Simultaneous Raman Microspectroscopy and Fluorescence Imaging of Bone Mineralization in Living Zebrafish Larvae

M Bennet,A Akiva,D Faivre,G Malkinson,K Yaniv,S Abdelilah-Seyfried,P Fratzl,A Masic

doi:10.1016/j.bpj.2014.01.002

Abstract

Confocal Raman microspectroscopy and fluorescence imaging are two well-established methods providing functional insight into the extracellular matrix and into living cells and tissues, respectively, down to single molecule detection. In living tissues, however, cells and extracellular matrix coexist and interact. To acquire information on this cell-matrix interaction, we developed a technique for colocalized, correlative multispectral tissue analysis by implementing high-sensitivity, wide-field fluorescence imaging on a confocal Raman microscope. As a proof of principle, we study early stages of bone formation in the zebrafish (Danio rerio) larvae because the zebrafish has emerged as a model organism to study vertebrate development. The newly formed bones were stained using a calcium fluorescent marker and the maturation process was imaged and chemically characterized in vivo. Results obtained from early stages of mineral deposition in the zebrafish fin bone unequivocally show the presence of hydrogen phosphate containing mineral phases in addition to the carbonated apatite mineral. The approach developed here opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces under (patho-)physiological conditions.

Highlights

Understanding fundamental biological processes relies on probing intra- and extracellular environments, targeted delivery inside living cells and tissues, and real-time detection and imaging of chemical markers and biomolecules [1,2]
Transcutaneous Raman spectroscopy has the potential as a tool for in vivo bone quality assessment [6], whereas the time- and space-resolved Raman spectroscopy allows the visualization in vivo of the distributions of molecular species in human and yeast cells [4,5,7]
As a proof of principle, we have studied the different mineral phases involved in bone formation of the zebrafish larvae

Summary

Introduction

Understanding fundamental biological processes relies on probing intra- and extracellular environments, targeted delivery inside living cells and tissues, and real-time detection and imaging of chemical markers and biomolecules [1,2]. Confocal Raman imaging alone does not allow live, high-resolution imaging of larger regions of interest in complex biological tissues. We developed a correlative Raman and fluorescence imaging method that combines the strengths and compensates for the shortcomings of each of these imaging modalities and allows studying in vivo processes in complex animal models such as zebrafish larvae.

Results

Conclusion