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This protocol focuses on measuring the microscope's lateral and axial resolution, essential for reporting size measurements of near-resolution limit objects or distances between them. Here resolution is expressed as the Full Width at Half Maximum (FWHM) of a measured Point Spread Function (PSF) of sub-resolution size beads. PSF is highly related to objective quality and condition, but also depends strongly upon other parameters ranging from sample preparation to signal detection. Monitoring PSF over time will identify possible aberrations in the system (e.g., damaged, unclean objective, defective or not adapted oil, etc.). We define test sample preparation, image acquisition, and data analysis protocols for point scanning and spinning disk confocal microscopes.

A common technique used to differentiate bacterial species and to determine evolutionary relationships is sequencing their 16S ribosomal RNA genes. However, this method fails when organisms exhibit high similarity in these sequences. Two such strains that have identical 16S rRNA sequences are Mycobacterium indicus pranii (MIP) and Mycobacterium intracellulare. MIP is of significance as it is used as an adjuvant for protection against tuberculosis and leprosy; in addition, it shows potent anti-cancer activity. On the other hand, M. intracellulare is an opportunistic pathogen and causes severe respiratory infections in AIDS patients. It is important to differentiate these two bacterial species as they co-exist in immuno-compromised individuals. To unambiguously distinguish these two closely related bacterial strains, we employed Raman and resonance Raman spectroscopy in conjunction with multivariate statistical tools. Phenotypic profiling for these bacterial species was performed in a kinetic manner. Differences were observed in the mycolic acid profile and carotenoid pigments to show that MIP is biochemically distinct from M. intracellulare. Resonance Raman studies confirmed that carotenoids were produced by both MIP as well as M. intracellulare, though the latter produced higher amounts. Overall, this study demonstrates the potential of Raman spectroscopy in differentiating two closely related mycobacterial strains. Graphical abstract.

In April 2020, the QUality Assessment and REProducibility for Instruments and Images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models, and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper 1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; 2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers, and observers of such; 3) outlines the current actions of the QUAREP-LiMi initiative, and 4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics., Comment: 17 pages, 3 figures, shortened abstract, Co-Lead Authors: Glyn Nelson and Ulrike Boehm, Corresponding author: Roland Nitschke

Chronic exposure to fluoride (F) beyond the permissible limit (1.5 ppm) is known to cause detrimental health effects by induction of oxidative stress-mediated DNA damage overpowering the DNA repair machinery. In the present study, we assessed F induced oxidative stress through monitoring biochemical parameters and looked into the effect of chronic F exposure on two crucial DNA repair genes Ogg1 and Rad51 having important role against ROS induced DNA damages. To address this issue, we exposed Swiss albino mice to an environmentally relevant concentration of fluoride (15 ppm NaF) for 8 months. Results revealed histoarchitectural damages in liver, brain, kidney and spleen. Depletion of GSH, increase in lipid peroxidation and catalase activity in liver and brain confirmed the generation of oxidative stress. qRT-PCR result showed that expressions of Ogg1 and Rad51 were altered after F exposure in the affected organs. Promoter hypermethylation was associated with the downregulation of Rad51. F-induced DNA damage and the compromised DNA repair machinery triggered intrinsic pathway of apoptosis in liver and brain. The present study indicates the possible association of epigenetic regulation with F induced neurotoxicity.

Iron(III) complexes of a vitamin B-6 Schiff base and NNN-donor ligands with pendant boron-dipyrromethene (BODIPY) moieties; namely, Fe(L1-3)(L-4,L-5)](NO3) (1-4), where L-1 is benzyl-bis(pyridin-2-yl)methyl]methanamine (bzdpa in 1), L-2 is a noniodinated BODIPY-appended dipicolylamine ligand (in 2, 3), L-3 is the diiodinated BODIPY analogue in 4, L-4 is a vitamin B-6 Schiff base, namely 3-hydroxy-5(hydroxymethyl)-4-{(2-hydroxyphenyl)imino]methyl}-2-meth ylpyridine (in 1, 3, and 4), and L-5 is 2-(2-hydroxyphenylimino)-methyl]phenol (in 2) as a nonpyridoxal Schiff base, were prepared, characterized, and their cellular localization and cytotoxic activity in light and in the dark were studied. The diiodo-BODIPY complex 4 displays remarkable photoinduced cytotoxicity in visible light (400-700 nm), with IC50 values within 0.11-0.25 mu m and about 200-fold lower dark toxicity. Complex 3, being fluorescent, was used for cellular imaging by confocal microscopy. Complex 4 shows supercoiled pUC19 DNA cleavage activity through the generation of singlet oxygen (O-1(2)) as the reactive oxygen species (ROS). Selective uptake of the complexes is observed from competitive cellular incorporation assays in cancer and noncancer cells. The complexes also show no apparent toxicity up to 100 mu m in the immortal human lung epithelial cells HPL1D in both light and the dark. Complex 3 shows preferential accumulation in lysosomes, giving a Pearson's correlation coefficient value of about 0.7.

Copper(II) complexes [Cu(L1/L2/L3)(A)] (1–3), where H2A·HCl is a vitamin B6 Schiff base, viz. 3-hydroxy-5-(hydroxymethyl)-4-(((2-hydroxyphenyl)imino)methyl)-2-methylpyridin-1-ium chloride, L1 and L2 are 2-(2-pyridyl)benzimidazole based borondipyrromethene (BODIPY) ligands and L3 is 2-(2-pyridyl)benzimidazole, were prepared, characterized and their visible light-induced anti-cancer activity was studied. Complex 3, characterized by X-ray crystallography, exhibits a distorted square-pyramidal geometry for copper (τ = 0.33). Complexes 1 and 2 showed absorption bands at 500 and 535 nm, respectively, in 20% DMSO/Dulbecco's phosphate buffered saline (DPBS) medium. Complex 1 exhibited emission at ∼510 nm (λexc = 480 nm) (ΦF = 0.1) in 20% DMSO/DPBS, while the non-emissive diiodo-BODIPY complex 2 is an efficient photosensitizer. The green fluorescent complex 1 enabled us to study its cellular uptake and localization. It showed selective uptake in proliferating cancer cells and significant mitochondrial localization (Pearson's coefficient = 0.7). Complex 2 showed excellent photocytotoxicity (400–700 nm, 10 J cm−2) in HeLa, MCF-7 and HepG2 cancer cells with IC50 values within 0.4–0.6 μM, while remaining less toxic in the dark and in non-cancerous HPL1D cells (photocytotoxic index ∼50). Complex 2, remarkable in targeting cancer cells over non-cancerous cells, showed photoinduced generation of singlet oxygen, causing apoptotic cell death, thus satisfying the major requirements of targeted photodynamic therapy.

An easily synthesized fluorescein-based luminescent dye has been utilized for the dual-mode detection of histamine at nanomolar concentrations at pH 7.0 in water. The specific response to histamine was achieved by imidazole-catalyzed 'imine formation' reaction. The protocol was subsequently applied for the estimation of histamine in complex biological milieu such as human blood serum and urine samples. Furthermore, the dose-dependent cellular uptake of histamine and de novo synthesis (by thapsigargin treatment) was visualized in RAW 264.7, a mouse macrophage cell line. We have also developed portable paper strips for rapid, on-site detection of histamine without involving costly instruments.

Copper(II) and zinc(II) beta-diketonates of N,N-donor ligands, viz. Cu(dppz)(cur)](NO3) (1), Cu(acdppz)(cur)](NO3) (2), Cu(acdppz) (acac)](NO3) (3), Zn(dppz)(cur)](NO3) (4) and Zn(acdppz)(cur)] (NO3) (5), where dppz is dipyrido3,2-a:2',3'-c]phenazine, acdppz is 11-(9-acridinyl)dipyrido3,2-a:2',3'-c]phenazine, cur and acac are mono-deprotonated curcumin (Hcur) and acetyl acetone (Hacac), were synthesized, characterized and their photocytotoxicity studied. An analogue of complex 3, viz. Cu(acdppz) (acac)(H2O)]Cl 1/2 (NO3) 1= 2 (3a), structurally characterized by X-ray crystallography, has a cationic complex in square-pyramidal geometry with CuN2O3 core with an axial aqua ligand. Complexes 1, 2, 4 and 5 displayed emission at similar to 520 nm (lambda(exc): 430 nm) in dimethyl sulfoxide (DMSO) giving a fluorescence quantum yield value within 0.01-0.06. The complexes, in contrast to free curcumin, were fairly stable in cellular media up to 36 h, with no apparent degradation of the bound curcumin. The complexes were photocytotoxic (IC50: 0.3 4.5 mu M) in human cervical cancer (HeLa), breast cancer (MCF-7) and liver cancer (HepG2) cells. The apoptotic cell death is due to reactive oxygen species formation. Complexes 2 and 5 showed significant uptake in HeLa cells, localizing predominantly in the cytoplasm. Mechanistic data from the pUC19 DNA photocleavage study suggest involvement of acridine and curcumin in photo-generation of singlet oxygen and hydroxyl radicals as the ROS in light of 400-700 nm.

Copper(ii) complexes [Cu(L

Copper(II) pyridoxal Schiff base complexes Cu(L-1/L-2)(B)]ClO4 (1-4), where HL1 is 4-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)-5-(hydroxymethyl)-2-methylp yridin-3-ol (in 1 and 2), HL2 is 2-(((2-(1H-imidazol-4-yl)ethyl)imino)methyl)phenol (in 3, 4), B is 11-(9-acridinyl)dipyrido3,2-a:2',3'-c]phenazine (acdppz in 1 and 3), dipyrido3,2-a:2',3'-c]phenazine (in 2) and 1,10-phenanthroline (in 4), were synthesized, characterized and their photocytotoxicity in visible light, intracellular localization, cellular uptake and DNA photocleavage activity were studied. Complex 4 was characterized by X-ray crystallography. Complexes 1 and 3 having acdppz as photosensitizer showed significant photocytotoxicity in visible light in HeLa and MCF7 cells giving IC50 value of

Selective low (15 mg sodium fluoride (NaF)/L) and relatively high (150 mg NaF/L) doses of in vivo fluoride (F) treatment to Swiss albino mice through drinking water elicited organ-specific toxicological response. All the F-exposed groups showed severe alterations in both liver and kidney architectures, but there was no significant change in the rate of water consumption and body weight. Vacuolar degeneration, micronecrotic foci in the hepatocytes, and hepatocellular hypertrophy were evident in the mice exposed to low dose (15 mg NaF/L for 30 days) while sinusoidal dilation with enlarged central vein surrounded by deep-blue erythrocytes were preponderant when treated with the same dose for a period of 90 days. Blood filled spaces, disintegration of tubular epithelium, and atrophy of glomeruli were also recorded in the kidney of the same treatment group. Change in reduced glutathione level (GSH), glutathione-s-transferase (GST) activity, malondialdehyde (MDA) production in both liver and kidney, disturbances in liver function, induction of heat shock protein 70 (Hsp 70) expression in kidney and its down regulation in liver were positively correlated with histopathological lesion.

Treatment of mice with 15 mg l−1 sodium fluoride (NaF) for 30 days increased the number of cell death, chromosomal aberrations (CAs) and ‘cells with chromatid breaks’ (aberrant cells) compared with control. The present study was intended to determine whether the fluoride (F)-induced genotoxicity could be reduced by substituting high F-containing water after 30 days with safe drinking water, containing 0.1 mg F ions l−1. A significant fall in percentage of CAs and aberrant cells after withdrawal of F-treatment following 30 days of safe water treatment in mice was observed which was highest after 90 days, although their levels still remained significantly high compared with the control group. This observation suggests that F-induced genotoxicity could be reduced by substituting high F-containing water with safe drinking water. Further study is warranted with different doses and extended treatment of safe water to determine whether the induced damages could be completely reduced or not. Copyright © 2011 John Wiley & Sons, Ltd.

Treatment of mice with 15 mg l(-1) sodium fluoride (NaF) for 30 days increased the number of cell death, chromosomal aberrations (CAs) and 'cells with chromatid breaks' (aberrant cells) compared with control. The present study was intended to determine whether the fluoride (F)-induced genotoxicity could be reduced by substituting high F-containing water after 30 days with safe drinking water, containing 0.1 mg F ions l(-1). A significant fall in percentage of CAs and aberrant cells after withdrawal of F-treatment following 30 days of safe water treatment in mice was observed which was highest after 90 days, although their levels still remained significantly high compared with the control group. This observation suggests that F-induced genotoxicity could be reduced by substituting high F-containing water with safe drinking water. Further study is warranted with different doses and extended treatment of safe water to determine whether the induced damages could be completely reduced or not.

A significant level of reactive oxygen species generation was observed in sodium fluoride (NaF) treated mouse bone marrow cells (BMCs). Reduced glutathione (GSH) as a free radical scavenger could be an important determining factor in F-induced genotoxicity. We therefore attempted to monitor GSH to understand the mechanism of NaF-induced genotoxicity. NaF was injected intra-peritoneally in normal, buthionine sulfoximine (BSO) or N-acetyl-L-cysteine (NAC) treated mice (n = 5). After 13 h of NaF-treatment BMCs were collected to harvest them at the same divisional cycle and processed for analysis of cell cycle, induction of apoptosis and chromosomal aberrations (CAs). Level of GSH was also measured concomitantly. NaF induced significant CAs in all treatment groups except at 2.5 mg NaF kg(-1) body weight. BSO-treatment alone induced significantly high frequency of CAs. BSO treatment prior to injection of 2.5-7.5 mg NaF kg(-1) b.w. was found to increase the frequency of CAs, significantly when compared with the positive control group, but the level was not significant in case of higher doses of NaF treatment (15 and 30 mg kg(-1) b.w.). NaF-treated cells also showed a higher population of Annexin-V positive cells. NAC pre-treatment significantly reduced the extent of NaF-induced CAs, which clearly indicates the involvement of GSH in the NaF response. However, further study is warranted to evaluate the low synergistic effect of BSO on higher doses of NaF-induced CAs.