microscopy Flashcards
Magnification =
Image size
——-———
Actual size
Magnification
How enlarged an image is
Resolution
How well you can distinguish two separate points to see in detail
Resolution: light-TEM-SEM
200nm-0.2nm-2nm
which microscope has the best resolution
TEM
Magnification light-TEM-SEM
1500-1,000,000-500,000
How does a light/optical microscope work?
Light passes from the condenser lens and through the specimen where certain wavelengths are filtered to produce an image. then light passes through the objective lens then eyepiece where magnification can be altered to be seen
What are the advantages of light microscopes?
They are cheap.
You can observe living organisms.
You can produce a coloured or stained image.
It is easy to use.
It is small and portable.
What are the advantages of electron microscopes?
More detail is seen because electrons have a smaller wavelength than light
They have a high magnification.
They have a higher resolution.
How does a transmission electron microscope TEM work?
Uses electromagnets to force a beam of electrons transmitted through the specimen to produce 2-D images. Denser parts of the specimen absorb more electrons which makes them look darker.
How does scanning electron microscope SEM work?
It Scans a beam of electrons across the surface of the specimen which bounces off it and are gathered in a cathode ray tube to form an image showing the surface of the specimen so it can be 3-D and has to be sliced to show the inside
Why can electron microscopes not observe living organisms?
The Microscope has to have a vacuum to ensure the electron beams travel in straight lines so the organism can’t survive
What are light microscopes used to observe?
Whole cells and tissues
What are electron microscopes used to observe?
Organelles
How can drawings be improved?
No shading
No arrowheads for labels
Add a scale and magnification
Add a title
How to make a stained sample for a light microscope
Place stain at the edge of sample
Lower covers slip at an angle using a mounted needle
Use blotting paper to remove excess stain
Two different stains are used while making a slide to observe through a light microscope one stains the cytoplasm, one stains the nucleus, Why are they both needed?
Stain is needed for the cytoplasm for it to be visible against the background and a stain is needed for the nucleus to differentiate it from the cytoplasm
How to view a sample through a light microscope
- Select the lowest powered objective lens
- Use the course adjustment knob to bring the stage up to just below the objective lens
- Look down the eye piece and use the course adjustment knobs to move the stage down until image is roughly in focus
- Adjust the focus with the fine adjustment knob until there is a clear image of the slide
- If greater magnification was required, refocused the microscope using a higher powered objective lens
What microscope would you use to observe the inside of a mitochondria and why 2 mark
An electron microscope because a high resolution and magnification is needed to study internal structures of mitochondria
How to put a stain on a wet mount side
Put a drop of stain next to one edge of the covers sip and a piece of paper towel next to the opposite edge. The paper towel will draw the stain under the slip across the specimen.
Safety precautions
Wear goggles/ gloves
take care not to break the glass
take care of the sharp tools
Identify the microscope used to produce this image (blank and white and 2D)
Tem/ transmission electron microscope
What is the difference between the images of light microscope and electron microscope
It has higher resolution so individual organelles can be observed better
How can you tell we used a TEM microscope
It has a higher resolution
More organelles can be seen in more detail (name organelles)
How to measure the diameter of the nucleus of a cell using a light microscope
- Use the stage micrometre to calibrate eyepiece graticule by aligning two scales to find the length of one division
- Measure the diameter using graticule units, take repeats, find a mean
- And Multiply that by the value of each division to calculate actual diameter
How can improvements be made to making a sample slide
- Use a sharp blade to cut sample thin so individual cells can be observed
- Choose a thinly sliced sample so maximum light penetrates sample
- Squash slide to see individual cells easily
Benefits of using stains in microscopy
- To identify different cell types
- To differentiate/view organelles easier
- For Higher contrast between cytoplasm and the background for a clearer image
Examples of stains
Methylene blue
Giesma stain
Haematoxylin
Eosin
What is a stage micrometre?
A microscope slide with an accurate scale that is used to work out the value of the divisions on the eye piece graticule at particular magnification
What are scanning electron microscopes used to observe?
The surface of cells
Coloured or not: light TEM SEM
Coloured, not, not
What would you use to make an accurate measurement of the length of something 200nm
Light microscope - eyepiece gratitude
Stage micrometre
Surface area and volume of the sphere
Sa - 4 Pie r2
Vol 4/3 pie r3
How can you tell this image was taken by a SEM
Black and white
3d/ shows a surface view
Has a pretty good resolution
Compared TEM and SEM
TEM has a resolution of 0.2nm and shows the image of cell interior organelles
SEM has a lower resolution of 20nm and shows a 3-D image of cell surface
