Laser Microscopy

The two microscopes categorised under Laser Microscopy both use lasers; however, they have very different functions. The two microscopes are categorised together for purely for administrative costing purposes.

Confocal

The confocal laser scanning microscope uses monochromatic lasers to excite fluorescent molecules. This is the same general principle used in fluorescence microscopy. The advantage of the confocal microscope is the removal of out-of-focus light (also called epifluorescent light) by the use of pinholes.

The laser is software controlled and can scan the sample at different Z-heights within the sample. This allows the imaging of one or more optical sections . Multiple optical sections can be merged to produce a three-dimensional stack to understand relationships within the sample of the proteins or components that are labelled. Multiple wavelengths representing different targets can be obtained simultaneously.

Light microscopy image of an Arabidopsis root tip. (Sample S. Kurup, image M. Le Bloa)

Epifluorescence of nuclear targeted GFP in the root tip. (Sample S. Kurup, image M. Le Bloa)

3D confocal stack of nuclear targeted GFP in the root tip. Individual nuclei can be delineated. (S. Kurup)

Laser Microdissection

The power of the laser on the laser microdissection system is utilised to excise individual cells or groups of cells. The focus and energy of the laser can be adjusted to cut through sections of different thicknesses and tissues of different compositions. The laser is then defocussed to spread the beam and quickly pulsed to 'flick' the excised cells into an Eppendorf tube for molecular biology techniques to extract DNA, RNA, proteins or metabolites.

Twelve micron thick cross section of a wheat leaf before and after laser microdissection. Some of the mesophyll cells have been excised. (E. Egidi).