Longitudinal label-free tracking of cell death dynamics in living engineered human skin tissue with a multimodal microscope.

Youbo Zhao,Benedikt W Graf,Ziad Mahmassani,Marni D Boppart,Stephen A Boppart,Marina Marjanovic,Eric J Chaney

doi:10.1364/boe.5.003699

Youbo Zhao, Benedikt W Graf + Show 5 more

Open Access

https://doi.org/10.1364/boe.5.003699

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Abstract

We demonstrate real-time, longitudinal, label-free tracking of apoptotic and necrotic cells in living tissue using a multimodal microscope. The integrated imaging platform combines multi-photon microscopy (MPM, based on two-photon excitation fluorescence), optical coherence microscopy (OCM), and fluorescence lifetime imaging microscopy (FLIM). Three-dimensional (3-D) co-registered images are captured that carry comprehensive information of the sample, including structural, molecular, and metabolic properties, based on light scattering, autofluorescence intensity, and autofluorescence lifetime, respectively. Different cell death processes, namely, apoptosis and necrosis, of keratinocytes from different epidermal layers are longitudinally monitored and investigated. Differentiation of the two cell death processes in a complex living tissue environment is enabled by quantitative image analysis and high-confidence classification processing based on the multidimensional, cross-validating imaging data. These results suggest that despite the limitations of each individual label-free modality, this multimodal imaging approach holds the promise for studies of different cell death processes in living tissue and in vivo organs.

Highlights

Cell death is a fundamental but complex process that occurs under normal physiological conditions as well as in pathological states
The magnitude of the changes is smaller in deeper layers than in more superficial layers. These results show the different dynamics in different epidermal layers, which is associated with different physiological states of keratinocytes, the effects related to drug diffusion could be considered as another possible explanation for these observations
Because of the crucial and distinctive roles of apoptosis and necrosis, detection and differentiation of these cell death processes are important for investigations of developing tissues and organisms under pertinent physiological and pathological conditions

Summary

Introduction

Cell death is a fundamental but complex process that occurs under normal physiological conditions as well as in pathological states. The other cell death pathway, is usually triggered by external factors. This so-called accidental cell death typically causes the release of intracellular content, leading to an immune response and an inflammatory reaction. Because of the different roles and consequences of these two cell death processes, having an effective method to monitor and differentiate them under different conditions is important for investigations of such diverse areas as normal tissue development, the health of developing and grafted engineered tissues, disease evolution, therapeutic efficacy, and drug delivery [5, 6]

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