What is the principle of confocal microscopy?

What is the principle of confocal microscopy?

The basic principle of confocal microscopy is that the illumination and detection optics are focused on the same diffraction-limited spot, which is moved over the sample to build the complete image on the detector.

What is the advantage of confocal microscopy?

Confocal microscopy offers several advantages over conventional widefield optical microscopy, including the ability to control depth of field, elimination or reduction of background information away from the focal plane (that leads to image degradation), and the capability to collect serial optical sections from thick …

How do you prepare slides for confocal microscopy?

Place one drop of Vectashield Mounting Media on each sheet of cells (1 for each chamber), and cover with a No. 1.5 thickness cover slip. Gently push out any air bubbles that form underneath the cover slip and seal the edges with clear nail polish.

What is confocal fluorescence microscopy?

Confocal fluorescence microscopy is a microscopic technique that provides true three-dimensional (3D) optical resolution. In confocal fluorescence microscopy, true 3D resolution is accomplished by ac- tively suppressing any signal coming from out-of-focus planes.

How long does confocal microscopy take?

Most laser scanning confocal microscopes (LSCMs) take approximately 1 second to acquire a single optical section, although several acquisitions are usually averaged by the software to improve signal-to-noise ratio.

How do you store confocal slides?

For the correct preservation of the slides, several criteria are important. The fluorochrome coupled to the antibody, the mounting medium, the storage temperature. We use the Alexa Fluor, Vectashield + DAPI, 4 ° C in the fridge for two months of storage in a humid chamber with PBS.

How do you fix a cell slide?

To fix with organic solvents, use ice-cold methanol, ethanol or a 1:1 mix of ethanol and methanol to cover the cells on your cover slips. Once covered, incubate your cells in the freezer (-20°C) for 5 to 7 minutes. Do not worry about keeping your cells sterile at this point – you are killing them!

What are the applications of confocal microscopy?

Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distribution of macromolecules in either fixed or living cells, the automated collection of 3D data, the imaging of multiple labeled specimens and the measurement of physiological events in living cells.

Why do we use fluorescence microscopy?

Fluorescent microscopy is often used to image specific features of small specimens such as microbes. It is also used to visually enhance 3-D features at small scales. When the reflected light and background fluorescence is filtered in this type of microscopy the targeted parts of a given sample can be imaged.