Quantitative Imaging of Biochemistry in Situ and at the Nanoscale

Abstract

Biochemical reactions in eukaryotic cells occur in subcellular, membrane-bound compartments called organelles. Each kind of organelle is characterized by a unique lumenal chemical composition whose stringent regulation is vital to proper organelle function. Disruption of the lumenal ionic content of organelles is inextricably linked to disease. Despite their vital roles in cellular homeostasis, there are large gaps in our knowledge of organellar chemical composition largely from a lack of suitable probes. In this Outlook, we describe how, using organelle-targeted ratiometric probes, one can quantitatively image the lumenal chemical composition and biochemical activity inside organelles. We discuss how excellent fluorescent detection chemistries applied largely to the cytosol may be expanded to study organelles by chemical imaging at subcellular resolution in live cells. DNA-based reporters are a new and versatile platform to enable such approaches because the resultant probes have precise ratiometry and accurate subcellular targeting and are able to map multiple chemicals simultaneously. Quantitatively mapping lumenal ions and biochemical activity can drive the discovery of new biology and biomedical applications.

Tissue specific targeting of DNA nanodevices in a multicellular living organism

“…Distinguished by lysosomal markers(Coburn & Gems, 2013; Dell’Angelica, Mullins, Caplan, & Bonifacino, 2000; Hermann et al, 2005), LROs play important roles in production and storage of melanin, immune defence and neurological function(Huizing, Helip-Wooley, Westbroek, Gunay-Aygun, & Gahl, 2008). We first sought to target a simple DNA nanodevice comprising a 38-bp double stranded DNA (dsDNA) bearing a 5’ Alexa 647 (A647), denoted D 38 , to label LROs in the intestinal epithelia ( Table 1 , Supplementary Information ). We reasoned that the biological barrier between the intestinal lumen and the pseudocoelom would effectively preclude D 38 from access to coelomocytes, as seen whenever DNA nanodevices are microinjected in the pseudocoelom (Bhatia, Surana, Chakraborty, Koushika, & Krishnan, 2011; Surana et al, 2011) .…”

“…We first sought to target a simple DNA nanodevice comprising a 38-bp double stranded DNA (dsDNA) bearing a 5’ Alexa 647 (A647), denoted D 38 , to label LROs in the intestinal epithelia ( Table 1 , Supplementary Information ). We reasoned that the biological barrier between the intestinal lumen and the pseudocoelom would effectively preclude D 38 from access to coelomocytes, as seen whenever DNA nanodevices are microinjected in the pseudocoelom (Bhatia, Surana, Chakraborty, Koushika, & Krishnan, 2011; Surana et al, 2011) .…”

“…We therefore utilized a liquid-feeding method to introduce 1 μM of D 38 into the intestinal lumen of one-day adult worms (M&M). We leveraged the high autofluorescence of LROs at shorter wavelengths to provided regions of interest (ROIs) to evaluate the efficacy of D 38 uptake.…”

“…We therefore utilized a liquid-feeding method to introduce 1 μM of D 38 into the intestinal lumen of one-day adult worms (M&M). We leveraged the high autofluorescence of LROs at shorter wavelengths to provided regions of interest (ROIs) to evaluate the efficacy of D 38 uptake. The mean intensity corresponding to D 38 uptake, compared to control mock-fed worms, was very low, indicating low or no intrinsic targetability of DNA nanodevices to IECs.…”

“…Nature solves the problem of transporting poorly permeable molecules across membrane barriers, either releasing or enriching them tissue-specifically, by membrane trafficking. DNA nanostructures are particularly amenable to endosomal trafficking, which has in part, led to an array of synthetic DNA nanostructures being presently deployed in living systems(Bujold, Lacroix, & Sleiman, 2018; Chakraborty et al, 2016; Krishnan, Zou, & Jani, 2020). Endosomal trafficking is ubiquitous: it regulates the internalisation, sorting and intracellular transport of diverse cargo, and is pivotal to development, signalling and homeostasis.…”