Confocal microscopy is an optical imaging technique allowing the acquisition of contrasted images of a single plane of a sample. A laser beam scans the specimen pixel by pixel and line by line and the emitted light is going through a pinhole located in front of the detector to eliminate the light emerging outside that plane. Only the light within the focal plane can reach the detector. The resulting image has a high contrast and resolution. This technique allows the acquisition of images in the X, Y and Z planes of a specimen that can be combined into a 3D representation.
The recent improvement of the equipment and the development of increasing array of fluorescent molecules offer the possibility to detect simultaneously several dyes or fluorophores at high sensitivity and to follow their dynamics in living samples.
Atomic force microscopy
In AFM imaging, a sharp tip is scanned over the surface of the sample, while sensing the so-called near-field physical interactions between the tip and the sample. This allows three-dimensional images to be generated directly in aqueous solution. The sample is mounted on a piezoelectric scanner which ensures three-dimensional positioning with high accuracy. While the tip is being scanned in the (x, y) directions, the force interacting between tip and specimen is monitored with piconewton sensitivity. This force is measured by the deflection of a soft cantilever which is detected by a laser beam focused on the free end of the cantilever and reflected into a photodiode.
In force spectroscopy modalities, the cantilever deflection is recorded as a function of the vertical displacement of the scanner, i.e. as the sample is pushed towards the tip and retracted. This results in a cantilever deflection vs scanner displacement curve, which can be transformed into a force-distance curve using appropriate corrections. The characteristic adhesion force between tip and sample observed during retraction may then be used to map chemical groups and receptor sites and to measure their binding forces.