LiveLattice: Real-time visualization of tilted light-sheet microscopy data using a memory-efficient transformation algorithm.

Light-sheet fluorescence microscopy (LSFM), a prominent fluorescence microscopy technique, offers enhanced temporal resolution for imaging biological samples in four dimensions (4D; x, y, z, time). Some of the most recent implementations, including inverted selective plane illumination microscopy (iSPIM) and lattice light-sheet microscopy (LLSM), rely on a tilting of the sample plane with respect to the light sheet of 30-45 degrees to ease sample preparation. Data from such tilted-sample-plane LSFMs require subsequent deskewing and rotation for proper visualization and analysis. Such transformations currently demand substantial memory allocation. This poses computational challenges, especially with large datasets. The consequence is long processing times compared to data acquisition times, which currently limits the ability for live-viewing the data as it is being captured by the microscope. To enable the fast preprocessing of large light-sheet microscopy datasets without significant hardware demand, we have developed WH-Transform, a novel GPU-accelerated memory-efficient algorithm that integrates deskewing and rotation into a single transformation, significantly reducing memory requirements and reducing the preprocessing run time by at least 10-fold for large image stacks. Benchmarked against conventional methods and existing software, our approach demonstrates linear scalability. Processing large 3D stacks of up to 15 GB is now possible within one minute using a single GPU with 24 GB of memory. Applied to 4D LLSM datasets of human hepatocytes, human lung organoid tissue, and human brain organoid tissue, our method outperforms alternatives, providing rapid, accurate preprocessing within seconds. Importantly, such processing speeds now allow visualization of the raw microscope data stream in real time, significantly improving the usability of LLSM in biology. In summary, this advancement holds transformative potential for light-sheet microscopy, enabling real-time, on-the-fly data processing, visualization, and analysis on standard workstations, thereby revolutionizing biological imaging applications for LLSM, SPIM and similar light microscopes.

Simultaneous determination of efavirenz, rifampicin and its metabolite desacetyl rifampicin levels in human plasma.

A simple and rapid isocratic, high performance liquid chromatography (HPLC) assay employing solid phase extraction (SPE) for the simultaneous determination of the anti HIV drug, efavirenz, the anti-tuberculosis drug, rifampicin and the desacetyl metabolite of rifampicin in plasma from HIV/tuberculosis infected patients has been developed. Using a Zorbax SB-Phenyl reverse-phase analytical column with UV detection, good separation and detection of the drugs was attained within a 10min run time. Intra- and inter-assay precision RSD values were found to be less than 15% at the concentrations examined (0.1-20µg/mL). The LOQ was found to be 0.1µg/mL for each agent and the assay was found to generate a linear response up to 20µg/mL. This low cost assay can accurately detect efavirenz and rifampicin concentrations within a clinically relevant concentration range using standard chromatography equipment, making it particularly applicable to resource-limited settings.

New approaches in the detection of calcium-containing microcrystals in synovial fluid.

BACKGROUND: The presence of calcium phosphate crystals such as basic calcium phosphate and calcium pyrophosphate dihydrate in intra-articular fluid is linked to a number of destructive arthropathies and detection of these deposits is often pivotal for early diagnosis and appropriate management of such disease. RESULTS: We describe the use of a calcium-sensitive dye, Fluo-4, to selectively label calcium-containing mineral deposits in synovial fluid, which can then be easily visualized using a standard fluorescence microscope. Furthermore, we have combined the fluorescent properties of the tagged crystals with flow cytometry as a fast and semi-quantitative method of detection. CONCLUSION: Dot-plots were used to quantify differences between various types of arthropathies and confirmed by visual observation of the crystals under a fluorescence microscope.

Detection of basic calcium phosphate crystals in osteoarthritis.

Osteoarthritis (OA) is the most common human joint disorder. Its complex pathogenesis remains poorly understood but appears multifactorial. To date, no specific pharmacological agent has been identified to alter the disease course of OA. Calcification of articular cartilage is a hallmark of OA and evidence suggests it contributes directly to joint degeneration. Calcium crystals are frequently found in OA synovial fluid but their exact role in the disease process is unclear. Basic calcium phosphate (BCP) crystals are the predominant crystal type found in OA and recent data indicate a pathogenic role for these crystals in OA. However, information on the exact frequency and distribution of BCP crystals vary considerably, mainly due to the lack of simple and reliable methods of detection. The purpose of this review is to describe the current available methods for detecting BCP crystals and to highlight their obvious advantages and limitations. Recent developments in the field are also discussed with particular reference to their potential clinical applicability. Improved methods of detection for BCP crystals could potentially aid the diagnosis of OA and the development of novel therapies.

Determination of calcium in synovial fluid samples as an aid to diagnosing osteoarthritis.

BACKGROUND: Microscopic inorganic crystals are commonly observed in the synovial fluid of patients suffering from arthritic diseases. Basic calcium phosphate (BCP) crystals are known to occur quite commonly in the joint fluid of osteoarthritis (OA) patients and are insoluble at physiological pH. Current analysis of patient synovial fluid depends on light microscopy and staining with Alizarin Red-S. Both methods cannot identify crystals < 1µm in size and are highly subjective. This article investigates the use of o-cresolphthalein complexone (OCP), a colorimetric reagent, to quantify calcium from crystals isolated from synovial fluid samples as a means of identifying the presence of BCP and, hence, improving the diagnosis of OA. RESULTS: Inorganic crystals were isolated following degradation of the biological sample matrix with hyaluronidase. 1-M HNO(3) was used for crystal dissociation into ions and the colorimetric response of OCP to calcium was measured in a basic environment of 2-amino-2-methyl-1-propanol. The average calcium content in OA patient samples was up to 40% higher than in rheumatoid arthritis (RA) patient samples. RA samples were used as a comparison, because they are generally accepted to be crystal free. Within the OA group, higher levels of calcium were detected in three out of 12 synovial fluid samples, which correlated with a significantly greater number of BCP crystals detected during microscopic examination. CONCLUSIONS: A simple method based on colorimetry for measurement of calcium content and semiquantification of BCP crystals in synovial fluid samples has been described. Sample pretreatment following addition of hyaluronidase proved to be effective in reducing viscosity and aiding the dissociation of BCP crystals in synovial fluid samples.

Simultaneous determination of anthracyclines and taxanes in human serum using online sample extraction coupled to high performance liquid chromatography with UV detection.

An online SPE-LC method that can determine both anthracyclines and taxanes simultaneously in human serum samples is reported. The entire method of extraction, separation and UV detection was achieved online by column switching between an SPE column (Biotrap 500 (20 x 4 mm)) and an analytical column (Zorbax XDB C18, 150 x 4.6 mm, 5 microm) with a 23 min total cycle time. The method is linear (r(2)>0.998) over the range of 0.5-25 microg/mL. The analytes of interest are retained on the SPE column with good recovery (84-117%), while proteins and other serum components elute to waste. This online clean-up is much faster (150 s) and less manual than traditional off-line extraction methods. Using 0.1 mL spiked serum samples, the LOQ was 0.5 microg/mL. Intra- and inter-day precision were acceptable (< or = 15% RSD) at and above the LOQ. The method was applied to the analysis of serum samples from patients undergoing chemotherapy with these agents.

Development of a high-performance liquid chromatographic-mass spectrometric method for the determination of cellular levels of the tyrosine kinase inhibitors lapatinib and dasatinib.

A highly sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed to quantify cellular levels of the tyrosine kinase inhibitors (TKIs) dasatinib (Sprycel) and lapatinib (Tykerb, Tyverb). Cellular samples were extracted with a tert-butyl methyl ether:acetonitrile (3:1, v/v):1 M ammonium formate pH 3.5 (8:1, v/v) mixture. Separation was achieved on a Hyperclone BDS C18 (150 mm x 2.0 mm 3 microm) column with isocratic elution using a mobile phase of acetonitirile-10 mM ammonium formate, pH 4 (54:46, v/v), at a flow rate of 0.2 mL/min. The TKIs were quantified using a triple quadrupole mass spectrometer which was operated in multi-reaction-monitoring mode employing positive electrospray ionisation. The limit of detection and limit of quantification for lapatinib was determined to be 15 and 31 pg on column, respectively. The limit of detection and quantification for dasatinib was 3 and 15 pg on column, respectively. The method allowed for sensitive and accurate determination of cellular levels of dasatinib and lapatinib. In addition, we examined the potential for this method to be utilised to quantitate other TKIs, using gefitinib, erlotinib, imatinib and sorafenib as examples. In principle, these agents were also quantifiable by this method, however, no drug specific validation studies were undertaken with these TKIs. The data indicates that in the cancer cell-line model, DLKP, significantly more lapatinib accumulates in cells in comparison to dasatinib. Additionally, over-expression of the membrane protein drug transporter, P-glycoprotein (P-gp) a common cancer drug resistance mechanism, greatly reduces the cellular accumulation of dasatinib but not of lapatinib.

A review of analytical methods for the determination of aminoglycoside and macrolide residues in food matrices.

The development of antibiotic resistance in bacteria has been attributed to the overuse of antimicrobials in human medicine. Another route by which humans are exposed to antibiotics is through the animal foods we eat. In modern agricultural practice, veterinary drugs are being used on a large scale, administered for treating infection or prophylactically to prevent infection. Hence, there is pressure on analytical scientists to detect and confirm the presence of antimicrobials in foods of animal origin. The aminoglycosides and macrolides are two families of antibiotics, each with important applications in veterinary medicine. These antibiotics are widely used in the treatment of bacterial disease, e.g., aminoglycosides for mastitis and macrolides for enteric infections. They have also been used as feed additives for growth promotion. As a result, legislation has been laid down by the European commission in which member states must meet strict criteria for monitoring residues (including antimicrobials). Testing for low levels of aminoglycosides and macrolides in foods is a priority and hence the development of fast, reliable, sensitive methods for their extraction and subsequent analysis is of great interest. This paper reviews analytical methods for both extracting and determining these classes of antibiotics in various food matrices focusing in particular on the last 10 years. Extraction and clean-up methods such as deproteinisation, and solid-phase extraction are described. Various screening methods are also covered including thin layer chromatography (TLC), enzyme immunoassay, capillary electrophoresis (CE) and microbiological assays. Finally, liquid chromatography (LC) methods are discussed which are combined with mass spectrometry (MS) when sensitivity requirements are stringent.

Isolation of calcium phosphate crystals from complex biological fluids using bisphosphonate-modified superparamagnetic beads.

The surface of superparamagnetic magnetic beads was modified with bisphosphonates to selectively capture calcium phosphate crystals from complex biological fluids (i.e. synovial fluid).

Detection of calcium phosphate crystals in the joint fluid of patients with osteoarthritis - analytical approaches and challenges.

Clinically, osteoarthritis (OA) is characterised by joint pain, stiffness after immobility, limitation of movement and, in many cases, the presence of basic calcium phosphate (BCP) crystals in the joint fluid. The detection of BCP crystals in the synovial fluid of patients with OA is fraught with challenges due to the submicroscopic size of BCP, the complex nature of the matrix in which they are found and the fact that other crystals can co-exist with them in cases of mixed pathology. Routine analysis of joint crystals still relies almost exclusively on the use of optical microscopy, which has limited applicability for BCP crystal identification due to limited resolution and the inherent subjectivity of the technique. The purpose of this Critical Review is to present an overview of some of the main analytical tools employed in the detection of BCP to date and the potential of emerging technologies such as atomic force microscopy (AFM) and Raman microspectroscopy for this purpose.

Rapid and sensitive liquid chromatography-tandem mass spectrometry for the quantitation of epirubicin and identification of metabolites in biological samples.

A highly sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method has been developed for the determination of epirubicin in serum and cell specimens using daunorubicin as an internal standard. Using atmospheric pressure chemical ionisation (APCI), the epirubicin metabolites were readily distinguishable by their fragmentation pattern in the mass spectrometer. Selected reaction monitoring (SRM) mode was employed for quantitation of epirubicin and the metabolites. Following extraction, chromatography was performed on a C18 column with a mobile phase consisting of water-acetonitrile-formic acid, pH 3.2, with a flow rate of 200mul/min. The limit of detection (LOD) and the limit of quantitation (LOQ) of this method in serum were determined to be 1.0 and 2.5ng/ml, respectively. Linearity of the method was verified over the concentration range of 2.5-2000ng/ml, with a high correlation coefficient (R(2)>/=0.998). For the extraction procedure, an aliquot of 500mul serum, spiked with internal standard, was extracted using a chloroform-2-isopropanol (2:1, v/v) mixture. The method has been applied to the analysis of epirubicin in cancer cell samples and the identification of known and unknown metabolites in clinical trial patient serum samples.

Peptide synthesis by recombinant Fasciola hepatica cathepsin L1.

Synthesis of the tripeptide Z-Phe-Arg-SerNH2 has been accomplished by a recombinant cysteine protease, cathepsin L1 from liver fluke (Fasciola hepatica), using Z-Phe-Arg-OMe as acyl acceptor and SerNH2 as nucleophile in 0.1 M ammonium acetate pH 9.0-12.5% v/v acetonitrile at 37 degrees C. LC-MS detection indicated tripeptide formation after 10 min, continuing up to 5.5 h. The ester Z-Phe-Arg-OMe was detected throughout the experiment but the hydrolysis product Z-Phe-Arg-OH appeared early and in quite large amounts. We believe that this is the first application of a parasite protease in enzymatic peptide synthesis.

Purification, identification and characterisation of seprase from bovine serum.

The study and identification for the first time of a soluble form of a seprase activity from bovine serum is presented. To date, this activity has only been reported to be an integral membrane protease but has been known to shed from its membrane. The activity was purified 30,197-fold to homogeneity, using a combination of column chromatographies, from bovine serum. Inhibition by DFP, resulting in an IC(50) of 100:nM confirms classification as a serine protease. The protease after separation and visualisation by native PAGE was subjected to tryptic digestion and the subsequent peptides sequenced. Each peptide sequenced was found to be present in the primary structure of seprase/fibroblast activation protein (FAP), a serine gelatinase specific for proline-containing peptides and macromolecules. Substrate specificity studies using kinetic, RP-HPLC and LC-MS analysis of synthetic peptides suggest that this peptidase has an extended substrate-binding region in addition to the primary specificity site S(1). This analysis revealed at least five subsites to be involved in enzyme-substrate binding, with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P(1) was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end of the scissile bond (P'(1)) was evident. The enzyme also showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, JTP-4819, Fmoc-Ala-pyrrCN and Z-Phe-Pro-BT. To date, no physiological substrate has clearly been defined for this protease but its ability to effectively degrade gelatin suggests a candidate protein substrate in vivo and a possible role in extracellular matrix protein degradation.

Characterisation of the active site of a newly-discovered and potentially significant post-proline cleaving endopeptidase called ZIP using LC-UV-MS.

There are enzymes that specifically recognise the amino acid proline within peptides and proteins that are called post-proline cleaving enzymes. Many of them are implicated in neurodegenerative disorders and psychiatric diseases. ZIP is one such newly-discovered peptidase. In this work, it has been purified from bovine serum and subjected to various analytical studies in order to characterise it. A series of reactions between synthesised peptides and ZIP were carried out in order to elucidate the size and specificity of the active site of the enzyme. On-line LC-MS was carried out on samples before and after incubation and the results obtained allowed us to detect if cleavage of the peptides was taking place, and if so, where in the peptide chain. It was found that the enzyme has a preference for another larger, bulky amino acid to follow the proline and that little or no cleavage was observed when polar acidic or other small amino acids were in that location. In terms of the size of the active site, the endopeptidase was found to have optimum activity when there were two more amino acids after the proline, with a fall-off in activity detected for the longer peptides. Data from kinetic studies confirmed the LC-MS results. The methodology described in this paper, which is a combination of LC separation and UV-MS detection, is required for the accurate monitoring of the reactions between the peptidase and its peptide substrates and for analysis of the products of such enzyme-peptide reactions. This work will assist in the design of site-directed inhibitors for new drug therapies.

Characterisation of the ester-substituted products of the reaction of p-t-butyl calix[4]arene and ethyl bromoacetate using LC-UV-MS and LC-DAD.

A series of derivatisation reactions between p-t-butyl calix[4]arene and ethyl bromoacetate were carried out in order to prepare 1,3 diester substituted calix[4]arene. Mass spectral data, obtained from direct injection of samples, indicated that the reactions were rich in the desired product. Since the ultra violet (UV) spectra of the desired product and possible impurities are very similar, liquid chromatography (LC) chromatographic data seemed to corroborate these results. However, when on-line LC-UV-MS was carried out and each LC peak subjected to MS analysis as it eluted, a very different picture emerged. It was found that many of these reactions actually contained high levels of the monoester product which, having less affinity for sodium in the MS, is therefore seriously underestimated in any direct injection assay. LC-diode array detection (DAD) methods were also used to help successfully identify and characterise the compounds being formed in these complex reactions. The overall results obtained in this paper allowed the optimal reaction conditions to be determined for this reaction. LC-MS analysis of the chromatographic peaks also identified the presence of two isomers of the diester substituted calix[4]arene (1,3 and 1,2 diesters). The combination of LC and UV/MS detection is required for accurate analysis of the products of such reactions.