Toxicological Analysis of Hepatocytes Using FLIM Technique: In Vitro versus Ex Vivo Models

The search for new criteria indicating acute or chronic pathological processes resulting from exposure to toxic agents, testing of drugs for potential hepatotoxicity, and fundamental study of the mechanisms of hepatotoxicity at a molecular level still represents a challenging issue that requires the selection of adequate research models and tools. Microfluidic chips (MFCs) offer a promising in vitro model for express analysis and are easy to implement. However, to obtain comprehensive information, more complex models are needed. A fundamentally new label-free approach for studying liver pathology is fluorescence-lifetime imaging microscopy (FLIM). We obtained FLIM data on both the free and bound forms of NAD(P)H, which is associated with different metabolic pathways. In clinical cases, liver pathology resulting from overdoses is most often as a result of acetaminophen (APAP) or alcohol (ethanol). Therefore, we have studied and compared the metabolic state of hepatocytes in various experimental models of APAP and ethanol hepatotoxicity. We have determined the potential diagnostic criteria including the pathologically altered metabolism of the hepatocytes in the early stages of toxic damage, including pronounced changes in the contribution from the bound form of NAD(P)H. In contrast to the MFCs, the changes in the metabolic state of hepatocytes in the ex vivo models are, to a greater extent, associated with compensatory processes. Thus, MFCs in combination with FLIM can be applied as an effective tool set for the express modeling and diagnosis of hepatotoxicity in clinics

The water vapour continuum in near-infrared windows – current understanding and prospects for its inclusion in spectroscopic databases

Spectroscopic catalogues, such as GEISA and HITRAN, do not yet include information on the water vapour continuum that pervades visible, infrared and microwave spectral regions. This is partly because, in some spectral regions, there are rather few laboratory measurements in conditions close to those in the Earth’s atmosphere; hence understanding of the characteristics of the continuum absorption is still emerging. This is particularly so in the near-infrared and visible, where there has been renewed interest and activity in recent years. In this paper we present a critical review focusing on recent laboratory measurements in two near-infrared window regions (centred on 4700 and 6300 cm−1) and include reference to the window centred on 2600 cm−1 where more measurements have been reported. The rather few available measurements, have used Fourier transform spectroscopy (FTS), cavity ring down spectroscopy, optical-feedback – cavity enhanced laser spectroscopy and, in very narrow regions, calorimetric interferometry. These systems have different advantages and disadvantages. Fourier Transform Spectroscopy can measure the continuum across both these and neighbouring windows; by contrast, the cavity laser techniques are limited to fewer wavenumbers, but have a much higher inherent sensitivity. The available results present a diverse view of the characteristics of continuum absorption, with differences in continuum strength exceeding a factor of 10 in the cores of these windows. In individual windows, the temperature dependence of the water vapour self-continuum differs significantly in the few sets of measurements that allow an analysis. The available data also indicate that the temperature dependence differs significantly between different near-infrared windows. These pioneering measurements provide an impetus for further measurements. Improvements and/or extensions in existing techniques would aid progress to a full characterisation of the continuum – as an example, we report pilot measurements of the water vapour self-continuum using a supercontinuum laser source coupled to an FTS. Such improvements, as well as additional measurements and analyses in other laboratories, would enable the inclusion of the water vapour continuum in future spectroscopic databases, and therefore allow for a more reliable forward modelling of the radiative properties of the atmosphere. It would also allow a more confident assessment of different theoretical descriptions of the underlying cause or causes of continuum absorption

Toxicological Analysis of Hepatocytes Using FLIM Technique: In Vitro versus Ex Vivo Models

The search for new criteria indicating acute or chronic pathological processes resulting from exposure to toxic agents, testing of drugs for potential hepatotoxicity, and fundamental study of the mechanisms of hepatotoxicity at a molecular level still represents a challenging issue that requires the selection of adequate research models and tools. Microfluidic chips (MFCs) offer a promising in vitro model for express analysis and are easy to implement. However, to obtain comprehensive information, more complex models are needed. A fundamentally new label-free approach for studying liver pathology is fluorescence-lifetime imaging microscopy (FLIM). We obtained FLIM data on both the free and bound forms of NAD(P)H, which is associated with different metabolic pathways. In clinical cases, liver pathology resulting from overdoses is most often as a result of acetaminophen (APAP) or alcohol (ethanol). Therefore, we have studied and compared the metabolic state of hepatocytes in various experimental models of APAP and ethanol hepatotoxicity. We have determined the potential diagnostic criteria including the pathologically altered metabolism of the hepatocytes in the early stages of toxic damage, including pronounced changes in the contribution from the bound form of NAD(P)H. In contrast to the MFCs, the changes in the metabolic state of hepatocytes in the ex vivo models are, to a greater extent, associated with compensatory processes. Thus, MFCs in combination with FLIM can be applied as an effective tool set for the express modeling and diagnosis of hepatotoxicity in clinics

Monitoring membrane viscosity in differentiating stem cells using BODIPY-based molecular rotors and FLIM

Membrane fluidity plays an important role in many cell functions such as cell adhesion, and migration. In stem cell lines membrane fluidity may play a role in differentiation. Here we report the use of viscosity-sensitive fluorophores based on a BODIPY core, termed “molecular rotors”, in combination with Fluorescence Lifetime Imaging Microscopy, for monitoring of plasma membrane viscosity changes in mesenchymal stem cells (MSCs) during osteogenic and chondrogenic differentiation. In order to correlate the viscosity values with membrane lipid composition, the detailed analysis of the corresponding membrane lipid composition of differentiated cells was performed by time-of-flight secondary ion mass spectrometry. Our results directly demonstrate for the first time that differentiation of MSCs results in distinct membrane viscosities, that reflect the change in lipidome of the cells following differentiation

Optical Biomedical Imaging Reveals Criteria for Violated Liver Regenerative Potential

To reduce the risk of post-hepatectomy liver failure in patients with hepatic pathologies, it is necessary to develop an approach to express the intraoperative assessment of the liver’s regenerative potential. Traditional clinical methods do not enable the prediction of the function of the liver remnant. Modern label-free bioimaging, using multiphoton microscopy in combination with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), can both expand the possibilities for diagnosing liver pathologies and for assessing the regenerative potential of the liver. Using multiphoton and SHG microscopy, we assessed the structural state of liver tissue at different stages of induced steatosis and fibrosis before and after 70% partial hepatectomy in rats. Using FLIM, we also performed a detailed analysis of the metabolic state of the hepatocytes. We were able to determine criteria that can reveal a lack of regenerative potential in violated liver, such as the presence of zones with reduced NAD(P)H autofluorescence signals. Furthermore, for a liver with pathology, there was an absence of the jump in the fluorescence lifetime contributions of the bound form of NADH and NADPH the 3rd day after hepatectomy that is characteristic of normal liver regeneration. Such results are associated with decreased intensity of oxidative phosphorylation and of biosynthetic processes in pathological liver, which is the reason for the impaired liver recovery. This modern approach offers an effective tool that can be successfully translated into the clinic for express, intraoperative assessment of the regenerative potential of the pathological liver of a patient