Module 2 - CLSMs Flashcards
Cells, Chemicals for Life, Transport and Gas Exchange
How CLSMs work… (1)
A CLSM moves a single spot of focused light across a specimen; this is known as point illumination.
How CLSMs work… (2)
The laser passes through an illumination pinhole onto a dichroic mirror which reflects the single wavelength of light from the laser. The dichroic mirror allows other wavelengths of light from the specimen to pass through i.e. the mirror filters the light.
How CLSMs work… (3)
The single wavelength of light that is reflected from the mirror causes the fluorochrome marker to fluorescence.
How CLSMs work… (4)
The light that is then emitted from the specimen passes through a confocal pinhole aperture and passes to a detector. Only light radiated from very close to the focal plane is detected.
How CLSMs work… (5)
If any light is emitted from other parts of the specimen it reduces the resolution and causes blurring of the image. The confocal pinhole prevents this unwanted radiation passing to the detector to ensure a sharp image is obtained.
Define fluorescence.
The specific light scattering and fluorescent characteristics of each cell as they pass the laser beam. The fluorescence is detected and can thereby by used for counting and sorting.
Optical Sectioning:
- Laser can scan the specimen at different depths.
- A series of optical sections can be combined to form an image stack made from many layers.
- Thus a 3D image is produced.
Fluorochromes:
- Fluorochromes or fluorescent markers can be used in CLSMs to tag a chemical which binds to a specific part of the cell surface membrane/DNA/organelles.
- They can also be attached to antibodies which are specific to antigens (on a particular cell).
How fluorochromes work…
- As the excitation laser light beam passes through the specimen, the light is reflected, and the fluorochromes give off different colours at different wavelengths.
- These wavelengths are then detected in the CLSM and are used to colour and construct the image.
