Spectrally and Time-Resolved Fluorescence Imaging of 22-NBD-Cholesterol in Human Peripheral Blood Mononuclear Cells in Chronic Kidney Disease Patients.

The interaction of the fluorescent probe 22-NBD-cholesterol with membranes of human peripheral blood mononuclear cells (PBMC) was tested by time- and spectrally resolved fluorescence imaging to monitor the disturbance of lipid metabolism in chronic kidney disease (CKD) and its treatment with statins. Blood samples from healthy volunteers (HV) and CKD patients, either treated or untreated with statins, were compared. Spectral imaging was done using confocal microscopy at 16 spectral channels in response to 458 nm excitation. Time-resolved imaging was achieved by time-correlated single photon counting (TCSPC) following excitation at 475 nm. The fluorescence of 22-NBD-cholesterol was mostly integrated into plasmatic membrane and/or intracellular membrane but was missing from the nuclear region. The presence of two distinct spectral forms of 22-NBD-cholesterol was uncovered, with significant variations between studied groups. In addition, two fluorescence lifetime components were unmasked, changing in CKD patients treated with statins. The gathered results indicate that 22-NBD-cholesterol may serve as a tool to study changes in the lipid metabolism of patients with CKD to monitor the effect of statin treatment.

Fluorescence responsiveness of unicellular marine algae Dunaliella to stressors under laboratory conditions.

We examined the responsiveness of unicellular green alga Dunalliela tertiolecta to selected stressors employing confocal- and time-resolved imaging of endogenous fluorescence. Our aim was to monitor cell endogenous fluorescence changes under exposure to heavy metal Cd, acidification, as well as light by laser-induced photobleaching. The accumulation of Cd in algae cells was confirmed by the secondary ion mass spectroscopy technique. For the first time, custom-made computational techniques were employed to evaluate separately the fluorescence in the flagella vs. the body region. In the presence of Cd, we recorded increase in the green fluorescence in the flagella region in the form of opacities, without change in the fluorescence lifetimes, suggesting higher availability of the fluorescent molecules. Under acidification, we noted significant rise in the green fluorescence in the flagella region, but associated with longer fluorescence lifetimes, pointing to changes in the algae environment. Photobleaching experiments corroborated gathered observations. Obtained data support a differential responsiveness of the flagella vs. the body region to stressors and enable us to better understand the pathophysiological changes of algal cells in culture under stress conditions.

Algal cell response to laboratory-induced cadmium stress: a multimethod approach.

We examined the response of algal cells to laboratory-induced cadmium stress in terms of physiological activity, autonomous features (motility and fluorescence), adhesion dynamics, nanomechanical properties, and protein expression by employing a multimethod approach. We develop a methodology based on the generalized mathematical model to predict free cadmium concentrations in culture. We used algal cells of Dunaliella tertiolecta, which are widespread in marine and freshwater systems, as a model organism. Cell adaptation to cadmium stress is manifested through cell shape deterioration, slower motility, and an increase of physiological activity. No significant change in growth dynamics showed how cells adapt to stress by increasing active surface area against toxic cadmium in the culture. It was accompanied by an increase in green fluorescence (most likely associated with cadmium vesicular transport and/or beta-carotene production), while no change was observed in the red endogenous fluorescence (associated with chlorophyll). To maintain the same rate of chlorophyll emission, the cell adaptation response was manifested through increased expression of the identified chlorophyll-binding protein(s) that are important for photosynthesis. Since production of these proteins represents cell defence mechanisms, they may also signal the presence of toxic metal in seawater. Protein expression affects the cell surface properties and, therefore, the dynamics of the adhesion process. Cells behave stiffer under stress with cadmium, and thus, the initial attachment and deformation are slower. Physicochemical and structural characterizations of algal cell surfaces are of key importance to interpret, rationalize, and predict the behaviour and fate of the cell under stress in vivo.

Spectrally and spatially resolved laser-induced photobleaching of endogenous flavin fluorescence in cardiac myocytes.

Naturally occurring endogenous fluorescence of flavins, arising in response to excitation by visible light, offers broad opportunity to investigate mitochondrial metabolic state directly in living cells and tissues, including in clinical settings. However, photobleaching, the loss of the autofluorescence intensity following prolonged exposure to light is an inherent phenomenon occurring during the fluorescence acquisition, which can have a negative impact on the recorded data, particularly in the context of measurement of metabolic modulations in pathophysiological conditions. In the presented study, we present a detailed analysis of endogenous flavins fluorescence photobleaching arising in living cardiac cells during spectrally-resolved confocal imaging. We demonstrate significant nonuniform photobleaching related to different bleaching rates of individual flavin components, resolved by linear spectral unmixing of the recorded signals. Induced photodamage was without effect on the cell morphology, but lead to significant modifications of the cell responsiveness to metabolic modulators and its contractility, suggesting functional metabolic alterations in the recorded cells. These findings point to the necessity of inducing limited photobleaching during metabolic screening in all studies involving visible light excitation and fluorescence acquisition in living cells. © 2018 International Society for Advancement of Cytometry.

The Impact of Vitamin D3 Supplementation on Mechanisms of Cell Calcium Signaling in Chronic Kidney Disease.

Intracellular calcium concentration in peripheral blood mononuclear cells (PBMCs) of patients with chronic kidney disease (CKD) is significantly increased, and the regulatory mechanisms maintaining cellular calcium homeostasis are impaired. The purpose of this study was to examine the effect of vitamin D3 on predominant regulatory mechanisms of cell calcium homeostasis. The study involved 16 CKD stages 2-3 patients with vitamin D deficiency treated with cholecalciferol 7000-14000 IU/week for 6 months. The regulatory mechanisms of calcium signaling were studied in PBMCs and red blood cells. After vitamin D3 supplementation, serum concentration of 25(OH)D3 increased (P < 0.001) and [Ca(2+)]i decreased (P < 0.001). The differences in [Ca(2+)]i were inversely related to differences in 25(OH)D3 concentration (P < 0.01). Vitamin D3 supplementation decreased the calcium entry through calcium release activated calcium (CRAC) channels and purinergic P2X7 channels. The function of P2X7 receptors was changed in comparison with their baseline status, and the expression of these receptors was reduced. There was no effect of vitamin D3 on P2X7 pores and activity of plasma membrane Ca(2+)-ATPases. Vitamin D3 supplementation had a beneficial effect on [Ca(2+)]i decreasing calcium entry via CRAC and P2X7 channels and reducing P2X7 receptors expression.

Time- and spectrally resolved characteristics of flavin fluorescence in U87MG cancer cells in culture.

Early detection of cancer is crucial for the successful diagnostics of its presence and its subsequent treatment. To improve cancer detection, we tested the progressive multimodal optical imaging of U87MG cells in culture. A combination of steady-state spectroscopic methods with the time-resolved approach provides a new insight into the native metabolism when focused on endogenous tissue fluorescence. In this contribution, we evaluated the metabolic state of living U87MG cancer cells in culture by means of endogenous flavin fluorescence. Confocal microscopy and time-resolved fluorescence imaging were employed to gather spectrally and time-resolved images of the flavin fluorescence. We observed that flavin fluorescence in U87MG cells was predominantly localized outside the cell nucleus in mitochondria, while exhibiting a spectral maximum under 500 nm and fluorescence lifetimes under 1.4 ns, suggesting the presence of bound flavins. In some cells, flavin fluorescence was also detected inside the cell nuclei in the nucleoli, exhibiting longer fluorescence lifetimes and a red-shifted spectral maximum, pointing to the presence of free flavin. Extra-nuclear flavin fluorescence was diminished by 2-deoxyglucose, but failed to increase with 2,4-dinitrophenol, the uncoupler of oxidative phosphorylation, indicating that the cells use glycolysis, rather than oxidative phosphorylation for functioning. These gathered data are the first step toward monitoring the metabolic state of U87MG cancer cells.