CONFOCAL MICROSCOPY - CFM
Over the years, confocal microscopy has become the method of choice for obtaining clear, three-dimensional optical images of sample structures. It has been used for imaging almost anything - from studying biological samples such as cells, fluorescence measurements, to examining physical structures such as semiconductor quantum dots, NEMS/MEMS devices and also for the emerging area of nano-optics. The confocal imaging system achieves out-of-focus rejection by two strategies (schematically illustrated in the figure below):
1. Illuminating a single point of the specimen with a focused beam. Thus, the illumination intensity drops rapidly above and below the plane of focus.
2. Using of a blocking pinhole in the conjugate plane to the specimen that eliminates the degrading out-of-focus information.
By scanning many thin sections through the sample a very clean three-dimensional image can be obtained. Confocal imaging can offer another advantage in favourable situations (small pinhole size, bright specimen): the obtained resolution can be better than with any microscope operated conventionally. In practice, the best horizontal resolution of a confocal microscope is about 0.4 μm, and the best vertical resolution is about 1.4 μm, assuming an excitation wavelength of 633 nm and a numerical aperture of 0.65.
Cryogenic Confocal Microscopy
To improve the image quality in high resolution microscopy, confocal microscopes are often used at cryogenic temperatures. At these conditions, a combination of high resolution power, clear optical spectra, and reduced thermal noise can be achieved. Spectral lines become sharper as thermal broadening is reduced due to the lower thermal energy present in the system. Optical signals become stronger as quantum efficiency is improved due to less scattering and non-radiative recombinations. For many optical microscopy applications cryogenic temperatures are therefore inevitably required.
These advantages are profitable particularly for high resolution optical spectroscopy of semiconductor structures or single molecule detection. Thus, investigation of the emitted optical energy of the sample due to changes in the surrounding material, applied voltages, or the deposited optical energy becomes feasible.
Additionally, high spatial resolution and sharp spectral lines are a prerequisite for investigating photon anti-bunching (single photon emitters).