Spectrally and spatially resolved laser-induced photobleaching of endogenous flavin fluorescence in cardiac myocytes.

Naturally occurring endogenous fluorescence of flavins, arising in response to excitation by visible light, offers broad opportunity to investigate mitochondrial metabolic state directly in living cells and tissues, including in clinical settings. However, photobleaching, the loss of the autofluorescence intensity following prolonged exposure to light is an inherent phenomenon occurring during the fluorescence acquisition, which can have a negative impact on the recorded data, particularly in the context of measurement of metabolic modulations in pathophysiological conditions. In the presented study, we present a detailed analysis of endogenous flavins fluorescence photobleaching arising in living cardiac cells during spectrally-resolved confocal imaging. We demonstrate significant nonuniform photobleaching related to different bleaching rates of individual flavin components, resolved by linear spectral unmixing of the recorded signals. Induced photodamage was without effect on the cell morphology, but lead to significant modifications of the cell responsiveness to metabolic modulators and its contractility, suggesting functional metabolic alterations in the recorded cells. These findings point to the necessity of inducing limited photobleaching during metabolic screening in all studies involving visible light excitation and fluorescence acquisition in living cells. © 2018 International Society for Advancement of Cytometry.

Chemical imaging of cardiac cell and tissue by using secondary ion mass spectrometry.

PURPOSE: Identification and localization of biomolecules in cells and tissue samples are important for understanding of subcellular structures and can be helpful in biomedical and pharmaceutical research. PROCEDURES: Isolated cardiac cells and tissue of rats are studied by using time-of-flight secondary ion mass spectrometry. This technique provides chemical composition of cardiac cell membrane and tissue surface in native form. RESULTS: The result is a spatially resolved chemical imaging of cell and tissue surfaces as a lateral distribution of biologically relevant molecules-phospholipids, along with fatty acids, and cholesterol. Phospholipids are represented by phosphatidylcholine and cardiolipin molecules and their fragments. Phosphatidylcholine polar head group at mass of 184.1 u has an origin in the cell membrane, and a two-dimensional distribution of this fragment provides clear chemical contours of the cell. The high-resolution contrast of the cell is observed within its environment represented with Na(+) ions. Images of PO(4)H(-) fragment and fatty acids with 16 or 18 C atoms are determined in cardiac tissue. Distributions of these 16 and 18 C fatty acids are the same within their groups, and interestingly, these two distribution groups are spatially complementary. Contours of phosphatidylcholine and cardiolipin fragments are also complementary, the distributions of 16 C fatty acids and phosphatidylcholine are identical, and the distributions of 18 C fatty acids and cardiolipin are also the same. This complementarity thus supports the chemical compositions of phosphatidylcholine and cardiolipin based on 16 C and 18 C fatty acids, respectively. CONCLUSION: The method provides information not only about cell and tissue morphology, shape, and condition but also about cellular membrane chemical composition and lateral distribution of biologically relevant molecules.