Nanofluidic Platform for Single Mitochondria Analysis Using Fluorescence Microscopy

Abstract

Using nanofluidic channels in PDMS of cross section 500 nm × 2 μm, we demonstrate the trapping and interrogation of individual, isolated mitochondria. Fluorescence labeling demonstrates the immobilization of mitochondria at discrete locations along the channel. Interrogation of mitochondrial membrane potential with different potential sensitive dyes (JC-1 and TMRM) indicates the trapped mitochondria are vital in the respiration buffer. Fluctuations of the membrane potential can be observed at the single mitochondrial level. A variety of chemical challenges can be delivered to each individual mitochondrion in the nanofluidic system. As sample demonstrations, increases in the membrane potential are seen upon introduction of OXPHOS substrates into the nanofluidic channel. Introduction of Ca2+ into the nanochannels induces mitochondrial membrane permeabilization (MMP), leading to depolarization, observed at the single mitochondrial level. A variety of applications in cancer biology, stem cell biology, apoptosis studies, and high throughput functional metabolomics studies can be envisioned using this technology.

Mitochondria, Bioenergetics and Apoptosis in Cancer

“…in mitochondria (many of which can be assayed individually in suspended lipid bilayers), the interrelationship among these and their contribution to the mtPTP is still largely unresolved. A lot of this uncertainty stems from a lack of tools to measure electrophysiology at the nanoscale with high temporal resolution (Figure II from 61 ). What is known is that when mitochondria depolarize in response to ROS (e.g.…”

The rise of mitochondria in medicine

“…This integration between the Western anatomical/molecular and Eastern bioenergetic perspectives in medicine should be facilitated the development and application of noninvasive and minimally invasive technologies to measure mitochondrial (dys)function (Goh et al, 2014; Minh Tdo et al, 2012; Roede et al, 2012; Wallace et al, 1988; Zand et al, 2013). Mitochondria are not all created equal but are functionally specialized, both in their composition (Pagliarini et al, 2008) and functions (Picard et al, 2012a).…”

Microwell array integrating nanoelectrodes for coupled opto-electrochemical monitorings of single mitochondria

“…Indeed, the most widely used techniques including fluorescence spectroscopy, chemiluminescence and electrochemistry (e.g. the Clark electrode) provide mean responses of mitochondria populations following bioenergetics activation/inhibition protocols (Allen et al 2009;Dikalov and Harrison 2014;Hall et al 2013;Quarato et al 2011;Vasdekis and Stephanopoulos 2015;Zand et al 2013). As a consequence, in order to decipher on mitochondrial heterogeneity and exact metabolic responses, studies at single organelle level have drawn huge interest (Appelhans and Busch 2017;Chang and Marshall 2017;Follain et al 2017;Norregaard et al 2017;Suraniti et al 2013;Vajrala et al 2014;Vajrala et al 2016).…”

“…On the other hand, fluorescence monitoring of single mitochondria evolved recently as a strategy for highly sensitive analyses of mitochondrial metabolic markers (Quarato et al 2011;Suraniti et al 2013;Vajrala et al 2014;Vajrala et al 2016;Zand et al 2013). Markers include the intrinsic autofluorescent NADH and FAD metabolites, as well as extrinsic dyes (rhodamine derivatives) for the mitochondrial membrane potential (ΔΨ), reactive oxygen species and other parameters (Kilgore et al 2013;Li et al 2016;Zhu et al 2016).…”

“…Markers include the intrinsic autofluorescent NADH and FAD metabolites, as well as extrinsic dyes (rhodamine derivatives) for the mitochondrial membrane potential (ΔΨ), reactive oxygen species and other parameters (Kilgore et al 2013;Li et al 2016;Zhu et al 2016). The microscopic platforms for single mitochondria monitoring are generally dependent on nanofluidics, nanohole arrays for trapping and surface functionalization for immobilization (Lim et al 2012;Zand et al 2013).…”