light source to an appropriate level
- Look through the eyepiece, and use the coarse focus knob to bring the specimen into view
- Then fine-tune with the fine focus knob until the image is clear
What power lens should you begin with?
Always start with the lowest power objective lens and gradually increase magnification as needed
What is the first step in preparing a microscope side using a temporary mount?
Begin by pipetting a small drop of water onto the centre of the slide
What is the second step in preparing a microscope side using a temporary mount?
You will then need to place a thin section of your specimen on top of the water drop through using tweezers
What is the third step in preparing a microscope side using a temporary mount?
Add a drop of stain. stains are used to highlight objects in a cell
What is the fouth step in preparing a microscope side using a temporary mount?
- Add a cover slip
- Do this by standing the slide upright on the slide, next to the water droplet
- Carefully tilt with a mounted needle and lower so specimen is covered at 45°
- Ensure no air bubbles are seen they’ll obstruct view of specimen
