BODIPY Probe Research Articles

Lipid Peroxidation Assay Using BODIPY-Phenylbutadiene Probes: A Methodological Overview.

The assessment of reactive oxygen species has increasing importance in biomedical sciences, due to their biological role in signaling pathways and induction of cell damage at low and high concentrations, respectively. Detection of lipid peroxidation with sensing probes such as some BODIPY dyes has now wide application in studies using fluorescent microplate readers, flow cytometry, and fluorescence microscopy. Two phenylbutadiene derivatives of BODIPY are commonly used as peroxidation probes, non-oxidized probes and oxidized products giving red and green fluorescence, respectively. Peculiar features of lipoperoxidation and BODIPY dye properties make this assessment a rather complex process, not exempt of doubts and troubles. Color changes and fluorescence fading that are not due to lipid peroxidation must be taken into account to avoid misleading results. As a characteristic feature of lipoperoxidation is the propagation of peroxyl radicals, pitfalls and advantages of a delayed detection by BODIPY probes should be considered.

Characterization of Oxidative Lipidomics and Autophagy Induction in Chlamydomonas reinhardtii Under Abiotic Stress.

Autophagy constitutes an essential process triggered by oxidative stress that enables cells to recycle damaged biomolecules and organelles, which is eventually traced by immunodetection with anti-ATG8. In parallel with autophagy induction, carbon metabolism in Chlamydomonas reinhardtii under abiotic stress is diverged toward lipid biosynthesis and lipid droplet accumulation, which can be analyzed by a simple thin-layer chromatography and in vivo staining with the fluorescent probe BODIPY 493/503. We show the responses in Chlamydomonas cells exposed to mercury or cadmium (0-50μM doses), as examples of oxidative stress-mediated changes in autophagy and lipid metabolism, monitored with the procedures described in this report.

Relationship between sperm telomere length and sperm quality in infertile men

Telomeres, noncoding and repetitive DNA sequences play a significant function in chromatin integrity. Telomere length is age-dependent in somatic cells, while it increases in sperm cell with age. Therefore, we aimed to assess sperm chromatin, leucocyte and sperm telomere length (LTL, STL) in spermatozoon of 38 infertile and 19 fertile men aged between 20 and 50years. Protamine deficiency (chromomycin A3 test), DNA fragmentation (TUNEL assay), lipid peroxidation (Bodipy probe) and telomere length (quantitative real-time PCR) were assessed. A significant decrease in mean of sperm concentration and motility and a significant increase in means of sperm abnormal morphology, DNA fragmentation, lipid peroxidation and protamine deficiency were observed in infertile compared with fertile men. In addition, the mean of LTL and STL were significantly shorter in infertile men compared with fertile individuals. We observed significant associations between telomere length with sperm concentration, DNA fragmentation and lipid peroxidation. We hypothesised that increased oxidative stress in spermatozoa of infertile men can result in abnormal packaging of chromatin, damage of DNA and shorter sperm telomere length. Together, these anomalies may account for fertility failure in these individuals.

A lysosome specific, acidic-pH activated, near-infrared Bodipy fluorescent probe for noninvasive, long-term, in vivo tumor imaging

Long-term, in vivo, fluorescent cell tracking probes are useful for understanding complex cellular processes including tissue regeneration, communication, development, invasion, and cancer metastasis. A near-infrared fluorescent, water-soluble probe is particularly important for studying these biological events and processes. Herein, a lysosome specific, near-infrared Bodipy probe with increased fluorescent intensity in the acidic, lysosome environment is reported. This Bodipy probe is packaged in a nanoparticle using DSPE-PEG2000. The resulting nanoparticle is intravenously delivered to a tumor xenograft, where the fluorescent Bodipy becomes useful for non-invasive, long-term, in vivo fluorescent tumor imaging for periods greater than 36days. These long-term, in vitro and in vitro tracking data indicate that the described Bodipy nanoparticles hold great potential for monitoring biological processes.

Detection of hydrogen sulfide using BODIPY based colorimetric and fluorescent on-off chemosensor

A colorimetric and fluorescence turn-off probe 10-(4-azido phenyl)-5,5-difluoro-5h-dipyrrolo[1,2-c:1’,2’-f][1,3,2] diazaborinin-4-ium-5-uide, 1, for selective detection of H2S is reported. The probe displayed a robust decrease in fluorescence intensity with high sensitivity, specificity and least toxicity to detect exogenous H2S and also in live normal human oral fibroblast cells loaded with probe 1. The detection limit of probe 1 being 0.17 µm for H2S. A colorimetric and fluorescence turn-off BODIPY probe 1, for selective detection of exogenous H2S in live normal human oral fibroblast cells loaded with probe 1 and poisonous H2S gas formed under biodegradation in sewage waters and water tanks and corroded pipe lines. The detection limit of probe 1 being 0.17 μM

Multi-state amine sensing by electron transfers in a BODIPY probe.

Amines are ubiquitous in the chemical industry and are present in a wide range of biological processes, motivating the development of amine-sensitive sensors. There are many turn-on amine sensors, however there are no examples of turn-on sensors that utilize the amine's ability to react by single electron transfer (SET). We investigated a new turn-on amine probe with a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore. BODIPY fluorescence is first preprogrammed into an off state by internal photoinduced electron transfer (PET) to an electron-deficient quinolinium ring, resulting in fluorescence quenching. At low concentrations of aliphatic amine (0 to 10 mM), this PET pathway is shut down by external SET from the amine to the photoexcited charge-transfer state of the probe and the fluorescence is turned on. At high concentrations of amine (50 mM to 1 M), we observed collisional quenching of the BODIPY fluorescence. The probe is selective for aliphatic amines over aromatic amines, and aliphatic thiols or alcohols. The three molecular processes modulate the BODIPY fluorescence in a multi-mechanistic way with two of them producing a direct response to amine concentrations. The totality of the three molecular processes produced the first example of a multi-state and dose-responsive amine sensor.

A new fluorescent probe for sensing of biothiols and screening of acetylcholinesterase inhibitors.

A new N2O-type BODIPY probe (LF-Bop) has been proposed for the selective and sensitive detection of biologically relevant small molecular thiols. This detection is based on the Michael addition reaction between the thiol and nitrostyrene groups in the probe, which decreases the quenching effect from the nitro group, thus resulting in the recovery of the deep-red fluorescence from the BODIPY structure. The results show that LF-Bop is able to detect all tested free thiols through a fluorescence turn-on assay. The lowest limit of detection (LOD) for glutathione was found to be down to nanomolar levels (220 nM). Based on this probe, we have developed a new fluorescence assay for the screening of acetylcholinesterase inhibitors. In total, 11 natural and synthetic alkaloids have been evaluated. Both experimental measurements and theoretical molecular docking results reveal that both natural berberine and its synthetic derivative dihydroberberine are potential inhibitors of acetylcholinesterase.

Imaging lipophilic regions in rodent brain tissue with halogenated BODIPY probes

The effect of halogen substitution in fluorescent BODIPY species was evaluated in the context of staining lipids in situ within brain tissue sections. Herein we demonstrate that the halogenated species maintain their known in vitro affinity when applied to detect lipids in situ in brain tissue sections. Interestingly, the chlorine substituted compound revealed the highest specificify for white matter lipids. Furthermore, the halogen substituted compounds rapidly detected lipid enriched cells, in situ, associated with a case of brain pathology and neuroinflammation.

The BODIPY-Based Chemosensor for Fluorometric/Colorimetric Dual Channel Detection of RDX and PA.

A fluorometric/colorimetric dual-channel chemosensor based on a hydrazine-substituted BODIPY probe has been successfully fabricated for the detection of RDX and PA. The chemosensor displays turn-on fluorescence behavior upon RDX with a detection limit of 85.8 nM, while showing a turn-off response to PA with a detection limit of 0.44 μM. Meanwhile, an obvious color difference is observed by the naked-eye after the reaction for RDX. Thus, in application, a two-to-two logic gate is constructed for potential application in explosives detection. Additionally, portable equipment is also developed for in situ determination of RDX.

Reduced sperm telomere length in individuals with varicocele is associated with reduced genomic integrity

Varicocele, defined as enlarged varicose veins in the scrotum, is the most common identifiable cause of male infertility. There are significant correlations between oxidative stress and varicocele-related infertility due to testicular hyperthermia, which can result in low sperm function. In addition, recent excessive oxidative stress can affect sperm telomere length and integrity of sperm DNA. Therefore, we assessed sperm telomere length as a potential marker of paternal genome integrity and leukocyte telomere length as an internal control (real-time PCR), along with sperm chromatin status (TUNEL and chromomycin A3 assay), and lipid peroxidation (Bodipy probe) in 18 infertile men with grade II or III varicocele, and 20 fertile men. Means of sperm parameters, sperm and leukocyte telomere length were significantly lower, while means of sperm DNA fragmentation, protamine deficiency, and lipid peroxidation were significantly higher in infertile men with varicocele compared to fertile men. Therefore, shortened telomere length in sperm and leukocytes is likely associated with increased oxidative stress related to the state of varicocele, which also accounts for increase in sperm DNA fragmentation. Thus, assessment of leukocyte telomere length could be taken as an indicator of antioxidant capacity in an individual, which also affects sperm function.

Macromolecular inversion-driven polymer insertion into model lipid bilayer membranes

Macromolecules of amphiphilic invertible polymers (AIPs) are capable of self-assembly into micellar assemblies of various morphologies in solvents of different polarities. The micellar assemblies in aqueous media are capable of encapsulating poorly aqueous soluble cargo and can undergo inverse conformational change and cargo release in contact with non-polar media, including potentially, cell membranes. Thus, invertible micellar assemblies have significant potential in drug delivery and related domains. However, to date there have been few investigations into their interactions with lipid membranes.Herein, we investigate the interactions of three recently developed AIPs of varying hydrophobicity/hydrophilicity balance with a highly fluidic microcavity supported 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer. We combined electrochemical impedance spectroscopy (EIS) with fluorescence correlation spectroscopy (FCS) to understand how the AIP micellar assemblies impacted bilayer permeability and fluidity respectively, across polymer concentrations above and below their critical micelle concentrations (cmcs). At concentration as above their cmcs, all of the AIPs explored increased permeability and decreased the fluidity of the lipid membrane. The extent of impact depended on the hydrophobicity of the AIP. PEG600-PTHF650, the most hydrophobic of the polymers, synthesized from PEG (molecular weight 600 g/mol) and PTHF (molecular weight 650 g/mol) exerted the greatest influence on the bilayer’s physical properties and fluorescence imaging and correlation data indicate that PEG600-PTHF650 micelles loaded with BODIPY probes adsorb and invert at the lipid membrane with release of cargo into the bilayer.This study should help inform future advancement of AIPs for membrane molecular delivery.

Linker length in fluorophore-cholesterol conjugates directs phase selectivity and cellular localisation in GUVs and live cells.

Lipid membrane fluorescent probes that are both domain-selective and compatible with demanding microscopy methods are crucial to elucidate the presence and function of rafts and domains in cells and biophysical models. Whereas targeting fluorescent probes to liquid-disordered (Ld) domains is relatively facile, it is far more difficult to direct probes with high selectivity to liquid-ordered (Lo) domains. Here, a simple, one-pot approach to probe–cholesterol conjugation is described using Steglich esterification to synthesise two identical BODIPY derivatives that differ only in the length of the aliphatic chain between the dye and cholesterol. In the first, BODIPY-Ar-Chol, the probe and cholesterol were directly ester linked and in the second BODIPY-Ahx-Chol, a hexyl linker separated probe from cholesterol. Uptake and distribution of each probe was compared in ternary, phase separated giant unilamellar vesicles (GUVs) using a commercial Ld marker as a reference. BODIPY-Ar-Chol targets almost exclusively the Ld domains with selectivity of >90% whereas by contrast introducing the C6 linker between the probe and cholesterol drove the probe to Lo with excellent selectivity (>80%). The profound impact of the linker length extended also to uptake and distribution in live mammalian cells. BODIPY-Ahx-Chol associates strongly with the plasma membrane where it partitioned preferably into opposing micron dimensioned do-mains to a commercial Ld marker and its concentration at the membrane was reduced by cyclodextrin treatment of the cells. By contrast the BODIPY-Ahx-Chol permeated the membrane and localised strongly to lipid droplets within the cell. The data demonstrates the profound influence of linker length in cholesterol bioconjugates in directing the probe.

A red fluorescent BODIPY probe for iridium (III) ion and its application in living cells.

A new red fluorescent probe 1 based on BODIPY skeleton has been successfully synthesized through introduction of 2-(thiophen-2-yl) quinoline moiety at meso- and 3-position, which exhibits excellent optical performance, including high fluorescence quantum yield, large pseudo Stokes' shift as well as high selectivity and sensitivity towards iridium (III) ion in aqueous solution and in living cells.

Recent advances in the application of BODIPY in bioimaging and chemosensing

Recent work on the use of BODIPY probes in bioimaging/bio-staining, as well as in chemosensing, is reviewed.

Lysosome-targeting turn-on red/NIR BODIPY probes for imaging hypoxic cells.

A small series of fluorescent lysosome-targeting probes based on the BODIPY fluorophore and containing morpholine and nitro groups were rationally designed. These probes emitted light from green to NIR wavelengths, and provided specificity for imaging the lysosomes of hypoxic cells. The electron-withdrawing nitrophenyl group at the meso position was found to lead to highly efficient nonradiative decay of the S1 state, and hence a recovery of fluorescence when reduction of the nitro group occurred under hypoxic conditions.

Isolation and Propagation of Single Inclusion-Derived Chlamydia Using Laser Microdissection.

Other than its routine application for capturing pure cell populations from fixed tissue sections for diverse downstream molecular assays, laser microdissection enables isolation of single live cells. Here we describe a method for the isolation of single Chlamydia trachomatis-infected cells using a laser microdissection system, in which the dissected samples are captured via gravity. Cells infected by C. trachomatis at low multiplicity of infection are marked with the fluorescent Golgi-specific probe BODIPY® FL C5-ceramide, to facilitate identification of the cells with chlamydial inclusions under the microscope. Individual C. trachomatis-infected cells are harvested into separate wells with a pregrown host cell monolayer. Inclusions in harvested cells maturate, and the released elementary bodies infect the host cell monolayer, thus initiating propagation of single inclusion-derived Chlamydia. The method can be used for generation of microbiological clones of C. trachomatis and recovery of transformants and mutants. Isolated single Chlamydia-infected cells can also be examined by diverse downstream molecular assays to reveal unknown features of the Chlamydia replication at a single inclusion level.

Evaluation of sperm telomere length in infertile men with failed/low fertilization after intracytoplasmic sperm injection

Telomeres are non-coding, repetitive DNA sequences (TTAGGG repeats) that play an important role in maintaining genome integrity. Unlike in somatic cells, telomere length in spermatozoa increases with male age and is considered as a molecular marker of sperm quality. The aetiology of failed fertilization following intracytoplasmic sperm injection (ICSI) is multifactorial; perhaps one of the reasons for this failure in these individuals is shortened sperm telomere length. This study therefore aimed to assess sperm telomere length in addition to DNA damage, lipid peroxidation and protamine deficiency in infertile men with previously failed/low fertilization post-ICSI. Semen samples were obtained from infertile men with previous failed/low fertilization rates (n = 10). Chromatin integrity (chromomycin A3 staining and TUNEL assay), lipid peroxidation (BODIPY probe) and telomere length (real-time PCR) for semen samples from these men were compared with samples obtained from fertile individuals (n=10). The results showed significantly higher mean values for sperm DNA damage, lipid peroxidation and reduced telomere length in spermatozoa of infertile men with previous failed/low fertilization compared with fertile individuals (P<0.05). Failed/low fertilization rates could be related to oxidative stress resulting in short telomere length, and also increased sperm chromatin damage and lipid peroxidation. From literature sources, shortened telomere length may lead to detachment of chromosomes from the nuclear membrane, the consequences of which are defects in the process of spermatogenesis, pronuclei formation, and delayed or arrested cell cycle post-ICSI.

A Photoactivatable BODIPY Probe for Localization‐Based Super‐Resolution Cellular Imaging

The synthesis and application of a photoactivatable boron-alkylated BODIPY probe for localization-based super-resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously unknown boron-photodealkylation reaction with a single low-power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization-based microscopy of biological systems. The probe can be easily linked to nucleophile-containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super-resolution imaging of microtubules is demonstrated. This is the first example of single-molecule localization-based super-resolution microscopy using a visible-light-activated BODIPY compound as a fluorescent probe.

A Photoactivatable BODIPY Probe for Localization‐Based Super‐Resolution Cellular Imaging

AbstractThe synthesis and application of a photoactivatable boron‐alkylated BODIPY probe for localization‐based super‐resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously unknown boron‐photodealkylation reaction with a single low‐power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization‐based microscopy of biological systems. The probe can be easily linked to nucleophile‐containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super‐resolution imaging of microtubules is demonstrated. This is the first example of single‐molecule localization‐based super‐resolution microscopy using a visible‐light‐activated BODIPY compound as a fluorescent probe.

Abstract 312: Cardiomyocyte-secreted Signaling Factors Modulate Adipocyte Differentiation And Function

Heart failure (HF) alters cardiomyocyte metabolism by altering energy substrate utilization and mitochondrial function, which has potential implications for global metabolic health. Recent studies demonstrated that cardiomyocyte-released signaling factors communicate with peripheral organ systems to alter metabolic function and that signaling pathways activated by HF control the release of these factors. However, the identity of these factors is still under investigation. Conditioned media (CM) collected from neonatal rat ventricular myocytes (NRVMs) was used to treat 3T3-L1 pre-adipocytes to 1) identify signaling molecules released by cardiomyocytes that could regulate global metabolic function and 2) determine how HF signaling pathways alter their release. NRVM-CM slowed adipocyte differentiation as measured by lipid accumulation using the fluorescent probe BODIPY 493/503 (57±8% decrease in normalized fluorescence compared to control media) and reduced the expression of adipocyte-related proteins. NRVM CM treatment also increased basal mitochondrial oxygen consumption (78 ± 3 vs 62±4 pmol/min) and maximal respiration (270±12 vs 220 ± 16 pmol/min) in mature adipocytes. CM protein fractionation revealed that released-factor activity was retained in &gt;100kDa fractions and this activity was abolished by trypsin digestion, suggesting a novel, high-MW cardiomyocyte-released signaling protein that targets adipocytes. To determine if NRVM-released factor activity is altered by HF signaling, the ability of G-protein kinase 2 (GRK2, upregulated by HF) signaling activity to alter NRVM-released factor activity was investigated. GRK2 overexpression in NRVMs increased the activity of this released-factor as measured by further decreased 3T3-L1 differentiation (lipid accumulation measured by BODIPY fluorescence was 70±8% compared to CM from GFP-overexpressing NRVMs), while overexpression of the GRK2 inhibitor βARKct increased adipocyte differentiation (140±13% compared to control NRVM-CM). These results suggest a novel, high-MW cardiomyocyte-released signaling protein under the control of HF signaling pathways communicates with adipocytes to alter adipose tissue function and potentially alter global metabolic function.