Environmental pollution by microplastics (MPs) represents a serious burden of the 21st century. Sensing the interactions of photosynthetic organisms with MPs is based on the study of their endogenous fluorescence derived from chlorophylls. Fluorescently labelled custom-made MPs were tested. We also recorded endogenous fluorescence of the moss in the presence of “naturally-occurring” MPs (polyethylene content of 2 mg/g, non fluorescent) in suspended matter (SM) from the river Rhine. Performed experiments evaluated the distribution of the MPs, as well as the sensitivity of endogenous fluorescence of chlorophylls to their presence. Understanding the interaction of living organisms with MPs will help to assess the impact of this environmental pollution and eventually to propose new approaches for its removal from water sources.
Fluorescence microscopy based on non-linear optical phenomena has become a new perspective optical tool in biological imaging. This contribution is aimed at synthesis and micro/spectroscopic characterization of novel S,N-heteroarene-functionalized BODIPY-class dyes with potential application as fluorescence and NLO markers in high-resolution multiphoton microscopy. The molecules have been classified with respect to their NLO response and most perspective ones has been tested for potential applications on cultured cancer cells. Supported by: EU-H2020 871124 (Laserlab-Europe) and 810701 (LAMatCU), Slovak research grant agencies APVV-17-0324, APVV-21-0503, VEGA No. 1/0669/22 and VEGA No. 2/0070/21.
Nanoparticles (NPs) from various metals (Zinc, Nickel, Cobalt, Copper) were designed and fabricated by direct synthesis using femtosecond laser ablation in liquids. Employing confocal microscopy with spectral detection and fluorescence lifetime imaging (FLIM), we have evaluated interaction of fabricated NPs with living Chlorella sp. algae by means of their naturally presented endogenous fluorescence. Live cell imaging was done in spectral region 500-550 nm and 650- 710 nm to evaluate the effect of NPs on both, the green and the red fluorescence that is derived from flavonoids/carotenoids and chlorophylls respectively. We observed fluorescence intensity decrease in the red spectral region by all but Ni NPs. The presence of NPs also lead to an increase in the blue fluorescence at 477-488 nm, possibly resulting from reflected light. Gathered observations constitute the first step towards creation of methodological approaches for fast natural biosensing of the effects of environmental pollution directly in live algae.
Our contribution is focused on broadening of the spectrum of available non-linear optical (NLO)-phores (contrast agents for nonlinear optical microscopy) by design and synthesis of new organic dyes with appropriate optical properties. One of the main pre-requisites of microscopy utilizing non-linear excitation is the existence of molecules that are able to provide NLO response for the second-harmonic generation (SHG) or for the two-photon excited fluorescence (TPEF). Many molecules naturally occurring in living tissue such as collagens or NAD(P)H were successfully used in this regard, but there is a natural interest in broadening of the spectrum of available NLO-phores. Gathered results confirm applicability of the newly synthesized dyes as new potential NLO-phores for confocal laser scanning microscopy with nonlinear excitation in rat aorta.
Multimodal optical imaging of suspected tissues is showing to be a promising method for distinguishing suspected cancerous tissues from healthy ones. In particular, the combination of steady-state spectroscopic methods with timeresolved fluorescence provides more precise insight into native metabolism when focused on tissue autofluorescence. Cancer is linked to specific metabolic remodelation detectable spectroscopically. In this work, we evaluate possibilities and limitations of multimodal optical cancer detection in single cells, collagen-based 3D cell cultures and in living organisms (whole mice), as a representation of gradually increasing complexity of model systems.
The last decade has witnessed a rapid growth of nanoscale-oriented biosensors that becomes one of the most promising and rapidly growing areas of modern research. Despite significant advancements in conception of such biosensors, most are based at evaluation of molecular, or protein interactions. It is however becoming increasingly evident that functionality of a living system does not reside in genome or in individual proteins, as no real biological functionality is expressed at these levels. Instead, to comprehend the true functioning of a biological system, it is essential to understand the integrative physiological behavior of the complex molecular interactions in their natural environment and precise spatio-temporal topology. In this contribution we therefore present a new concept for creation of biosensors, bio-inspired from true functioning of living cells, while monitoring their endogenous fluorescence, or autofluorescence.
Design and fabrication of appropriate biocompatible microstructures that ensure fixation and control of experimental conditions for live cell and bacteria observations is an important prerequisite for number of real time experiments. Our approach is to design engineered microfabricated 3D structures for growth of cells in culture without significant modification of their metabolic state. Presented approach is aimed at evaluation of the potential applicability of biocompatible constructs in the biomedical field and thus live cell monitoring in controlled conditions. Design and evaluation of properties of materials and structures with mesoscopic arrangement and their interaction with biological objects is a prerequisite for establishment of physiologically relevant in vitro models of pathologies as well as for development of a new generation of nano / micro / bio-sensors.
Fabricated micro- and nano-structured surfaces were evaluated for use with living cells. Metabolic state was tested by means of endogenous flavin fluorescence of living peripheral blood mononuclear cells (PBMC) positioned on a coverslip, non-covered, or covered with micro- or nano-structured surfaces (OrmoComp polymer structures produced by 2-photon photopolymerisation, or Zinc Oxide (ZnO) layer fabricated by pulsed laser deposition). Confocal microscopy and Fluorescence Lifetime Imaging Microscopy (FLIM) were employed to gather flavin fluorescence lifetime images of living PBMC on structured surfaces. Gathered data are the first step towards monitoring of the live cell interaction with different micro/nano-structured surfaces and thus evaluate their potential applicability in the biomedical field.
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.
Precise evaluation of intracellular cholesterol distribution is crucial for improving diagnostics of diseased states associated with cholesterol alteration. Time-resolved fluorescence techniques are tested for non-invasive investigation of cholesterol in living cells. Fluorescent probe NBD attached to cholesterol was employed to evaluate cholesterol distribution in peripheral blood mononuclear cells (PBMC) isolated from the human blood. Fluorescence Lifetime Imaging Microscopy (FLIM) was successfully applied to simultaneously monitor the spatial distribution and the timeresolved characteristics of the NBD-cholesterol fluorescence in PBMC. Gathered data are the first step in the development of a new perspective non-invasive diagnostic method for evaluation of cholesterol modifications in diseases associated with disorders of lipid metabolism.
Lipid peroxidation is a major biochemical consequence of the oxidative deterioration of polyunsaturated lipids in cell membranes and causes damage to membrane integrity and loss of protein function. 4-hydroxy-2-nonenal (HNE), one of the most reactive products of n-6 polyunsaturated fatty acid peroxidation of membrane phospholipids, has been shown to be capable of affecting both nicotinamide adenine dinucleotide (phosphate) reduced [NAD(P)H] as well as NADH production. However, the understanding of its effects in living cardiac cells is still lacking. Our goal was to therefore investigate HNE effects on NAD(P)H noninvasively in living cardiomyocytes. Spectrally resolved lifetime detection of endogenous fluorescence, an innovative noninvasive technique, was employed. Individual fluorescence components were resolved by spectral linear unmixing approach. Gathered results revealed that HNE reduced the amplitude of both resolved NAD(P)H components in a concentration-dependent manner. In addition, HNE increased flavoprotein fluorescence and responsiveness of the NAD(P)H component ratio to glutathione reductase (GR) inhibitor. HNE also increased the percentage of oxidized nucleotides and decreased maximal NADH production. Presented data indicate that HNE provoked an important cell oxidation by acting on NAD(P)H regulating systems in cardiomyocytes. Understanding the precise role of oxidative processes and their products in living cells is crucial for finding new noninvasive tools for biomedical diagnostics of pathophysiological states.
Spectral analysis of the autofluorescence images of isolated cardiac cells was performed to evaluate and to classify the metabolic state of the cells in respect to the responses to metabolic modulators. The classification was done using machine learning approach based on support vector machine with the set of the automatically calculated features from recorded spectral profile of spectral autofluorescence images. This classification method was compared with the classical approach where the individual spectral components contributing to cell autofluorescence were estimated by spectral analysis, namely by blind source separation using non-negative matrix factorization. Comparison of both methods showed that machine learning can effectively classify the spectrally resolved autofluorescence images without the need of detailed knowledge about the sources of autofluorescence and their spectral properties.
KEYWORDS: Luminescence, Bacteria, Modulators, Time resolved spectroscopy, Electron transport, Systems modeling, Confocal microscopy, Temperature metrology, Photon counting, Semiconductor lasers
The genus Gluconobacter is frequently used for biotechnological and/or nanotechnological applications. We studied
endogenous fluorescence of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), indicator of the oxidative
metabolic state in mammalian cells, in Gluconobacter oxydans (G. oxydans). Time-resolved measurements (excitation by 375nm pulsed diode laser) were employed to record the bacterial fluorescence intensity, as well as its modifications by metabolic modulation. Results were gathered on fresh bacteria, on de-frozen ones, as well as on bacteria encapsulated
in alginate beads. NAD(P)H fluorescence increased linearly with the concentration of bacteria. Freezing, which has little effect on the viability of bacteria or the concentration-dependent fluorescence rise, affected the temperature-dependence
of NAD(P)H fluorescence. Sodium cyanide (10 mM) provoked significant rise in the NAD(P)H fluorescence, while dinitrophenol (200 μM) induced its decrease, confirming the bacterial NAD(P)H fluorescence sensitivity to modulators of electron transport chain. Gathered results demonstrate that endogenous NAD(P)H fluorescence can be successfully
recorded in the bacterial strain G. oxydans using time-resolved measurements.
Time-resolved spectrometry of endogenous nicotinamide dinucleotide phosphate [NAD(P)H] fluorescence is a useful method to evaluate metabolic oxidative state in living cells. Ouabain is a well-known pharmaceutical drug used in the treatment of cardiovascular disease, the effects of which on myocardial metabolism were recently demonstrated. Mechanisms implicated in these actions are still poorly understood. We investigate the effect of ouabain on the metabolic oxidative state of living cardiac cells identified by time-resolved fluorescence spectroscopy of mitochondrial NAD(P)H. Spectral unmixing is used to resolve individual NAD(P)H fluorescence components. Ouabain decreased the integral intensity of NAD(P)H fluorescence, leading to a reduced component amplitudes ratio corresponding to a change in metabolic state. We also noted that lactate/pyruvate, affecting the cytosolic NADH gradient, increased the effect of ouabain on the component amplitudes ratio. Cell oxidation levels, evaluated as the percentage of oxidized NAD(P)H, decreased exponentially with rising concentrations of the cardiac glycoside. Ouabain also stimulated the mitochondrial NADH production. Our study sheds a new light on the role that ouabain plays in the regulation of metabolic state, and presents perspective on a noninvasive, pharmaceutical approach for testing the effect of drugs on the mitochondrial metabolism by means of time-resolved fluorescence spectroscopy in living cells.
Cardiac cells are highly structured with a non-uniform morphology. Although precise estimation of their volume is
essential for correct evaluation of hypertrophic changes of the heart, simple and unified techniques that allow
determination of the single cardiomyocyte volume with sufficient precision are still limited. Here, we describe a novel
approach to assess the cell volume from confocal microscopy 3D images of living cardiac myocytes. We propose a fast
procedure based on segementation using active deformable contours. This technique is independent on laser gain and/or
pinhole settings and it is also applicable on images of cells stained with low fluorescence markers. Presented approach is
a promising new tool to investigate changes in the cell volume during normal, as well as pathological growth, as we
demonstrate in the case of cell enlargement during hypertension in rats.
NAD(P)H fluorescence was investigated by spectrally-resolved lifetime detection, while individual NAD(P)H
fluorescence components were resolved by spectral linear unmixing approach. Photobleaching was induced by excitation
of a defocused elliptical spot with a 375nm picosecond laser for 30s repeated every 60s for 7min. Our data indicate
presence of three individual components in cardiac cell autofluorescence (AF), and we recorded comparable
photobleaching of the two resolved NAD(P)H components ("bound" and "free"). Decrease in photon counts during
photobleaching was induced by lowering of the component amplitudes, without modification in the fluorescence
lifetimes, while the ratio of the two amplitudes remained unchanged. Gathered results are crucial for choosing
appropriate light excitation and fluorescence acquisition for prolonged studies of endogenous fluorescence aiming to
investigate changes in metabolic oxidative state in living cardiac cells during their contraction.
Tissue autofluorescence is one of the most versatile non-invasive tools for mapping the metabolic state in living tissues.
Increasing interest in the imaging and diagnosis of living cells and tissues, based on their intrinsic fluorescence rather than fluorescence labeling, is closely connected to the latest developments in high-performance spectroscopic and microscopic techniques. We investigate metabolic state of cardiac cells isolated from one additional human biopsy from transplanted pediatric patients presenting either no rejection (R0) or mild rejection (R1). Two different approaches for isolation of human cardiac myocytes are also compared. Spectrally-resolved fluorescence lifetime detection of NAD(P)H fluorescence (excitation by pulsed 375 nm picosecond laser) is tested as a promising new tool for quantitative analysis of intrinsic cellular autofluorescence signals in living cardiomyocytes. This work opens new horizons in the evaluation of cardiac transplant rejection using latest fluorescence imaging approaches.
Autofluorescence is one of the most versatile non-invasive tools for mapping the metabolic state of living tissues, such as
the heart. We present a new approach to the investigation of changes in endogenous fluorescence during cardiomyocyte
contraction - by spectrally-resolved, time correlated, single photon counting (TCSPC). Cell contraction is stimulated by
external platinum electrodes, incorporated in a home-made bath and triggered by a pulse generator at a frequency of 0.5
Hz (to stabilize sarcoplasmic reticulum loading), or 5 Hz (the rat heart rate). Cell illumination by the laser is
synchronized with cell contraction, using TTL logic pulses operated by a stimulator and delayed to study mitochondrial
metabolism at maximum contraction (10-110 ms) and/or at steady state (1000-1100 ms at 0.5 Hz). To test the setup, we
recorded calcium transients in cells loaded with the Fluo-3 fluorescent probe (excited by 475 nm pulsed picosecond
diode laser). We then evaluated recordings of flavin AF (excited by 438 nm pulsed laser) at room and physiological
temperatures. Application of the presented approach will shed new insight into metabolic changes in living, contracting
myocytes and, therefore, regulation of excitation-contraction coupling and/or ionic homeostasis and, thus, heart
excitability.
We present a new approach for analysis of multi-wavelength time-resolved spectroscopy data, based on sequential
spectral unmixing. Principal component analysis was used to identify the number and spectral profiles of the main
components of intrinsic flavin signal in multi-wavelength time-resolved fluorescence recordings from isolated living
cardiac myocytes. To determine these components, natural variations in the cardiomyocyte autofluorescence spectra
were induced by modulators of mitochondrial metabolism and respiration. Using aforementioned approach we have
identified two main components of intrinsic flavin emission in cardiac myocytes. The first component show emission
maximum at 486-504 nm and mean lifetime of 1.2 nanoseconds, the second component with peak at 522 nm has two-exponential
decay with fluorescence lifetimes of 0.3 and 3.1 nanoseconds. Comparison of gathered new results to our
previous studies of flavins in vitro and in cardiac cells clearly points to the fact that the estimated spectral components
correspond to flavin adenine dinucleotide (FAD) bound to enzyme(s) of mitochondrial metabolic chain, and to free FAD,
respectively.
Rejection of transplanted hearts remains an important reason for death of transplanted children. Finding diagnostic tools
for its detection can therefore improve the prognosis in this population of patients. Endomyocardial biopsy (EMB) by
cardiac catheterization is currently accepted as the "gold standard" for the diagnosis of rejection. Here, we investigate
new approach to monitor mitochondrial metabolic state of cardiac cells using spectrally-resolved autofluorescence
lifetime detection of nicotinamide adenine dinucleotide (phosphate), or NAD(P)H, the principal electron donor in
mitochondrial oxidative energy metabolism responsible for vital ATP supply of cardiomyocytes. NAD(P)H
autofluorescence is long used for non-invasive fluorescent probing the metabolic state of the heart. In this contribution
we report dynamic characteristics of NAD(P)H fluorescence decays in living human cardiomyocytes from EMB,
following excitation by UV-pulsed laser diode and detection by spectrally-resolved time-correlated single photon
counting. At least a 3-exponential decay model, with 0.5-0.7 ns, 1.9-2.4 ns and 9.0-15.0 ns lifetimes, is necessary to
describe cardiomyocyte autofluorescence in human cells. When gathered data were compared to those recorded under
same conditions in rats, autofluorescence in human hearts was found significantly lower in comparison to rat ones.
Rotenone, the inhibitor of the Complex I of the respiratory chain, increased the fluorescence in human cardiac cells,
making them more comparable to experimental rat model. These results suggest that human cardiac cells are more
metabolically active than the rat ones in the same conditions. Presented work proposes a new tool for evaluation of
oxidative metabolism changes in transplanted hearts.
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