probing-metabolism-viscosity-cancer-cells-using-fluorescence-lifetime

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and ...

Privolzhsky Research Medical University

Becker & Hickl GmbH

Single Photon Avalanche Diode (SPAD) arrays are increasingly exploited and have demonstrated potential in biochemical and biomedical research, both for imaging and single-point spectroscopy applications. In this study, we explore the application of SPADs together with fiber-optic-based delivery and collection geometry to realize fast and simultaneous single-point time-, spectral-, and depth-resolved fluorescence measurements at 375 nm excitation light. Spectral information is encoded across the columns of the array through grating-based dispersion, while depth information is encoded across the rows thanks to a linear arrangement of probe collecting fibers. The initial characterization and validation were realized against layered fluorescent agarose-based phantoms. To verify the practicality and feasibility of this approach in biological specimens, we measured the fluorescence signature of formalin-fixed rabbit aorta samples derived from an animal model of atherosclerosis. The initial results demonstrate that this detection configuration can report fluorescence spectral and lifetime contrast originating at different depths within the specimens. We believe that our optical scheme, based on SPAD array detectors and fiber-optic probes, constitute a powerful and versatile approach for the deployment of multidimensional fluorescence spectroscopy in clinical applications where information from deeper tissue layers is important for diagnosis.

Multispectral Depth-Resolved Fluorescence Lifetime Spectroscopy Using SPAD Array Detectors and Fiber Probes.

Classic time-correlated single photon counting (TCSPC) technique involves detection of single photons of a periodic optical signal, registration of the photon arrival time in respect to the reference pulse, and construction of photon distribution with regard to the detection times. This technique achieves extremely high time resolution and near-ideal detection efficiency. Modern TCSPC is multi-dimensional, i.e., in addition to the photon arrival time relative to the excitation pulse, spatial coordinates within the image area, wavelength, time from the start of the experiment, and many other parameters are determined for each photon. Hence, the multi-dimensional TCSPC allows generation of photon distributions over these parameters. This review describes both classic and multi-dimensional types of TCSPC microscopy and their application for fluorescence lifetime imaging in different areas of biological studies.

Multiparametric Time-Correlated Single Photon Counting Luminescence Microscopy.

Conventional techniques are insufficient precisely to describe the internal structure, the heterogeneous cell populations, and the dynamics of biological processes occurring in diseased liver during surgery. There is a need for a rapid and safe method for the successful diagnosis of liver disease in order to plan surgery and to help avoid postoperative liver failure. We analyze the progression of both acute (cholestasis) and chronic (fibrosis) liver pathology using multiphoton microscopy with fluorescence lifetime imaging and second-harmonic generation modes combined with time-of-flight secondary ion mass spectrometry chemical analysis to obtain new data about pathological changes to hepatocytes at the cellular and molecular levels. All of these techniques allow the study of cellular metabolism, lipid composition, and collagen structure without staining the biological materials or the incorporation of fluorescent or other markers, enabling the use of these methods in a clinical situation. The combination of multiphoton microscopy and mass spectrometry provides more complete information about the liver structure and function than could be assessed using either method individually. The data can be used both to obtain new criteria for the identification of hepatic pathology and to develop a rapid technique for liver quality analysis in order to plan surgery and to help avoid postoperative liver failure in clinic.

Metabolic imaging and secondary ion mass spectrometry to define the structure and function of liver with acute and chronic pathology.

Although fluorescence lifetime imaging microscopy (FLIM) has been extensively applied to study cellular metabolism in the liver, there is neither an established approach to analyze the data, nor have appropriate protocols been developed to maintain the optical metabolic characteristics in the  liver tissue sample. Here, we show that a tri-exponential decay fitting model for the fluorescence signal from nicotinamide adenine dinucleotide (NAD(P)H) and the use of  samples allows the most appropriate processing of the FLIM data. Moreover, we determine the medium that maintains the initial metabolic state of hepatocytes (liver cells), most effectively. Our results should be particularly relevant for the interrogation of liver samples, not only in laboratory research, but also in clinical settings in the future.

Mapping metabolism of liver tissue using two-photon FLIM.

Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival time. Real-time pathology assisted, or image guided surgical procedures that eliminate tumors promise to improve the clinical outcome and prolong the lives of patients. Our work is focused on the development of a rapid and sensitive assay for intraoperative diagnostics of glioma and identification of optical markers essential for differentiation between tumors and healthy brain tissues. We utilized fluorescence lifetime imaging (FLIM) of endogenous fluorophores related to metabolism of the glioma from freshly excised brains tissues. Macroscopic time-resolved fluorescence images of three intracranial animal glioma models and surgical samples of patients' glioblastoma together with the white matter have been collected. Several established and new algorithms were applied to identify the imaging markers of the tumors. We found that fluorescence lifetime parameters characteristic of the glioma provided background for differentiation between the tumors and intact brain tissues. All three rat tumor models demonstrated substantial differences between the malignant and normal tissue. Similarly, tumors from patients demonstrated statistically significant differences from the peritumoral white matter without infiltration. While the data and the analysis presented in this paper are preliminary and further investigation with a larger number of samples is required, the proposed approach based on the macroscopic FLIM has a high potential for diagnostics of glioma and evaluation of the surgical margins of gliomas.

Vladislav Shcheslavskiy

Institute of Experimental Oncology and Biomedical Technologies

Privolzhsky Research Medical University

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy

Vladislav Shcheslavskiy

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Experimental Oncology and Biomedical Technologies

Privolzhsky Research Medical University

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy

Institute of Experimental Oncology and Biomedical Technologies