The Kanbar Scientific Equipment Center

The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Israel

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Basics of Confocal Microscopy

 

Confocal microscopy has advantages over widefield microscopy.

The reduction of out-of-focus light from the focal plane, which leads to images only from the in-focus planes, reduces haziness, and increases the resolution of images. This also allows the option to collect serial optical sections (tomography) from thick samples and project them in 3D.

There are three main things that turn a confocal into a confocal.
1.  A point light source in the form of a laser, that raster scans the defined field of view. (In a widefield, the light is projected on the entire field of view). 
2.  A pinhole that removes out of focus light.
3.  A light detector in the form of a PMT, that records the intensity of emitted fluorescence light, at every defined pixel, one pixel at a time. (In a widefield, the light is detected by a camera, all pixels simultaneously.)

The fact that only one small area is being excited and only it's emission recorded, enables the use of the pinhole. Altogether, this provides a higher resolution image, both in the lateral (x/y) plane and the axial (z) plane.

(Note: There also exists other types, such as spinning disk, laser-less confocals that do use cameras)

 

wide

 

 irit hyv0106

 

 

We have 4 laser scanning confocal microscopes. One with integrated HyVolution (automated software deconvolution which increases resolution) and live capabilities, a STED super-resolution, an Olympus FV1000 and a single PMT Leica SPE. 

See also: 

Leica confocal
Leica super-resolution