Microsocopic 3D phase imaging Unstained imaging method

In conventional microscopy, when using an incoherent illumination-detection system, the recorded image results from a complex interaction between the incoherent illumination and the specimen (object). The contrast observed enables morphology to be studied effectively, but does not directly provide quantitative information on the characteristics of a specimen.

To overcome these problems, particularly in biology, the techniques employed often require functional labeling of samples, as in fluorescence, confocal, STED (Stimulated-Emission-Depletion), PALM (Photo-Activated Localization Microscopy), SIM (Structured Illumination Microscopy) microscopy, or their variants. Imaging based on fluorescent markers is today the technique of choice for microscopic imaging, thanks to the wide variety of fluorophores developed and the resolutions achieved. In particular, STED and PALM were awarded the Nobel Prize in 2014. However, these imaging modes can sometimes pose problems (phototoxicity, photobleaching, high energy density) and for scientists unable to use these systems, other types of label-free microscopy must be used. There are various types of label-free imaging: CARS (Coherent Anti-Stokes Raman Scattering), Raman effect, second harmonic generation and phase measurement. Harmonic generation or Raman-based techniques enable contrast to be observed based on the optical properties of the specimen, but require costly instrumentation and delicate implementation (femtosecond laser).

Holography and diffractive tomography are based on a measurement of the phase change induced on the illumination wave by the sample, but unlike DIC (Differential Interference Contrast) or phase contrast, they provide access to a quantitative measurement of refractive index and/or phase. This article presents the concepts required to understand this type of measurement, and how they can be implemented with interferometric and tomographic setups. Based on the principles of digital holography, and after a digital processing step, it is possible to obtain a series of tomographic data required to solve an inverse problem linked to the image formation mechanism in a microscope. The solution to this inverse problem enables the reconstruction of an image of the absorption and local optical index of a 3D specimen.

In order to understand the mechanism of image formation, the notions of light wave propagation using Fourier space (spatial spectrum) and the principle of the associated angular spectrum are presented in the first part, leading to the formulation of direct models (projective and diffractive).

Holographic microscopy is presented, and the resolution limit is studied using frequency content (bandwidth). Tomography is described with an experimental set-up...