We present the results of combined fluorescence and optoacoustic monitoring of tumor treatment using novel photoactivatable multi-inhibitor liposomes with BPD and Irinotecan providing a synergetic effect of PDT and chemotherapeutic impact.
3D-models based on patient-specific induced pluripotent stem cells are widely used for biomedical research. Thus, non-invasive and continuous comprehensive description of these of 3D-models using FLIM and multiphoton microscopy requires.
The aim of our work was to investigate energy metabolism of patient’s tumor ex vivo using fluorescence lifetime imaging (FLIM). Tumor metabolism was examined by detecting the fluorescence lifetime of metabolic cofactor NAD(P)H. In glioma tumor samples was shown a different energy metabolism with white matter. Colorectal tumors displayed marked intertumor heterogeneity and shift towards oxidative energy metabolism. Thereby, the possibility of assessing the energy metabolism in postoperative tumor samples of patients was shown.
KEYWORDS: Fluorescence lifetime imaging, Oxygen, 3D modeling, Luminescence, Biological research, 3D displays, Tissues, Stem cells, Microscopy, Medical research
3D models based on cells differentiated from patient-specific induced pluripotent stem cells (iPSCs) are widely used to identify disease phenotypes, to accurately analyze dysfunctions at the level of human tissues and organs, to screen new drugs, which makes them more promising tool for biomedical research tasks than monolayer cultures, which is associated with their proximity to in vivo. The metabolic activity with oxygenation level of cells, assessed by optical imaging methods, can be used as markers of cell viability, proliferative activity and the degree of differentiation in 3D culture conditions. In this paper we used fluorescence and phosphorescence lifetime imaging microscopies (FLIM and PLIM) to study the metabolic status and the oxygenation level of derived from iPSCs neural stem cells (NSC) cultured in 3D condition. An analysis of the fluorescence intensities and FLIM data showed that NSCs in monolayer and at the periphery of large spheroids have more glycolytic phenotypes, NSCs in the center of large spheroids and NSCs grouped into small spheroids have more oxidative state. For determination of the relative oxygen level in spheroids PLIM of BPTDM stained neurospheres was carried out. As it was supposed, oxygen transport in the spheroid depended on it size. In neurospheres with an average size 600 μm O2 distribution is radial, with the lowest concentration in the center. Thus, the metabolic status and oxygenation level of the NSC in the spheroid composition was assessed in a life-time and noninvasive manner.
Genetically encoded photosensitizers are a unique instrument for investigation of cellular mechanisms of photodynamic therapy (PDT). Fluorescent flavoprotein miniSOG (mini Singlet Oxygen Generator) generates singlet oxygen with a high yield and demonstrates strong phototoxic properties in vitro in cancer cells. However, the effective approaches for PDT with miniSOG have not been developed so far. The purpose of the study is to investigate phototoxic effects of miniSOG induced by continuous wave (CW) or pulsed laser irradiation in tumor spheroids. We found that maximum photobleaching of miniSOG without temperature effects was achieved at 120 mW/cm2 in CW mode or pulse periodic mode. PDT in pulse periodic mode provided more pronounced increase in the number of dead cells in comparison with CW mode and, moreover, induced apoptosis more efficiently. Therefore, we report for the first time on an effective regimen for PDT with miniSOG in a tumor spheroid model using pulsed periodic laser irradiation.
This presentation describes the development of the optical macroscanner and its application for metabolic imaging of large areas of tumors in mice. The scanner allows to interrogate areas as large as 15x15mm with the lateral resolution on the order of 15 micrometers. Acquisition times range from a few seconds for low pixel numbers to
several minutes for high-resolution images. We present data for NAD(P)H imaging of tumor with genetically encoded mKate2. In addition, using macroscanner we demonstrated the possibility of visualizing caspase-3 activity using the FRET-biosensor TR23K, which is based on a pair of proteins - a red fluorescent protein as a donor and a chromoprotein as an acceptor. The in vivo assay was noninvasive and could be applied in strongly and weakly fluorescent subcutaneous xenografts in mice using the FLIM-FRET method.
Cisplatin is an effective anticancer drug commonly used in the treatment of solid tumors. Although DNA is considered as the primary target, the cisplatin action at the cellular level remains unknown. Advanced fluorescence microscopy techniques allow probing various physiological and physicochemical parameters in living cells and tissues with unsurpassed sensitivity in real time. This study was focused on the investigation of cellular bioenergetics and cytosolic pH in colorectal cancer cells during chemotherapy with cisplatin. Special attention was given to the changes in cisplatininduced apoptosis that was identified using genetically encoded FLIM/FRET sensor of caspase-3 activity. Metabolic measurements using FLIM of the metabolic cofactor NAD(P)H showed decreased contribution from free NAD(P)H (a1, %) in all treated cells with more pronounced alterations in the cells undergoing apoptosis. Analysis of cytosolic pH using genetically encoded fluorescent sensor SypHer1 revealed a rapid increase of the pH value upon cisplatin exposure irrespective of the induction of apoptosis. To the best of our knowledge, a simultaneous assessment of metabolic state, cytosolic pH and caspase-3 activity after treatment with cisplatin was performed for the first time. These findings improve our understanding of the cell response to chemotherapy and mechanisms of cisplatin action.
Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.