Label-free cytotoxicity screening assay by digital holographic microscopy

Abstract We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.

Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy.

Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.

Propofol anesthesia impairs the maturation and survival of adult-born hippocampal neurons.

BACKGROUND: Adult neurogenesis occurs in the hippocampus of most mammals, including humans, and plays an important role in hippocampal-dependent learning. This process is highly regulated by neuronal activity and might therefore be vulnerable to anesthesia. In this article, the authors investigated this possibility by evaluating the impact of propofol anesthesia on mouse hippocampal neurons generated during adulthood, at two functionally distinct maturational stages of their development. METHODS: Adult-born hippocampal neurons were identified using the cell proliferation marker bromodeoxyuridine or a retroviral vector expressing the green fluorescent protein in dividing cells and their progenies. Eleven or 17 days after the labeling procedure, animals (n = 3-5 animals per group) underwent a 6-h-long propofol anesthesia. Twenty-one days after labeling, the authors analyzed the survival, differentiation, and morphologic maturation of adult-born neurons using confocal microscopy. RESULTS: Propofol impaired the survival and maturation of adult-born neurons in an age-dependent manner. Anesthesia induced a significant decrease in the survival of neurons that were 17 days old at the time of anesthesia, but not of neurons that were 11 days old. Similarly, propofol anesthesia significantly reduced the dendritic maturation of neurons generated 17 days before anesthesia, without interfering with the maturation of neurons generated 11 days before anesthesia. CONCLUSIONS: These results reveal that propofol impairs the survival and maturation of adult-born hippocampal neurons in a developmental stage-dependent manner in mice.

Genetic manipulation of adult-born hippocampal neurons rescues memory in a mouse model of Alzheimer's disease.

In adult mammals, neural progenitors located in the dentate gyrus retain their ability to generate neurons and glia throughout lifetime. In rodents, increased production of new granule neurons is associated with improved memory capacities, while decreased hippocampal neurogenesis results in impaired memory performance in several memory tasks. In mouse models of Alzheimer's disease, neurogenesis is impaired and the granule neurons that are generated fail to integrate existing networks. Thus, enhancing neurogenesis should improve functional plasticity in the hippocampus and restore cognitive deficits in these mice. Here, we performed a screen of transcription factors that could potentially enhance adult hippocampal neurogenesis. We identified Neurod1 as a robust neuronal determinant with the capability to direct hippocampal progenitors towards an exclusive granule neuron fate. Importantly, Neurod1 also accelerated neuronal maturation and functional integration of new neurons during the period of their maturation when they contribute to memory processes. When tested in an APPxPS1 mouse model of Alzheimer's disease, directed expression of Neurod1 in cycling hippocampal progenitors conspicuously reduced dendritic spine density deficits on new hippocampal neurons, to the same level as that observed in healthy age-matched control animals. Remarkably, this population of highly connected new neurons was sufficient to restore spatial memory in these diseased mice. Collectively our findings demonstrate that endogenous neural stem cells of the diseased brain can be manipulated to become new neurons that could allow cognitive improvement.

Adiposity in children born small for gestational age.

Epidemiological studies indicate that children born small for gestational age (SGA) have an increased risk of metabolic and cardiovascular disorders as adults. This suggests that foetal undernutrition leads to permanent metabolic alterations, which predispose to metabolic abnormalities upon exposure to environmental factors such as low physical activity and/or high-energy intake in later life (thrifty phenotype hypothesis). However, this relationship is not restricted to foetal undernutrition or intrauterine growth retardation, but is also found for children born premature, or for high birth weight children. Furthermore, early post-natal nutrition, and more specifically catch-up growth, appear to modulate cardiovascular risk as well. Intrauterine growth retardation can be induced in animal models by energy/protein restriction, or ligation of uterine arteries. In such models, altered glucose homeostasis, including low beta-cell mass, low insulin secretion and insulin resistance is observed after a few weeks of age. In humans, several studies have confirmed that children born SGA have insulin resistance as adolescents and young adults. Alterations of glucose homeostasis and increased lipid oxidation can indeed be observed already in non-diabetic children born SGA at early pubertal stages. These children also have alterations of stature and changes in body composition (increased fat mass), which may contribute to the pathogenesis of insulin resistance. Permanent metabolic changes induced by foetal/early neonatal nutrition (metabolic inprinting) may involve modulation of gene expression through DNA methylation, or alterations of organ structure. It is also possible that events occurring during foetal/neonatal development lead to long-lasting alterations of the hypothalamo-pituitary-adrenal axis or the hypothalamo-pituitary-insulin-like growth factor-1 axis.

Simultaneous Optical Recording in Multiple Cells by Digital Holographic Microscopy of Chloride Current Associated to Activation of the Ligand-Gated Chloride Channel GABA(A) Receptor.

Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A) mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A) receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A) receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

Measurement of absolute cell volume, osmotic membrane water permeability, and refractive index of transmembrane water and solute flux by digital holographic microscopy.

ABSTRACT. A dual-wavelength digital holographic microscope to measure absolute volume of living cells is proposed. The optical setup allows us to reconstruct two quantitative phase contrast images at two different wavelengths from a single hologram acquisition. When adding the absorbing dye fast green FCF as a dispersive agent to the extracellular medium, cellular thickness can be univocally determined in the full field of view. In addition to the absolute cell volume, the method can be applied to derive important biophysical parameters of living cells including osmotic membrane water permeability coefficient and the integral intracellular refractive index (RI). Further, the RI of transmembrane flux can be determined giving an indication about the nature of transported solutes. The proposed method is applied to cultured human embryonic kidney cells, Chinese hamster ovary cells, human red blood cells, mouse cortical astrocytes, and neurons.

Selection methods regulate evolution of cooperation in digital evolution.

A key, yet often neglected, component of digital evolution and evolutionary models is the 'selection method' which assigns fitness (number of offspring) to individuals based on their performance scores (efficiency in performing tasks). Here, we study with formal analysis and numerical experiments the evolution of cooperation under the five most common selection methods (proportionate, rank, truncation-proportionate, truncation-uniform and tournament). We consider related individuals engaging in a Prisoner's Dilemma game where individuals can either cooperate or defect. A cooperator pays a cost, whereas its partner receives a benefit, which affect their performance scores. These performance scores are translated into fitness by one of the five selection methods. We show that cooperation is positively associated with the relatedness between individuals under all selection methods. By contrast, the change in the performance benefit of cooperation affects the populations' average level of cooperation only under the proportionate methods. We also demonstrate that the truncation and tournament methods may introduce negative frequency-dependence and lead to the evolution of polymorphic populations. Using the example of the evolution of cooperation, we show that the choice of selection method, though it is often marginalized, can considerably affect the evolutionary dynamics.