Mitochondria functions as a major power source in diverse cell physiological processes. Mitochondria dysfunction is associated with many diseases, such as neurodegenerative diseases and various cancers. It is crucial to understand mitochondrial metabolism in live cells and related dysfunction in diseases. Currently, three-dimensional (3D) cultured tumor spheroids have been widely used in drug development in cancer therapy fields, due to their similarities with the animal model compared with 2D cultured cells. However, characterization methods to monitor metabolism status in tumor spheroids are limited by the lack of techniques to measure mitochondrial metabolic fluxes in a noninvasive and real-time way. Here, we proposed a Fluorescence Lifetime Imaging Microscopy (FLIM) based method to monitor the mitochondria status in 3D cultured cells. MCF-7 tumor spheroids or 2D cultured cells were labelled with a mitochondria specific dye Rhodamine123 (R123). Images of cultured cells were captured with a FLIM system and analyzed with software SPCImage, providing the size and shape of each tumor spheroid as well as the fluorescence lifetime values of mitochondria. In 2D cultured cells, the fluorescence lifetime values of each dye had little fluctuation, indicating similar physiological status. In tumor spheroids labelled with R123, the fluorescence lifetime values of the inner part of spheroids were uneven compared with those of the outer edge. The reason for this uneven lifetime span of mitochondria may be associated with the difference of status between the inner and outer layers of tumor spheroids. In conclusion, this FLIM based method has shown great advantages to characterize the mitochondria status in 3D cultured tumor spheroids, providing a powerful quantitative method in cellular physiology research fields.
As one of the most fatal cancers, pancreatic cancer generated nearly a half million of new cases world-widely in 2021. The cure rate of pancreatic cancer is extremely low, whose five-year survival rate after surgery is less than 5%, leading to a great demand of early diagnosis. Currently, the aspiration biopsy with hematoxylin-eosin (H&E) staining is considered as one golden standard for clinical cancer diagnosis. However, the accuracy of the identification of the biopsy is unsatisfied, which is highly affected by the experience of doctors. Here, we have used fluorescence lifetime imaging microscopy (FLIM) method to analyze H&E stained sections, providing quantitative data for further identification. Mice were randomly selected, divided into the experimental group injected with pancreatic cancer cells and the control group. Then the pancreatic tissues of both groups were obtained and stained with H&E dye solution. Next, the sections were imaged with FLIM system, providing fluorescence lifetime data for analysis. The results showed that the average fluorescence lifetime value of normal tissues was around 30.5% less than that of cancerous tissues. Moreover, phasor plot software was applied to distinguish certain regions (such as desmoplasia), which were not identified in either the bright-field-image mode nor the fluorescence lifetime mode. In conclusion, FLIM technique on H&E stained sections has generated quantitative information to identify cancerous tissues as well as desmoplasia, leading to an improved diagnostic accuracy. This FLIM-based method in analyzing H&E stained sections has a high potential in further quantitative diagnosis of different types of cancers.
Esophageal carcinoma is a common clinical cancer and with sixth occurrence frequency in the world, has a low five-year survival rate. Among different types of esophageal carcinoma, esophageal squamous cell carcinoma (ESCC) has the highest incidence rate and has a poor prognosis after surgery. For clinical diagnosis, hematoxylin and eosin (H&E) stained sections of diseased tissues are considered as the “golden standard”. However, determination of the tumor regions is usually relied on professional experience, which is time consuming and has a high misdiagnosis rate. Currently, novel optical imaging tools such as multi-photon excitation imaging and fluorescence lifetime imaging microscopy (FLIM) have been applied in clinical diagnosis. FLIM contains advantages of accurate measurement and high sensitivity to microenvironment. In this work, we constructed mice orthotopic esophageal cancer model to investigate the characteristics of esophageal tumor cells. Then FLIM technique were used to investigate H&E stained sections from both healthy control mice and ESCC mice, providing difference between the fluorescence lifetime values of normal tissues and those of the pathological tissues. Results also revealed an alteration of the fluorescence lifetime values of esophageal stratum corneum, which might be generated through tumor extrusion. Furthermore, the fluorescence lifetime values of tumor cells are distinctly smaller than those of the surrounding stroma, indicating an accurate identification the lesion area. In conclusion, the fluorescence lifetime images obtained by FLIM could provide a quantitative method in future pathological identification.
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