Three-dimensional Holographic Microscopy Research Articles

Haloarchaea swim slowly for optimal chemotactic efficiency in low nutrient environments

Archaea have evolved to survive in some of the most extreme environments on earth. Life in extreme, nutrient-poor conditions gives the opportunity to probe fundamental energy limitations on movement and response to stimuli, two essential markers of living systems. Here we use three-dimensional holographic microscopy and computer simulations to reveal that halophilic archaea achieve chemotaxis with power requirements one hundred-fold lower than common eubacterial model systems. Their swimming direction is stabilised by their flagella (archaella), enhancing directional persistence in a manner similar to that displayed by eubacteria, albeit with a different motility apparatus. Our experiments and simulations reveal that the cells are capable of slow but deterministic chemotaxis up a chemical gradient, in a biased random walk at the thermodynamic limit.

Sterol synthesis and cell size distribution under oscillatory growth conditions in Saccharomyces cerevisiae scale-down cultivations.

Physiological responses of yeast to oscillatory environments as they appear in the liquid phase in large-scale bioreactors have been the subject of past studies. So far, however, the impact on the sterol content and intracellular regulation remains to be investigated. Since oxygen is a cofactor in several reaction steps within sterol metabolism, changes in oxygen availability, as occurs in production-scale aerated bioreactors, might have an influence on the regulation and incorporation of free sterols into the cell lipid layer. Therefore, sterol and fatty acid synthesis in two- and three-compartment scale-down Saccharomyces cerevisiae cultivation were studied and compared with typical values obtained in homogeneous lab-scale cultivations. While cells were exposed to oscillating substrate and oxygen availability in the scale-down cultivations, growth was reduced and accumulation of carboxylic acids was increased. Sterol synthesis was elevated to ergosterol at the same time. The higher fluxes led to increased concentrations of esterified sterols. The cells thus seem to utilize the increased availability of precursors to fill their sterol reservoirs; however, this seems to be limited in the three-compartment reactor cultivation due to a prolonged exposure to oxygen limitation. Besides, a larger heterogeneity within the single-cell size distribution was observed under oscillatory growth conditions with three-dimensional holographic microscopy. Hence the impact of gradients is also observable at the morphological level. The consideration of such a single-cell-based analysis provides useful information about the homogeneity of responses among the population.

Electrochemistry of Single Nanodomains Revealed by Three-Dimensional Holographic Microscopy.

Interest in nanoparticles has vigorously increased over the last 20 years as more and more studies show how their use can potentially revolutionize science and technology. Their applications span many different academically and industrially relevant fields such as catalysis, materials science, health, etc. Until the past decade, however, nanoparticle studies mostly relied on ensemble studies, thus leaving aside their chemical heterogeneity at the single particle level. Over the past few years, powerful new tools appeared to probe nanoparticles individually and in situ. This Account describes how we drew inspiration from the emerging fields of nanoelectrochemistry and plasmonics-based high resolution holographic microscopy to develop a coupled approach capable of analyzing in operando (electro)chemical reaction over one single nanoparticle. A brief overview of selected optical strategies to image NPs in situ with emphasis on scattering based methods is presented. In an electrochemical context, it is necessary to track particle behavior both in solution and near a polarized electrode, which is why 3D optical observation is particularly appealing. These approaches are discussed together with strategies to track NPs beyond the diffraction limit, allowing a much finer description of their trajectories. Then, the holographic setup is used to study electrochemically triggered Ag NP oxidation reaction in the presence of different electrolytes. Holography is shown to be a powerful technique to track and analyze the trajectory of individual NPs in situ, which further sheds light on in operando behaviors such as electrogenerated NP transport, aggregation, or adsorption. We then show that spectroscopy and scattering-based optical methods are reliable and sensitive to the point of being used to investigate and quantify NP (electro)chemical reactions in model cases. However, since real chemical reactions usually take place in an inherently complex environment, approaches based exclusively on optical imaging only reach their limitations. The strategy is then taken one step further by merging together electrochemical nanoimpact experiments with 3D optical monitoring. Previous strategies are validated by showing that in simple cases, these two independent ways of probing NP size and reactivity yield the same results. For more complicated reactions (e.g., multistep reactions), one must go beyond either technique by showing that the two approaches are perfectly complementary and that the two signals contain information of different natures, thus providing a much better characterization of the reaction. This point is illustrated by studying Ag NP oxidation (single or agglomerates) in the presence of a precipitating agent, where the actual oxidation is uncoupled from the dissolution of the particle, thus proving the point of our symbiotic approach.

Non-scanning motionless fluorescence three-dimensional holographic microscopy

Demonstration of an imaging system that can capture high-resolution 3D fluorescent images of biological speciments without the need for any moving parts.

Three-dimensional identification of stem cells by computational holographic imaging

We present an optical imaging system and mathematical algorithms for three-dimensional sensing and identification of stem cells. Data acquisition of stem cells is based on holographic microscopy in the Fresnel domain by illuminating the cells with a laser. In this technique, the holograms of stem cells are optically recorded with an image sensor array interfaced with a computer and three-dimensional images of the stem cells are reconstructed from the Gabor-filtered digital holograms. The Gabor wavelet transformation for feature extraction of the digital hologram is performed to improve the process of identification. The inverse Fresnel transformation of the Gabor-filtered digital hologram is performed to reconstruct the multi-scale three-dimensional images of the stem cells at different depths along the longitudinal direction. For recognition and classification of stem cells, a statistical approach using an empirical cumulative density function is introduced. The experiments indicate that the proposed system can be potentially useful for recognizing and classifying stem cells. To the best of our knowledge, this is the first report on using three-dimensional holographic microscopy for automated identification of stem cells.