Microscopy Flashcards
How does a light microscope work?
There are two lenses;
objective lens- placed near the specimen
eyepiece lens- the specimen is viewed through this
The objective lens provides a magnified image, which is magnified again by the eyepiece lens - this allows for a much higher magnification
Illumination is provided by light underneath the sample
Describe a dry mount
Solid samples are cut into thin slices (sectioning)
The specimen is placed in the middle of the slide and a cover slip is placed on top
Describe a wet mount
Specimens are suspended in a liquid. A cover slip is placed on top, at an angle (to prevent air bubbles forming)
Describe a squash slide
A wet mount is prepared then two slides are used to press down the cover slip
Describe a smear slide
The edge of a slide is used to smear a sample, to create a thin coating on the slide. A cover slip is then placed on the sample.
Why is staining useful?
Stains increase contrast as different components of a cell take up stains to different degrees. Increase in contrast- components become more visible and easy to view.
No staining- low contrast- cells do not absorb a lot of light- harder to view components of a cell
What is negative staining?
Negatively charged dyes are repelled from the negatively charged cytosol of the cells- therefore they stay out of the cells and leave them unstained. This makes the cell stand out against the stained background.
What is differential staining?
Used to distinguish between two types of organisms/ different organelles which would otherwise be hard to identify.
What is a gram stain technique?
Separates bacteria into two groups- gram positive and gram negative.
Crystal violet, then iodine (fixes the dye)
Gram positive retain the crystal violet- appearing purple
Gram negative lose the crystal violet as they have thinner cell walls- they are stained with a counterstain and appear red
What is an acid fast technique?
Differentiate species of myobacterium from other bacteria
What is magnification?
How much bigger the image is than the actual specimen
What is resolution?
The ability to distinguish between two points that are close together.
What is the formula for magnification?
Magnification=image size/real size
How do you calibrate a light microscope?
Line up the stage micrometer with the eyepiece graticule
Why is the resolution better in an electron microscope?
More detail of cell ultrastructure can be seen because electrons have a shorter wavelength than light
What are the limitations of electron microscopy?
- Expensive
- Specimens can be damaged with the electron beam
Transmission electron microscopes
A beam of electrons is transmitted through a specimen and focused to produce an image.
Best resolution.
Scanning electron microscopes
A beam of electrons is sent across the surface of a specimen and reflected electrons are collected. Resolution is not as good as a TEM but 3D images are produced.
What are the differences between light and electron microscopes?
Electron microscope:
- is expensive to buy/operate
- is large/needs to be installed
- has complex sample preparation (distorts/ creates artefacts)
- requires a vacuum
- black and white image produced
- specimen must be dead
What is an artefact?
Visible structural detail caused by processing the specimen- not a feature of the specimen. Eg air bubble trapped when you put the cover slip on the slide.
What is a laser scanning confocal microscope?
It moves a single spot of focused light across a specimen. This causes fluorescence from the components with a dye. The emitted light from the fluorescence is filtered through a pinhole aperture
Why does all light not pass through the aperture and be detected?
Light emitted from other parts of the specimen would cause blurring and reduce the resolution
Why is a laser used in LSCF?
To obtain higher intensities and improve the illumination
Why are fluorescent tags used in LSCF?
To target specific features and get more precision.
Advantage OF LSCF?
Can be used on 3D specimen
High resolution
What is atomic force microscopy?
Feels the surface of a specimen with a mechanical probe to generate a 3D image of its surface.
Adv- specimen can be viewed in normal cell conditions, even living (fixation/staining not required)
High resolution- atomic level
