The principle of laser microdissection
To perform microdissection, an upright or inverted microscope is coupled to a laser. Selected areas or even individual cells are then excised by moving the focused laser beam along their contours. This procedure guarantees gentle specimen handling – in particular, it does not transfer any heat to the specimen – and provides exactly the material that is needed for analysis.
The dissectate is then transported to a collection device in a number of different ways. Depending on the system used, dissected tissue either falls by the force of gravity into a reaction vessel (Leica Microsystems LMD system), is catapulted against the force of gravity into a reaction vessel (PALM system) or removed indirectly together with a membrane covering the samples (Arcturus and MMI system). The Leica Microsystems LMD and the PALM system can also be used for laser micromanipulation, e.g. to cut through spindle fibers during cell division.
Laser microdissection is an established method for a large number of applications, mainly in molecular biology, particularly nucleic acid research, neurosciences, developmental biology, cancer research, forensics, proteomics, plant research, for cutting cell cultures and for single cell isolation. Laser microdissection is even used in climate research, especially for examining the annual rings of trees.
Methods and technology of non-contact laser microdissection
Laser microdissection offers a precise and contamination-free solution for the isolation and selection of single cells or tissue. The tissue samples can be embedded, sectioned and stained according to conventional methods of preparation. Paraffin sections, frozen sections, smear preparations, chromosome specimens and cell cultures are all suitable for laser microdissection. The area selected for dissection is drawn on the PC screen and automatically separated from the surrounding tissue with a laser beam. Fluorescence-labeled specimens can also be dissected using special filter cubes which transmit the full spectrum of the laser light. The dissectate is then immediately transported to a collection device (in different ways, depending on the manufacturer of the system) for further examination.
There are different techniques of laser microdissection. In the PALM system, for example, the specimen is moved in a fixed laser beam. Here, the chosen region is cut away from the surrounding tissue, after which non-contact transport is activated by a single laser pulse. The selected tissue is lifted from the specimen slide and catapulted against the force of gravity into a collection device.
The force of gravity is the method of choice of the Leica system. Here the sample is fixed and the laser beam is moved over it. This allows easy viewing of the sample, and the dissectate falls into a collection device by the force of gravity alone. The precision of the cutting process is optically coupled to the chosen magnification. A higher magnification automatically results in a finer step width, as the laser beam and its movement are reduced by the same degree. No other work steps are required in this case. Not only single cells, but also larger areas can be excised in a single pass. Despite the different methods for transferring the dissectate to a collection device, there is no risk of contact or contamination.
However, some other non-contact-free methods for laser microdissection are also available (Arcturus, MMI).