Module 2: section 1 - Microscopes Flashcards
What is sample preparation?
When samples and specimens are prepared for examination through light microscopy
What are the 4 methods that can be used for sample preparation?
Dry mount
Wet mount
Squash slides
Smear slides
Dry mount dis/ad?
Quick & easy
Specimen is dried out so dies
Wet mount ad?
Prevents dehydration and distortion
Can view live specimen
Squash slides ad?
Can view all cell content at once
Dry mount method?
Solid specimen is cut into slices through ‘sectioning’
Once prepared, it is placed on a slide and covered with a cover slip
Wet mount method?
Specimens are suspended in liquid (water or immersion oil)
Then, the cover slip is placed at an angle
Squash slides method?
A wet mount is prepared
Then, a lens tissue is used to press down cover slip to slide
Smear slides method?
The edge of the slide is used to smear sample into a thin coat
Then the cover slip is placed on top
What are 4 steps to producing a slide?
Fixing
Sectioning
Staining
Mounting
4 types of stains?
Basic stains
Negative stains
Gram stain
Acid-fast stain
How to stain a slide?
Air dry slide’s stain
Heat-fix by passing thru a flame
Specimen will adhere & absorb stain
What do stains do?
Increase contrast (of cell components)
How do negative stains work?
Repel negative material in cells and sit around cell, making components stand out
What is the scale on a stage meter?
Micrometers
How do you calculate magnification?
image size ÷ actual size
How do you get from millimeters to micrometers?
×1000
How do you get from micrometers to nanometers?
×1000
What if the function of an eyepiece graticule?
To find the calibration factor of the substance/microorganism being observed in the microscope
What is resolution?
The ability to see individual objects separated in a microscope
What is diffraction?
Spread of light
How + why is resolution limited?
By diffraction since the spread of light varies, causing overlapping so objects cannot be separated (blur).
Why do electron microscopes have a higher resolution?
Because electrons have a smaller wavelength than visible light. This allows them not to overlap when close together
What is magnification
The process of enlarging an object in appearance
What are the types of microscopes
Optical light microscopes, transmission electron microscopes, scanning electron microscopes and laser scanning confocal microscopes
How do optical light microscopes work
Visible light passes and is bent through the lens system to enable the viewer to see the specimens
Advantages for optical light microscopes
-specimen can be alive
- small and lightweight
- high levels of observation quality
Disadvantages for optical light microscopes
-Low resolution (around 0.2 micrometers)
- lower magnification
-can’t be used to observe smaller organelles
How do transmission electron microscopes work
Use electromagnets to focus a beam of electrons through the specimen
What are the advantages of transmission electron microscopes
High magnification, high resolution images so sub cellular organelles can be seen
What are the disadvantages of transmission electron microscopes
- no colour
- only thin specimens can be observed
- only observe dead specimens
How do scanning electron microscopes work
Scan a beam of electrons across the specimen, this beam bounces off the surface of the specimen and the electrons are detected forming an image
Advantages of scanning electron microscopes
Can be used on thick or 3D specimens, high resolution, external 3D structures can be observed
Disadvantages of scanning electron microscopes
Lower resolution than using transmission electron microscopes, no colour, only observed specimen
How do laser scanning confocal microscopes work
Stain cells with florescent dyes, thick sections of tissues or small living organisms are scanned with the laser beam
Advantages of laser scanning confocal microscopes
Thick or 3D specimen observed, external 3D structures observed,high resolution
Disadvantages of laser scanning confocal microscopes
Slow process takes a long time to obtain an image, laser can cause photo damage to cells
Explain how to measure the diameter of the nucleus of one of the white blood cells, when observing the cells
through a light microscope.
use eyepiece graticule ✓
calibrate graticule, using stage micrometer / detail of
calibration / calculate the length of one epu ✓
measure the diameter of the nucleus in, epu / graticule
units ✓
take repeat measurements and calculate a mean
diameter (in epu) ✓
use calibrated epu to calculate diameter (of nucleus) (in
μm) / described ✓
How do you use a light microscope
1)Clip the slide with the specimen you want to observe onto the stage
2) select the lowest power objective lens and use the coarse adjustment knob to move the stage upwards
3)Then look through the eyepiece and use the coarse adjustment knob to move the stage downwards until it is roughly focused
4)Then use the fine adjustment , till you get a clear image of what is happening
