Multispectral in-line hologram reconstruction with aberration compensation applied to Gram-stained bacteria microscopy.

In multispectral digital in-line holographic microscopy (DIHM), aberrations of the optical system affect the repeatability of the reconstruction of transmittance, phase and morphology of the objects of interest. Here we address this issue first by model fitting calibration using transparent beads inserted in the sample. This step estimates the aberrations of the optical system as a function of the lateral position in the field of view and at each wavelength. Second, we use a regularized inverse problem approach (IPA) to reconstruct the transmittance and phase of objects of interest. Our method accounts for shift-variant chromatic and geometrical aberrations in the forward model. The multi-wavelength holograms are jointly reconstructed by favouring the colocalization of the object edges. The method is applied to the case of bacteria imaging in Gram-stained blood smears. It shows our methodology evaluates aberrations with good repeatability. This improves the repeatability of the reconstructions and delivers more contrasted spectral signatures in transmittance and phase, which could benefit applications of microscopy, such as the analysis and classification of stained bacteria.

Single-cell scattering and auto-fluorescence-based fast antibiotic susceptibility testing for gram-negative and gram-positive bacteria.

In this study, we assess the scattering of light and auto-fluorescence from single bacterial cells to address the challenge of fast (<2 h), label-free phenotypic antimicrobial susceptibility testing (AST). Label-free flow cytometry is used for monitoring both the respiration-related auto-fluorescence in two different fluorescence channels corresponding to FAD and NADH, and the morphological and structural information contained in the light scattered by individual bacteria during incubation with or without antibiotic. Large multi-parameter data are analyzed using dimensionality reduction methods, based either on a combination of 2D binning and Principal Component Analysis, or with a one-class Support Vector Machine approach, with the objective to predict the Susceptible or Resistant phenotype of the strain. For the first time, both Escherichia coli (Gram-negative) and Staphylococcus epidermidis (Gram-positive) isolates were tested with a label-free approach, and, in the presence of two groups of bactericidal antibiotic molecules, aminoglycosides and beta-lactams. Our results support the feasibility of label-free AST in less than 2 h and suggest that single cell auto-fluorescence adds value to the Susceptible/Resistant phenotyping over single-cell scattering alone, in particular for the mecA+ Staphylococcus (i.e., resistant) strains treated with oxacillin.

Automatic numerical focus plane estimation in digital holographic microscopy using calibration beads.

We present a new method to achieve autofocus in digital holographic microscopy. The method is based on inserting calibrated objects into a sample placed on a slide. Reconstructing a hologram using the inverse problems approach makes it possible to precisely locate and measure the inserted objects and thereby derive the slide plane location. Numerical focusing can then be performed in a plane at any chosen distance from the slide plane of the sample in a reproducible manner and independently of the diversity of the objects in the sample.

Assessing the Functional Heterogeneity of Monocytes in Human Septic Shock: a Proof-of-Concept Microfluidic Assay of TNFα Secretion.

Objective: The development of advanced single-cell technologies to decipher inter-cellular heterogeneity has enabled the dynamic assessment of individual cells behavior over time, overcoming the limitation of traditional assays. Here, we evaluated the feasibility of an advanced microfluidic assay combined to fluorescence microscopy to address the behavior of circulating monocytes from septic shock patients. Methods: Seven septic shock patients and ten healthy volunteers were enrolled in the study. Using the proposed microfluidic assay we investigated the production over time of LPS-elicited TNFα by single monocytes encapsulated within droplets. Cellular endocytic activity was assessed by internalization of magnetic nanoparticles. Besides, we assessed HLA-DR membrane expression and LPS-induced TNFα production in monocytes through classical flow cytometry assays. Results: Consistent with the flow cytometry results, the total number of TNFα molecules secreted by encapsulated single monocytes was significantly decreased in septic shock patients compared to healthy donors. TNFα production was dampened as soon as 30 and 60 minutes after LPS stimulation in monocytes from septic patients. Furthermore, the microfluidic assay revealed heterogeneous individual behavior of monocytes from septic shock patients. Of note, monocytes from both healthy donors and patients exhibited similar phagocytic activities over time. Conclusion: The microfluidic assay highlights the functional heterogeneity of monocytes, and provides in-depth resolution in assessing the hallmark monocyte deactivation encountered in post-septic immunosuppression.

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap.

Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8-10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.

On the isotropic Raman spectrum of Ar2 and how to benchmark ab initio calculations of small atomic clusters: Paradox lost.

This is the long-overdue answer to the discrepancies observed between theory and experiment in Ar2 regarding both the isotropic Raman spectrum and the second refractivity virial coefficient, BR [Gaye et al., Phys. Rev. A 55, 3484 (1997)]. At the origin of this progress is the advent (posterior to 1997) of advanced computational methods for weakly interconnected neutral species at close separations. Here, we report agreement between the previously taken Raman measurements and quantum lineshapes now computed with the employ of large-scale CCSD or smartly constructed MP2 induced-polarizability data. By using these measurements as a benchmark tool, we assess the degree of performance of various other ab initio computed data for the mean polarizability α, and we show that an excellent agreement with the most recently measured value of BR is reached. We propose an even more refined model for α, which is solution of the inverse-scattering problem and whose lineshape matches exactly the measured spectrum over the entire frequency-shift range probed.

Collision-induced Raman scattering by rare-gas atoms: The isotropic spectrum of Ne-Ne and its mean polarizability.

We report the room-temperature isotropic collision-induced light scattering spectrum of Ne-Ne over a wide interval of Raman shifts, and we compare it with the only available experimental spectrum for that system as well as with spectra calculated quantum-mechanically with the employ of advanced ab initio-computed data for the incremental mean polarizability. The spectral range previously limited to 170 cm(-1) is now extended to 485 cm(-1) allowing us to successfully solve the inverse-scattering problem toward an analytic model for the mean polarizability that perfectly matches our measurements. We also report the depolarization ratio of the scattering process, lingering over the usefulness of this property for more stringent checks between the various polarizability models.

Laser-induced plasmas in ambient air for incoherent broadband cavity-enhanced absorption spectroscopy.

The emission from a laser-induced plasma in ambient air, generated by a high power femtosecond laser, was utilized as pulsed incoherent broadband light source in the center of a quasi-confocal high finesse cavity. The time dependent spectra of the light leaking from the cavity was compared with those of the laser-induced plasma emission without the cavity. It was found that the light emission was sustained by the cavity despite the initially large optical losses of the laser-induced plasma in the cavity. The light sustained by the cavity was used to measure part of the S(1) ← S(0) absorption spectrum of gaseous azulene at its vapour pressure at room temperature in ambient air as well as the strongly forbidden γ-band in molecular oxygen: b(1)Σ(g)(+)(ν'=2)←X(3)Σ(g)(-)(ν''=0).

Collision-induced Raman scattering and the peculiar case of neon: anisotropic spectrum, anisotropy, and the inverse scattering problem.

Owing in part to the p orbitals of its filled L shell, neon has repeatedly come on stage for its peculiar properties. In the context of collision-induced Raman spectroscopy, in particular, we have shown, in a brief report published a few years ago [M. Chrysos et al., Phys. Rev. A 80, 054701 (2009)], that the room-temperature anisotropic Raman lineshape of Ne-Ne exhibits, in the far wing of the spectrum, a peculiar structure with an aspect other than a smooth wing (on a logarithmic plot) which contrasts with any of the existing studies, and whose explanation lies in the distinct way in which overlap and exchange interactions interfere with the classical electrostatic ones in making the polarizability anisotropy, α∥ - α⊥. Here, we delve deeper into that study by reporting data for that spectrum up to 450 cm(-1) and for even- and odd-order spectral moments up to M6, as well as quantum lineshapes, generated from SCF, CCSD, and CCSD(T) models for α∥ - α⊥, which are critically compared with the experiment. On account of the knowledge of the spectrum over the augmented frequency domain, we show how the inverse scattering problem can be tackled both effectively and economically, and we report an analytic function for the anisotropy whose quantum lineshape faithfully reproduces our observations.

Molecular iodine (I2) emission from two Laminaria species (Phaeophyceae) and impact of irradiance and temperature on I2 emission into air and iodide release into seawater from Laminaria digitata.

Kelps of the genus Laminaria accumulate iodine at high concentrations, but the iodine retaining capacity can be affected by emersion and physiological stress. In this study, I2 emission into the atmosphere from Laminaria digitata and Laminaria hyperborea was compared under controlled low irradiances and temperatures. The two species exhibited different I2 emission rates as blades of L. digitata emitted I2 at rates five times higher than those from newly-grown blades (current growth season) of L. hyperborea. I2 emission was not detectable from old blades (previous growth season) of L. hyperborea. Additionally, effects of irradiance and temperature on both I2 emission into air and net I(-) release into seawater where assessed for L. digitata while monitoring photo-physiological parameters as stress indicators. Irradiances between 30 and 120 µmol photons m(-2) s(-1) had only marginal effects on both I2 emission and I(-) release rates, but physiological stress, indicated by photoinhibition, was observed. The results suggest that the irradiances applied here were not stressful enough to impact on the iodine release. By contrast, at elevated temperatures (20 °C), photoinhibition was accompanied by an increase in I2 emission rates, but net I(-) release rates remained similar at 10-20 °C. High I2 emission rates into air and I(-) release into seawater observed from L. digitata underpin the fundamental function of this kelp as mediator of coastal iodine fluxes.

Molecular iodine emission rates and photosynthetic performance of different thallus parts of Laminaria digitata (Phaeophyceae) during emersion.

The emission of molecular iodine (I(2)) from the stipe, the meristematic area and the distal blade of the brown macroalga Laminaria digitata (Hudson) Lamouroux (Phaeophyceae) was monitored under low light and dark conditions. Photosynthetic parameters were determined to investigate both the extent of stress experienced by different thallus parts and the effects of emersion on photosynthesis. Immediately after air exposure, intense I(2) emission was detectable from all thallus parts. I(2) emission declined continuously over a period of 180 min following the initial burst, but was not affected by the light regime. The total number of mole of I(2) emitted by stipes was approximately 10 times higher than those emitted from other thallus parts. Initial I(2) emission rates (measured within 30 min of exposure to air) were highest for stipes (median values: 2,999 and 5,222 pmol g(-1) dw min(-1) in low light and dark, respectively) and lower, by one order of magnitude, for meristematic regions and distal blades. After exposure to air for between 60 and 180 min, I(2) emission rates of all thallus parts were reduced by 70-80%. Air exposure resulted in a decrease of the maximum photosystem II (PSII) efficiency (F(v)/F(m)) by 3%, and in a 25-55% increase of the effective PSII quantum efficiency (F(v)/F'(m)); this was caused by a higher fraction of open reaction centres (q(P)), whereas the efficiency of the latter in capturing energy (F'(v)/F'(m)) remained constant. The results indicate the presence of an iodine pool which is easily volatilised and depleted due to air exposure, even under apparently low stress conditions.

Long optical cavities for open-path monitoring of atmospheric trace gases and aerosol extinction.

An incoherent broadband cavity-enhanced absorption spectroscopy setup employing a 20 m long optical cavity is described for sensitive in situ measurements of light extinction between 630 and 690 nm. The setup was installed at the SAPHIR atmospheric simulation chamber during an intercomparison of instruments for nitrate (NO(3)) radical detection. The long cavity was stable for the entire duration of the two week campaign. A detection limit of approximately 2 pptv for NO(3) in an acquisition time of 5 s was established during that time. In addition to monitoring NO(3), nitrogen dioxide (NO(2)) concentrations were simultaneously retrieved and compared against concurrent measurements by a chemiluminescence detector. Some results from the campaign are presented to demonstrate the performance of the instrument in an atmosphere containing water vapor and inorganic aerosol. The spectral analysis of NO(3) and NO(2), the concentration dependence of the water absorption cross sections, and the retrieval of aerosol extinction are discussed. The first deployment of the setup in the field is also briefly described.