|3D microscopy is mandatory for biological investigation. Among various physical mechanisms for imaging such as electronic, X-Ray, magnetic resonance, and acoustic processes, optics is able to provide simultaneously the highest spatial resolution with the minimum of invasivity. Nevertheless, optical imaging needs to cope with light scattering, and the turbidity of biological tissues is still a problem.
For 3D imaging, sectioning is necessary. As the oldest method, this method is already used with a great success in anatopathology. It relies on a real physical sectioning, whereby excellent 3D images are constructed with a collection of thin slices (3µm). However, the method is destructive. We present several methods, which are able to provide a minimally invasive optical sectioning toward an imaging in the three directions of space. Besides the recent progress in confocal microscopy and optical coherent tomography (OCT), the use of nonlinear optics will be emphasized. Two new optical methods will be introduced.
i) The stimulated emission depletion (STED) selective plane illumination (SPIM) microscope based on a single diode-pumped solid state (DPSS) laser that simultaneously delivers nanosecond-pulses at two wavelengths, and the use of a chromatic beam shaping demonstrates a 300% reduction of the light sheet thickness together with an enhancement of the sheet uniformity over larger field of view.
ii) A novel optical resolution photoacoustic microscopy concept in order to obtain an axial resolution only by optical methods. The photoacoustic signal is generated through a non-radiative relaxation from a level of a dye that is populated by excited state absorption. This two-step excitation process of a single laser enables to achieve an optical sectioning without any acoustic selectivity, whereby a full optical resolution photoacoustic microscopy is obtained.