ConfocalThe method of optical sectioning is made possible through the rejection of out of focus light using a collection of pinholes and a single point detector along with pixel-by-pixel illumination of the sample.

Light from a light source (i.e. a laser) passes through an aperture and is then reflected off a dichromatic mirror and directed onto an assembly of computer motorised scanning mirrors. These mirrors allow for the laser light to scan the specimen across the x and y planes. This axial controlled motion allows for the whole field of view to be illuminated uniformly on a point-by-point basis. Emitted light then passes through the same scanning mirrors and the dichromatic mirror to a pinhole in front of the PMT (photomultiplier tube) detector. The emitted light from the excitation point is therefore collected. Photons collected by the PMT are converted into an ‘avalanche’ of electrons which can later be read as a digital signal. The collection pinhole prevents light from above or below the excitation plane from being collected by the PMT. This improves optical sectioning over traditional microscopy to produce improved z-resolution in the final image. 

 

ConfocalvWidefieldCLSM has advantages over widefield microscopy: mainly a higher resolution in the z axis due to the reduction of out-of-focus light reaching the detector and the ability to collect serial optical sections from thick specimens. The most notable advantage is the removal of out of focus light that often blurs widefield images from 3D specimens. This can be demonstrated above by comparing samples imaged under widefield illumination (left top 3 images) and confocal laser scanning microscopy (left lower 3 images).

The downside to a higher resolution image is a reduction in speed of acquisition due to the requirement to scan the image on a point-by-point basis, making CLSM more suitable for obtaining 3D optical sections than the tracking of high speed objects.

ESRIC houses a Lecia SP5 SMD gated-STED confocal laser scanning microscope for CLSM.

You can find more information on the basics of confocal microscopy through this Bitesize Bio Webinar, presented by Dr Ali Dun (ESRIC manager) and sponsored by Leica Microsystems.