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Imaging

Under the Nanoscope

The use of nano-imaging techniques to observe biological tissues is now letting scientists visualize molecular phenomena involved in medical
disorders such as cataract and Parkinson's disease, with a precision never achieved before.

Nano-imaging techniques are slowly making their way into the medical arena, bringing scientists from different fields–biology, physics, and chemistry–to work together on common goals. Recently, two studies led by CNRS researchers have demonstrated that high-resolution microscopy could be used to visualize subcellular material in much greater detail than before. While Atomic Force Microscopy (AFM) enables to study a cell’s topography at the level of a single molecule, particle accelerators called synchrotrons can help localize chemical elements inside different cellular compartments.

nanoscope

© N. Buzhynskyy and S. Scheuring

High-resolution atomic force microscopy topography of a membrane from a cataract lens.




AFM, which was invented in the late 80s, relies on probing the forces established between the atom at the tip of its probe and the atoms on the surface it scans. So far, because of its complexity, the technique was restricted to state-of-the-art applications. But a recent study reveals it has now been used to observe diseased tissue.1 “A technique that lets us visualize individual molecules is very helpful to understand the molecular origins of diseases,” explains Simon Scheuring, from the PCC unit.2 “So far in the medical field, the most precise technique we had to observe the topography of tissues was electronic microscopy. But the AFM’s signal-to-noise ratio is much higher–you really can visualize individual molecules.”
The researchers used AFM to observe cell membranes from a cataract-afflicted eye. Cataract is a loss of transparency that develops in the eye’s crystalline, characterized by a blurry vision. Experts believe this phenomenon is related to a deficiency in membrane proteins called “aquaporins” and “connexons” inside the crystalline’s lens cells. These proteins are essential for nutrition, waste removal, and the tight connection between neighboring lens cells. By comparing assembly of connexons and aquaporins inside the membranes of healthy and diseased lens cells, the team observed that cataract is associated with a lack of connexons inside the membranes of the diseased tissue.
The researchers hope to use AFM to further investigate other tissues, such as neuronal endings. “But it’s not a push-down button technique like optical imaging–it’s not enough to have a sample of the tissue. You need expertise to use the machine and interpret its results. It takes time,” says Scheuring.
In another study3 published in September in PLoS ONE, an international team of scientists used another high-resolution imaging technique–a synchrotron
X-ray nanoprobe–to visualize the distribution of iron in dopaminergic neurons, in vitro. “Because of dopamine’s role in Parkinson’s disease and iron’s chemical affinity for dopamine, scientists had long suspected that iron could play a toxic role in this neurodegenerative process. So far no technique was sharp enough to observe iron distribution inside neurons,” explains Guillaume Devès, an engineer at the CNAB laboratory.4
The synchrotron nanoprobe is the first technique that lets scientists probe chemical elements present in trace quantities inside a cell. It functions by scanning a tissue sample and emitting X-rays that excite its atoms. In response, the atoms emit their own characteristic fluorescent signal. By measuring the signal’s wavelength, the scientists can identify which chemical element they are dealing with.

dopa fluo

© A. Carmona/CNRS

Visible light microscopy (A) and epifluorescence microscopy (B) of the same freeze-dried cells identifies dopamine distribution, while synchrotron X-ray fluorescence nano-imaging reveals the distribution of iron in vesicles (C, D).


Using a synchrotron, the authors observed that, in vitro, in dopaminergic neurons, iron binds to dopamine inside specific compartments –known as dopamine vesicles. “The hypothesis is that iron is stored and somewhat neutralized within these vesicles,” says Devès. When inhibiting the synthesis of dopamine, the researchers witnessed a parallel decrease in iron level in these compartments.
In vivo, in the case of Parkinson’s disease, dopamine production is impaired and its level inside vesicles is strongly decreased in dopaminergic neurons of specific brain areas. At the same time, iron accumulates within these areas. From their findings, the authors suggest that dopamine impairment might lead to less iron being stored inside the vesicles, leading to toxic iron dispersion elsewhere inside the cells, and subsequent deterioration of neuronal circuitries. This is only the first of many studies. Synchrotrons can now be used to investigate the subcellular distribution of other metal ions involved in neurodegenerative diseases or in physiological neuronal functions.
Clémentine Wallace

Notes :

1. N. Buzhynskyy et al., “Human cataract lens membrane at subnanometer resolution,” J. Mol. Biol. 374: 162-9. 2007.
2. Unité physico-chimie Curie (CNRS / Institut Curie).
3. R. Ortega, “Iron Storage within Dopamine Neurovesicles Revealed by Chemical Nano-Imaging,” PLoS ONE. 2: e925. 2007.
4. Chimie Nucléaire Analytique Bioenvironnementale (CNRS / Université Bordeaux-I).

Contacts :

Simon Scheuring, PCC, Paris, simon.scheuring@curie.fr

Guillaume Devès, CNAB, Draguignan,deves@cenbg.in2p3.fr


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