KEYWORDS: Tumors, Polymers, 3D modeling, Two photon polymerization, Confocal microscopy, Polymerization, Laser energy, Scanning electron microscopy, Cancer, In vitro testing
Interactions between tumor cells and immune cells are sparsely explored in 3D models, although occasional studies with 3D cell culture technologies have confirmed that tumor architecture influences cancer cell-immune system interactions. The development of technologies enabling controlled analysis of tumor-immune system interactions especially in 3D is highly challenging. Two photon polymerization (2PP) as a method for fabrication of various microstructures suitable for preparation of 3D tumor models finds applications in this context. In the present study 2PP technology was used to fabricate polymeric microfibers in 3D microspace that mimic fibers of extracellular matrix. UV-curable polymer OrmoComp (Micro Resist Technology GmbH) was irradiated by Newport Spirit ultrafast amplified laser operating at 520 nm. The fibers were made with one side anchored to the substrate with the supporting structure, whereas the other side was freely movable in space. The shape and curliness of fibers were adjusted by alternating parameters of fabrication, namely energy intensities and speed of fabrication. Before exposing fabricated fibers to live cells, microstructures were processed with KOH based treatment to enhance adhesion of cancer cells. Arrangement of the cancer cells in the network of polymeric fibers was visualized by confocal scanning microscopy. Cancer cells were able to proliferate and form spatial cellular clusters among polymeric fibers after several days of cultivation. At the same time they were available to immune cells that could be supplemented to the culture at any time. The results of the present study document feasibility to use 2PP technology to develop in vitro 3D models suitable for studies of tumor-immune cell interactions.
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.
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.
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.
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.
Klaudia Czaníková, Igor Krupa, Markéta Ilcíková, Peter Kasák, Dusan Chorvat, Marian Valentin, Miroslav Slouf, Jaroslav Mosnácek, Matej Micušík, Mária Omastová
KEYWORDS: Diodes, Composites, Polymers, Chemical elements, Carbon nanotubes, Atomic force microscopy, Scanning electron microscopy, Transmission electron microscopy, Carbon, Nanocomposites
The photo-actuating behavior of new polymeric nanocomposite materials based on a commercial elastomer, an ethylene-vinylacetate copolymer (EVA), filled with multiwalled carbon nanotubes (MWCNT) was investigated. A good dispersion of the MWCNT within the elastomeric matrix was ensured by using a novel, specific compatibilizer consisting of pyrenyl and cholesteryl groups. A uniaxial orientation of the MWCNT within the matrix was induced with shear forces by employing a special custom-made punch/die system. Good dispergation and alignment of the MWCNT within the matrix were demonstrated by scanning electron microscopy. Transmission electron microscopy showed a good dispersion of the MWCNT within the composite. Photo-actuation was qualitatively characterized by atomic force microscopy and quantitatively characterized by nanoindentation. The samples prepared in the form of Braille element showed expansion upon illumination by light diodes. The maximal height deformation changes about 15% was detected when a blue diode was used.
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.
Photomechanical actuation is preferred to electromechanical transduction because of some advantages including
wireless connection, a producing low noise, fast response etc. However, only few materials actually exhibit photoactuation.
Recently, nanotube-enriched elastomeric polymers have shown photo-actuating properties; the best results
were reported for liquid crystals elastomers. In the present paper we discuss photo-actuating behavior of new photoactuating
materials based on the commercial elastomer such as ethylene-vinylacetate copolymer (EVA) filled with
multiwalled carbon nanotubes (MWCNT) modified with novel, specific surfactant consisting of pyrenenyl and
cholesteryl groups. Orientation of MWCNT within the matrix was created by shear forces employing a special punch
and die system. Photo-actuation was characterized using AFM.
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.
In this contribution we show application of multispectral confocal laser scanning microscopy for visualisation and
characterisation of polyelectrolyte microcapsules. Fluorescent labels of different free charges, specifically bound by
electrostatic interactions to the polymer chains in polyelectrolyte microcapsules, were used for questioning the spatial
distribution of polymer components of the microcapsules. To separate the overlapping emission spectra of the fluorescent
labels spectral imaging and linear unmixing approaches was used.
Since August 2003 the K008 camera /1,2/ being coupled with a MS 3504i monochromator/spectrogaph /3/ has been used in the International Laser Center in Bratislava (Slovak Republic) for investigations in the field of non-stationary spectroscopy, in particular, for the study of fluorescence processes in different dyes. When putting the camera into operation its limiting temporal resolution was prelinarily checked and was found to be 20ps. The results of trial experiments on the study of dynamics of Rhodamin B fluorescence are given below.
KEYWORDS: Optical coherence tomography, Skin, Near infrared, Tissues, Luminescence, Diagnostics, Tissue optics, Information fusion, Laser induced fluorescence, In vivo imaging
Optical coherence tomography (OCT) is one of the most promising recently developed methods for non-invasive in vivo characterization of biological highly scattering tissues. However, one of the drawbacks of the pure OCT imaging is that it is not sensitive to changes in metabolism. This may impact derived information and consecutive diagnostics, because pathological changes of tissue structure are accompanied with changes in metabolic activity or functional state in these areas even if there is not yet strongly evident structural change. Therefore, it is desirable to combine early detection of tissue malformations by OCT with other techniques, capable to detect and evaluate their functional state. One of suitable candidates for such non-invasive optical functional imaging is detection of laser induced autofluorescence, which could provide information about rate of biological and chemical processes in living cells. As an example, the cells in proliferative state (with increased metabolic activity or during mitosis) show more intensive NIR fluorescence than the cells that are not proliferative, because of increased concentration of free porphyrins. In presented study we used OCT and laser induced NIR autofluorescence imaging for detection and evaluation of changes in areas of naevus and injuries in group of volunteers. The fusion of information on structural and functional state of biotissues provided by the two mentioned complementary methods may enhance the diagnostics power of their prospective clinical use. Firstly the fluorescence of area of naevus and injuries, excited by 630 nm was taken by CCD camera and then was naevus scanned by OCT. The findings of both methods were compared and correlated. In the case of naevus the obtained results were reviewed with histological treatment of the same area.
Methods of polarized fluorescence and fluorescence lifetime imaging microscopy were used to characterize the orientation and rotational diffusion capability of merocyanine 540 molecule bind to the model and biological membranes of single isolated cells and multilamellar liposomes. To estimate the binding properties of merocyanine in gel and fluid phases of the lipid bilayer, temperatures below and over phase-transition point of the lipid were used. Using a fluorescence microscope equipped with excitation and emission polarizers an effect of geometrically selective excitation was observed, indicating that the orientation of merocyanine chromophore is dominantly perpendicular to the membrane surface of synthetic liposomes. Using the microscope setup combined with pulsed laser excitation and gated MCP image amplifier a time-resolved fluorescence images of the phospholipid vesicles stained by merocyanine 540 were obtained, showing no apparent spatial domains of different fluorescence lifetimes in contrast to highly structured images of fluorescence intensity.
The contribution deals with the diagnostics of laser induced plasma (LIP) by optical emission spectroscopy. Laser induced plasma occurs during laser welding and reveals important information about technological process. The performed experiments were concerned with in-time spectral analysis (wavelength range: 200-800 nm) of laser induced plasma of austenitic steel (18% Cr, 9% Ni) during cw CO2 laser welding. Circumstances of conversion from conducting mode to keyhole mode (and vice versa) were investigated. A comparison of the characteristics of the spectrum emission, penetration characteristics and input welding parameters was also studied.
There are many fields in medicine and biology where optical coherence tomography (OCT) is starting to be used for diagnostics imaging. In our work, OCT imaging has been applied to obtain 3D structure and geometry of mouse aorta and atherosclerotic plaques in it. Differences in plaque formation have been detected between mice fed with cholesterol rich food, and mice kept on special diet. The results of OCT measurements have been confirmed with optical microscopy.
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