Search Results (5)

Background/Objectives: Chromosome research is essential for advancing our understanding of cytogenetics, gene regulation and numerous aspects of organismal health. Staining chromosomes with 4′,6-diamidino-2-phenylindole (DAPI) and applying Fluorescence Lifetime Imaging Microscopy (FLIM) enables the assessment of structural changes in pericentromeric and heterochromatin-rich region of chromosomes 1, with a shorter fluorescence lifetime (FLT) in the pericentromeric regions compared to the arms. Methods: We used FLIM to optimise sample preparation conditions for more robust imaging and furthermore to measure the impact of low-dose X-ray ionising radiation on chromosome structure when labelled with DAPI. Results: We applied this method to different DNA stains bound to chromosomes where only DAPI led to a clear FLT difference between the chromosome arms (p,q) with 2.98 ± 0.12 ns and 2.65 ± 0.07 ns at the pericentromeric region, while similar stains, such as Hoechst 33258 and NucBlue^TM did not highlight these regions as clearly following FLIM analysis. Our data showed that chromosomes of cells irradiated with 0.1 Gy and 1 Gy did not show a significant change in FLTs (2.94 ± 0.09 ns on the arms and 2.60 ± 0.06 ns on the pericentromeric region) of chromosome 1. Whilst irradiation with 0.5 Gy led to a noticeable and significant reduction in FLT with 2.42 ± 0.13 ns on the arms and 2.12 ± 0.06 ns on the pericentromeric region of HeLa chromosomes. The same pattern could also be seen on X-ray-irradiated T-cell chromosomes. Conclusions: These findings indicate that DAPI FLT may be a useful tool to measure chromosomal structural changes and further suggests that chromosomes undergo distinct structural changes at the pericentromeric region following low-dose irradiation. Full article

Intracellular monitoring of pH and polarity is crucial for understanding cellular processes and functions. This study employed pH- and polarity-sensitive nanomaterials such as carbon dots (CDs) for the intracellular sensing of pH, polarity, and viscosity using integrated time-resolved fluorescence anisotropy (FA) imaging (TR-FAIM) and fluorescence lifetime (FLT) imaging microscopy (FLIM), thereby enabling comprehensive characterization. The functional groups on the surface of CDs exhibit sensitivity to changes in the microenvironment, leading to variations in fluorescence intensity (FI) and FLT according to pH and polarity. The FLT of CDs in aqueous solution changed gradually from 6.38 ± 0.05 ns to 8.03 ± 0.21 ns within a pH range of 2–8. Interestingly, a complex relationship of FI and FLT was observed during measurements of CDs with decreasing polarity. However, the FA and rotational correlation time (θ) increased from 0.062 ± 0.019 to 0.112 ± 0.023 and from 0.49 ± 0.03 ns to 2.01 ± 0.27 ns, respectively. This increase in FA and θ was attributed to the higher viscosity accompanying the decrease in polarity. Furthermore, CDs were found to bind to three locations in Escherichia coli: the cell wall, inner membrane, and cytoplasm, enabling intracellular characterization using FI and FA decay imaging. FLT provided insights into cytoplasmic pH (7.67 ± 0.48), which agreed with previous works, as well as the decrease in polarity in the cell wall and inner membrane. The CD aggregation was suspected in certain areas based on FA, and the θ provided information on cytoplasmic heterogeneity due to the aggregation and/or interactions with biomolecules. The combined TR-FAIM/FLIM system allowed for simultaneous monitoring of pH and polarity changes through FLIM and viscosity variations through TR-FAIM. Full article

Fluorescence Lifetime (FLT) of intrinsic fluorophores may alter under the change in metabolic state. In this study, the FLT of rabbit retina was investigated in vivo after laser irradiation using fluorescence lifetime imaging ophthalmoscopy (FLIO). The retina of the Chinchilla bastard rabbits was irradiated with a 514 nm diode laser. FLIO, fundus photography, and optical coherence tomography (OCT) were conducted 30 min and 1 to 3 weeks after treatment. After strong coagulation, the FLT at laser spots was significantly elongated immediately after irradiation, conversely shortened after more than a week. Histological examination showed eosinophilic substance and melanin clumping in subretinal space at the coagulation spots older than one week. The FLT was also elongated right around the coagulation spots, which corresponded to the discontinuous ellipsoid zone (EZ) on OCT. This EZ change was recovered after one week, and the FLT became the same level as the surroundings. In addition, there was a region around the laser spot where the FLT was temporarily shorter than the surrounding area. When weak pulse energy was applied to selectively destroy only the RPE, a shortening of the FLT was observed immediately around the laser spot within one week after irradiation. FLIO could serve as a tool to evaluate the structural and metabolic response of the retina to laser treatments. Full article

We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime (FLT) detection, to perform high-throughput screening (HTS) in living cells for discovery of potential heart-failure drugs. Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump (SERCA2a), often due to excessive inhibition by phospholamban (PLB), a small transmembrane protein. We sought to discover small molecules that restore SERCA2a activity by disrupting this inhibitory interaction between PLB and SERCA2a. Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer (FRET) to detect changes in binding or structure of the complex. To optimize sensitivity to these changes, we engineered a biosensor that concatenates the two fluorescently labeled proteins on a single polypeptide chain. This SERCA2a-PLB FRET biosensor construct is functionally active and effective for HTS. By implementing 2-wavelength FLT detection at extremely high speed during primary HTS, we culled fluorescent compounds as false-positive Hits. In pilot screens, we identified Hits that alter the SERCA2a-PLB interaction, and a newly developed secondary calcium uptake assay revealed both activators and inhibitors of Ca-transport. We are implementing this approach for large-scale screens to discover new drug-like modulators of SERCA2a-PLB interactions for heart failure therapeutic development. Full article

Tissue-like phantoms are widely used as a model for mimicking the optical properties of live tissue. This paper presents the results of a diffusion reflection method and fluorescence lifetime imaging microscopy measurements of fluorescein-conjugated gold nanorods in solution, as well as inserted in solid tissue-imitating phantoms. A lack of consistency between the fluorescence lifetime results of the solutions and the phantoms raises a question about the ability of tissue-like phantoms to maintain the optical properties of inserted contrast agents. Full article