Automated Fourier space region-recognition filtering for off-axis digital holographic microscopy

Automated label-free quantitative imaging of biological samples can greatly benefit high throughput diseases diagnosis. Digital holographic microscopy (DHM) is a powerful quantitative label-free imaging tool that retrieves structural details of cellular samples non-invasively. In off-axis DHM, a proper spatial filtering window in Fourier space is crucial to the quality of reconstructed phase image. Here we describe a region-recognition approach that combines shape recognition with an iterative thresholding to extracts the optimal shape of frequency components. The region recognition technique offers fully automated adaptive filtering that can operate with a variety of samples and imaging conditions. When imaging through optically scattering biological hydrogel matrix, the technique surpasses previous histogram thresholding techniques without requiring any manual intervention. Finally, we automate the extraction of the statistical difference of optical height between malaria parasite infected and uninfected red blood cells. The method described here pave way to greater autonomy in automated DHM imaging for imaging live cell in thick cell cultures

Host red blood cell modifications induced by the malaria parasite Plasmodium falciparum

This project investigated the molecular host cell modifications behind the virulence of the Plasmodium falciparum parasite through the functional characterisation of two exported parasite proteins previously implicated to play a role in parasite virulence: PFB0115w and PFI1780w. Additionally we aimed to develop a flow-based high throughput technique to measure the loss of deformability in infected erythrocytes, a characteristic feature for parasite virulence in vivo. PFB0115w, previously stated to be essential for the expression of the main virulence factor, erythrocyte membrane protein 1 (PfEMP1), in pregnancy associated malaria (PAM), was investigated here with immunoprecipitation of GFP-tagged PFB0115w and loss-of-function analysis. Immunoprecipitation of detergent solubilised PFB0115w-GFP revealed that PFB0115w co-precipitates with exported proteins mature erythrocyte surface antigen (MESA), PFD0080c and glycophorin binding protein (GBP). Analysis of HA-tagged MESA, in native conditions, strongly indicates that MESA and PFB0115w are present in the same complex however an additional reciprocal pulldown with MESA is required to confirm MESA as an interacting partner. We also characterised the loss-of-function phenotype of PFB0115w by assaying the surface presentation of PfEMP1. The disruption of PFB0115w did not disrupt the surface presentation of PFEMP1. This indicates that PFB0115w is not essential for PfEMP1 expression. Therefore, future experiments on the co-precipitates of PFB0115w and loss-of-function studies are still required to determine the function of PFB0115w. PFI1780, a predicted export protein, was recently stated in an ex vivo study to interact with the cytoplasmic segment of the main virulence factor, PfEMP1. Here we investigated the putative interaction of PFI1780w and PfEMP1 through the immunoprecipitation of GFP-tagged PFI1780w. However, immunoprecipitation of detergent solubilised PFI1780w-GFP revealed that PFI1780w and PfEMP1 do not interact in vivo. We also developed a flow-based technique to measure deformability of individual cells using a quantitative phase imaging technique called digital holographic microscopy (DHM). DHM can quantify fluctuations in the height and volume of cells, giving us an indirect quantification of cell deformability. We quantified displacement of infected or uninfected cells that were bound to the base of a fluidic channel and under external fluidic pressure, as a measure of the cell deformability. We measured centre of mass (COM) displacement in both the direction of flow (x-axis) and in height (z-axis) as a unit for total cell deformability and membrane deformability respectively. The method was validated with a deformability model using uninfected erythrocytes artificially stiffened with glutaraldehyde. The method was able to distinguish total cell deformation of cells treated with concentrations ranging from 0% to 0.02%. We could also distinguish height deformation of cells treated with 0.005% glutaraldehyde as compared to untreated (0%) cells however not for concentrations above 0.005%. We then compared the deformability of trophozoite infected cells against infected cells and observed a significantly lower rate of deformation of the total cell for infected cells against uninfected cell (p < 0.0001). These results indicated that the method could distinguish total cell deformability of infected cells against uninfected cells. Although the results are promising, this technique would require further optimisation to increase throughput efficiency and improve quantification of membrane deformability. Thus, we were able to disprove previous studies on PFB0115w and PFI170w on its function in context of the main virulence factor PfEMP1 and showed PFB0115w may instead contribute towards the deformability phenotype. Additionally, we developed a flow-based method to quantify deformability using DHM that can infer deformability of single cells at in situ conditions

Containment of Polynitroaromatic Compounds in a Hydrogen Bonded Triarylbenzene Host

Co-crystallization of energetic materials has emerged as an important technique to modify their critical properties such as stability, sensitivity, etc. Using 1,3,5-tris­(4′-aminophenyl)­benzene (TAPB) as a novel co-crystal former, we have prepared co-crystals of 2,4,6-trinitrotoluene (TNT), 2,4,6-trinitrophenol (TNP), and <i>m</i>-dinitrobenzene (<i>m</i>DNB). Molecular structures of the co-crystals have been determined from single crystal X-ray diffraction data. The diffraction data analysis reveals that strong intermolecular π–π interaction directs the intercalation of polynitroaromatic explosives (PNACs) between the layers of TAPB molecules, which leads to the formation of vertically overlapped -A-B-A-B- types of π-stacks. Both TNT and TNP form π-interactions with the center of TAPB with 1:1 molar ratios, while <i>m</i>DNB forms a complex in a 1:3 stoichiometry through stacking between peripheral rings. The crystal lattices are further stabilized through interstack hydrogen bonds (N–H···N and N–H···O) between amino groups of TAPB and nitro groups of PNACs. NMR and Fourier transform infrared spectra further provide the information about the presence of various interactions in the crystal systems. Owing to the π electron-rich nature and ease of synthesis, triphenylbenzene systems are promising host candidates for co-crystallization of PNAC analytes

Adaptive spatial filtering for off-axis digital holographic microscopy based on region-recognition approach with iterative thresholding

Here we propose a region-recognition approach with iterative thresholding, which is adaptively tailored to extract the appropriate region or shape of spatial frequency. In order to justify the method, we tested it with different samples and imaging conditions (different objectives). We demonstrate that our method provides a useful method for rapid imaging of cellular dynamics in microfluidic and cell cultures

Automated fourier space region-recognition filtering for off-axis digital holographic microscopy

Automated label-free quantitative imaging of biological samples can greatly benefit high throughput diseases diagnosis. Digital holographic microscopy (DHM) is a powerful quantitative label-free imaging tool that retrieves structural details of cellular samples non-invasively. In off-axis DHM, a proper spatial filtering window in Fourier space is crucial to the quality of reconstructed phase image. Here we describe a region-recognition approach that combines shape recognition with an iterative thresholding method to extracts the optimal shape of frequency components. The region recognition technique offers fully automated adaptive filtering that can operate with a variety of samples and imaging conditions. When imaging through optically scattering biological hydrogel matrix, the technique surpasses previous histogram thresholding techniques without requiring any manual intervention. Finally, we automate the extraction of the statistical difference of optical height between malaria parasite infected and uninfected red blood cells. The method described here paves way to greater autonomy in automated DHM imaging for imaging live cell in thick cell cultures