Abstract

Programmed cell death, or apoptosis, is an essential process in development and homeostasis, and disruptions in associated pathways are responsible for a wide variety of diseases such as cancer, developmental abnormalities, and Alzheimer's disease. On the other hand, cell death, in many cases, is the desired outcome of therapeutic treatments targeting diseases such as cancer. Recently, metabolic imaging based on two-photon fluorescence microscopy has been developed and shown to be highly sensitive to certain cell death processes, most notably apoptosis, thus having the potential as an advanced label-free screening tool. However, the typically low acquisition rates of this imaging technique have resulted in a limited throughput approach, allowing only a small population of cells to be tracked at well-separated time points. To address this limitation, a high-speed two-photon fluorescence lifetime imaging microscopy (2P-FLIM) platform capable of video-rate imaging is applied to study and further characterize the metabolic dynamics associated with cell death. Building upon previous work demonstrating the capabilities of this system, this microscope is utilized to study rapid metabolic changes during cell death induction, such as dose-dependency of metabolic response, response in invasive vs. noninvasive cancer cells, and response in an apoptosis-resistant cell line, which is further shown to undergo autophagy in response to toxic stimuli. Results from these experiments show that the early apoptosis-related metabolic dynamics are strongly correlated with important cellular parameters including responsiveness to apoptosis-inducing stimuli. The high speed and sensitivity of the presented imaging approach enables new investigations into this highly dynamic and complex process.

Highlights

  • Programmed cell death is a vital process responsible for homeostasis in the human body [1]

  • This limitation primarily arises due to the reliance of many currently utilized 2P-FLIM platforms on a technique known as time-correlated single-photon counting (TCSPC)

  • Due to the slow acquisition rates of the TCSPC instruments used in these studies, the fast dynamics associated with apoptosis induction could not be fully captured

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Summary

Introduction

Programmed cell death is a vital process responsible for homeostasis in the human body [1]. While higher-throughput 2P-FLIM approaches have been developed [16,17,18,19,20], few have focused on the application of label-free metabolic imaging for tracking dynamic cellular processes This limitation primarily arises due to the reliance of many currently utilized 2P-FLIM platforms on a technique known as time-correlated single-photon counting (TCSPC). Due to the slow acquisition rates of the TCSPC instruments used in these studies, the fast dynamics associated with apoptosis induction could not be fully captured Investigating these early dynamics is important to better understand the intricate relationship between metabolism and cell death, as well as to assess the potential of metabolic imaging as a high-throughput therapeutic screening tool. This study explores the unique capabilities of this high-speed metabolic imaging platform for investigating the complex role of metabolism in cell death

High-speed two-photon fluorescence lifetime imaging microscopy
Analysis of high-speed 2P-FLIM data
Video-rate imaging
Large field-of-view imaging
Cell culture
Cell death induction
Alamar Blue cell viability assay
Video-rate and longitudinal imaging of STS-induced apoptosis
High-throughput screening of cell death
Apoptosis-related metabolic dynamics in cancer and non-tumorigenic cell lines
Metabolic dynamics of apoptosis-resistant cells
Conclusions

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