Two-photon deep-tissue spatially resolved mitochondrial imaging using membrane potential fluorescence fluctuations.

Kayvan Forouhesh Tehrani,Emily G Pendleton,William M Southern,Jarrod A Call,Luke J Mortensen

doi:10.1364/boe.9.000254

Abstract

Cell metabolism and viability are directly reflected in their mitochondria. Imaging-based analysis of mitochondrial morphological structure, size and dynamic characteristics can therefore provide critical insight into cell function. However, mitochondria are often very abundant, and due to their close to diffraction-limit size, it is often non-trivial to distinguish a tubular or large mitochondrion from an ensemble of punctate mitochondria. In this paper, we use membrane potential dependent fluorescence fluctuations of individual mitochondria to resolve them using an approach similar to single molecule localization microscopy. We use 2-photon microscopy to image mitochondrial intensity fluctuations at 200 μm deep inside an intact in-vivo mouse soleus muscle. By analyzing the acquired images, we can reconstruct images with an extra layer of information about individual mitochondria, separated from their ensemble. Our analysis shows a factor of 14 improvement in detection of mitochondria.

Highlights

Mitochondria are organelles that provide energy for the cell, and have a regulatory role for cellular metabolism
Cell metabolism and viability are directly reflected in their mitochondria
Our analysis shows a factor of 14 improvement in detection of mitochondria

Summary

Introduction

Mitochondria are organelles that provide energy for the cell, and have a regulatory role for cellular metabolism. Cell metabolism and viability are directly reflected in their mitochondria.

Results

Conclusion