More Microscopy Flashcards
What’s better, a light microscope or an electron microscope?
Electron microscope
Why do electron microscopes have a better resolution?
Because a beam of electrons with a wavelength of less than 1nm is used to illuminate the specimen. More detail of the cells ultrastructure can be viewed because electrons have smaller wavelengths than light waves. This means that individual beams can be much closer before they overlap (so less diffraction blurs the image).
How many times more can electron microscopes magnify specimen so that they still have a clear resolution?
x500,000
What are artefacts?
Structures that are produced due to the preparation process. They appear in both light and electron microscopes.
When preparing a specimen for electron microscopy, changes in the ultrastructure of cells are inevitable during the process that the samples must undergo.They are seen as the loss of continuity in membranes, distortion of organelles and empty spaces in the cytoplasm of cells.
What are the two types of electron microscopes?
- Transmission electron microscopes (TEM)
* Scanning electron microscope (SEM)
What is a scanning electron microscope (SEM)?
As the electron beam traces over the object, it interacts with the surface of the object, dislodging secondary electrons from the surface of the specimen in unique patterns. A secondary electron detector attracts those scattered electrons and, depending on the number of electrons that reach the detector, registers different levels of brightness on a monitor. Additional sensors detect backscattered electrons (electrons that reflect off the specimen’s surface) and X-rays (emitted from beneath the specimen’s surface). Dot by dot, row by row, an image of the original object is scanned onto a monitor for viewing (hence the “scanning” part of the machine’s name).
Why do electron microscopes contain vacuums?
To ensure the electron beams travel in straight lines.
How do you prepare a sample for a transmission electron microscope?
- First is fixation, were using chemicals or freezing and then staining with heavy metals and dehydration with solvents.
- Next, the sample is set in resin and may be stained again.
How do you prepare a sample for a scanning electron microscope microscope?
- First is fixation, were using chemicals or freezing and then staining with heavy metals and dehydration with solvents.
- Next, the sample is fractured (broken) to expose the inside and will then need to be coated with heavy metals.
What is an example of identifying artefacts?
‘Mesosome’ was the name for invaginations (inward foldings) of cell membranes that were observed using an electron microscope after bacterial specimen had been chemically fixed. They were thought to be a normal structure, or organelle, found within prokaryotes. However, when specimens were fixed by the more recently developed, non-chemical technique called croyfixation, the mesosomes were no longer visible. It is now thought that the mesosomes observed were actually artefacts.
How does a conventional optical microscope work?
They use visible light to illuminate specimens and a lens to produce a magnified image. In fluorescent microscopes a higher light intensity is used to illuminate a specimen that has been treated with a flourescent dye. Fluorescence is the absorption and re-radiation of light. Light of a longer wavelength and lower energy is emitted and used to produce a magnified image.
How does a laser scanning confocal light microscope work?
It moves a single spot of focused light across a specimen (called point illumination). This causes florescence from the components labelled with a ‘dye’. The emitted light from the specimen is filtered through a pinhole aperture. Only light radiated from very close to focal pane is detected.
Light emitted from other parts of the specimen would reduce the resolution and cause blurring. This unwanted radiation does not pass through the pinhole and is not detected. A laser is used instead of light to get higher intensities, which improves the illumination.
As very thin sections of specimen are examined and light from elsewhere is removed, very high resolution images can be obtained.
The spot illuminating the specimen is moved across the specimen and a two dimensional image is produced. A three dimensional image can be produced by creating images at different focal planes.
What is laser scanning confocal microscopy used for?
- The diagnoses of diseases in the eye
- endoscopic procedures
- The development of new drugs
How many lenses does a compound light microscope have?
2
What are the names of the two lenses of a compound light microscope?
objective lens and the eyepiece lens.
