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Speaker Detail

Professor Christian Depeursinge

King Abdullah University of Science & Technology, Saudi Arabia

Biography

​Professor Christian Depeursinge is one of the forefathers of holography applied to microscopy and seasoned partners of Nanolive. During his long carrier he hold several academic and industry positions, amongst others  as the leader of the Microvision and Micro-Diagnostics group at the Advanced Photonics Laboratory of the Institute of Microengineering at Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland, and currently at King Abdullah University of Science and Technology (KAUST), Saudi Arabia. His research and expertise in biomedical engineering and optics is internationally acknowledged. Professor Depeursinge is the head of the scientific advisory board at Nanolive and his role comprises scientific strategy and general guidance.

Abstract

Label free microscopy for quantitative assessment of biophysical data: cell growth and metabolism


LCIE / BESE /  KAUST

Historically  optical  microscopy  has  been one  of  the  most productive  scientific  instrument in technology and medicine. Nevertheless, some limitations of optical microscopy have appeared due in particular to the lack of resolution formulated by the well-known Abbe law, as well as the lack of quantitative data provided by conventional optical microscopes.
Although fluorescence microscopy has become an overwhelming imaging approach, mainly because of its capability to label cell components with high specificity, bulky fluorescent probes often perturb the biological activities of small biomolecules such as metabolites. Further on, because of the unavoidable quenching of fluorophores, quantitative data are also difficult to obtain. Microscopy had therefore to be developed in new directions: Quantitative Phase microscopy (QPM) which allows indirectly the determination of refractive index of cell and tissue components and more generally the development of label-free quantification and imaging techniques.
The images of cells or tissues called "phase contrast" were obtained several decades ago with the primary aim of improving the visibility of cell profiles. With the exception of certain cells such as erythrocytes and chloroplasts, the biological cells are mostly transparent and lack natural chromophores, which could allow contrasting them significantly. The first techniques for introducing the phase as a contrast agent were proposed at the end of the forties by Zernike (1947) and by Nomarski (1959). They are both based on an interferometric approach, i.e. using the phenomenon of interference between two waves passing through different paths. While the two methods contribute to a sensitive and well-resolved image of the cellular silhouettes, they are in fact of no help for the quantitative measurement of the phase itself.
However, over the past three decades it has gradually appeared that the phase measured quantitatively was a signal of great interest if it were to be interpreted in the light of cellular biophysical data. Indeed, it is well known that the phase shift results from the difference in the refractive index between the cellular components such as the cytoplasm and the intercellular space. Each of these biophysical parameters forms the basis of an objective assessment of cellular activity and more particularly of its metabolism.
The idea of reconstructing a wave front from the hologram, yielding potentially the exact replica of the object and then, deductively, of its shape and dielectric composition, was a great breakthrough of the development of holography. It also opened the way to the microscopic observation of transparent media, especially attractive for quantitative microscopy (E.Wolf 1969).  Its practical implementation based on digital processing methods brought fundamentally new perspectives in microscopy.
The perspective to introduce digital computing and bring quantitative data in optical microscopy on one side, and to reach the nanometer scale on the other side, has appeared clearly as an incentive to invent and develop new concepts in optical microscopy. Further on, the complex wavefront reconstructed from a hologram has been applied to microscopic images. For the first time the microscopic image was obtained both in amplitude and phase by mean of a holographic setup and from a single hologram taken off-axis The first quantitative phase image of a living neuron has been obtained by Etienne Cuche in 1999  (Optics Letter cited 612 x, Applied Optics cited 620 x) and Pierre Marquet (2005 Optics Letter cited 610 x). The development of quantitative microscopy based on digital holography has helped us in extracting meaningful data from images. The determination of the precise topology (with nanometer accuracy) is henceforth possible by Digital Holographic Microscopy (DHM). Other characteristics of the microscopic object have also been measured precisely: the Refractive Index (RI) (Rappaz 2008, Boss 2012,2013) and birefringence properties (Colomb 2005) in particular.
A clear trend towards super-resolution microscopy is another strand of phase imaging. It is the fruit of a better knowledge of physical principles underlying microscopy. The use of coherence properties of light waves has henceforth permitted altogether breaking the diffraction limit and providing a full 3D image of microscopic or nanometric objects. Super-resolution is a commonly accepted term to designate imaging objects beyond the diffraction limit (defined by Abbe law). Objects can be observed practically in the range of a few tens or hundreds of nanometers. Nanoscopy has been proposed to coin these innovative developments. Super-resolution technologies have been extended to non-fluorescent microscopies. Different approaches based on the diffraction tomography theorem have been developed in the DHM team (Charrière 2006).Further on, by exploiting the opportunity of achieving a correction of the microscope objective aberration by a complex wave deconvolution, together with aperture synthesis, Y.Cotte (Nature Photonics 2013 cited 155 x) and F.Toy pushed DHM based tomography to its limits. Resolution even better than 100 nm has been demonstrated. Furthermore, it is applicable for real-time analysis in living tissues. Time lapse studies are also easily performed
The quantitative phase signal provides valuable information on cell morphology and its content. Their interpretation in terms of biophysical parameters is problematic. The development of high-resolution and near-real-time optical diffraction tomography makes it possible to overcome the potential interpretation difficulties of the phase signal. It allows determining the three-dimensional distribution of the refractive index (Charrière et al., 2006, Charrière et al., 2006, Choi et al., 2007). The practical realization of a high resolution 3D map of the intracellular refractive index provides valuable information on cell cytoachitecture and related biomechanical data. Cellular physiology deserves a better understanding in view of a better observation of the cytoplasm and its compartmentalization. The localization and importance of protein generation and resorption is a decisive step forward in the understanding of basic cellular mechanisms.
Quantitative label-free imaging is also achieved today by the recourse to Raman spectroscopy which has the advantage of delivering specific data for biochemicals. In particular non-linear response of biological matter to high intensity laser illumination provides a quantitative and potentially a fast access to species involved in metabolic activities like lipids, glucose and some proteins or nucleic acids. As such this imaging approach appears complementary to quantitative phase imaging. Vibrational imaging technique known as Coherent Antistokes Raman Scattering (CARS) microscopy (Begley 1974, Evans 2008) and Stimulated Raman Scattering (SRS) (Freudiger 2008), are capable of chemically selective, highly sensitive, and high-speed imaging of biomolecules with submicron resolution. Their use in brain tissue imaging, in particular as far as metabolic activity is concerned is just in its early stage.
In summary, label-free imaging provides a critical advantage in microscopy. The interpretation of the quantitative phase signal has proved very fruitful for the follow-up of physiological parameters such as the movement of fluids, and in particular the transmembrane flux depending, in particular, on ionic concentrations. Protein concentrations and growth can be precisely quantified. The morphologies of cells and organelles can be established by phase tomography. Their study provide invaluable information on the biomechanical characteristics of cell structures and membranes. These data are indicative of biomolecular activity, which can be affected significantly by pathology. These benefits are particularly noticeable for the study of brain metabolism where the need for quantitative data is welcome.



All sessions by Professor Christian Depeursinge

  • TuesdayApril 10
3:25 PM

"Label free microscopy for quantitative assessment of biophysical data: cell growth and metabolism"