11 research outputs found
Quantitative Imaging With DNA-PAINT for Applications in Synaptic Neuroscience
Super-resolution (SR) microscopy techniques have been advancing the understanding of neuronal protein networks and interactions. Unraveling the arrangement of proteins with molecular resolution provided novel insights into neuron cytoskeleton structure and actin polymerization dynamics in synaptic spines. Recent improvements in quantitative SR imaging have been applied to synaptic protein clusters and with improved multiplexing technology, the interplay of multiple protein partners in synaptic active zones has been elucidated. While all SR techniques come with benefits and drawbacks, true molecular quantification is a major challenge with the most complex requirements for labeling reagents and careful experimental design. In this perspective, we provide an overview of quantitative SR multiplexing and discuss in greater detail the quantification and multiplexing capabilities of the SR technique DNA-PAINT. Using predictable binding kinetics of short oligonucleotides, DNA-PAINT provides two unique approaches to address multiplexed molecular quantification: qPAINT and Exchange-PAINT. With precise and accurate quantification and spectrally unlimited multiplexing, DNA-PAINT offers an attractive route to unravel complex protein interaction networks in neurons. Finally, while the SR community has been pushing technological advances from an imaging technique perspective, the development of universally available, small, efficient, and quantitative labels remains a major challenge in the field
Review
Innovation in genomics, transcriptomics, and proteomics research has created a plethora of state-of-the-art techniques such as nucleic acid sequencing and mass-spectrometry-based proteomics with paramount impact in the life sciences. While current approaches yield quantitative abundance analysis of biomolecules on an almost routine basis, coupling this high content to spatial information in a single cell and tissue context is challenging. Here, current implementations of spatial omics are discussed and recent developments in the field of DNA-barcoded fluorescence microscopy are reviewed. Light is shed on the potential of DNA-based imaging techniques to provide a comprehensive toolbox for spatial genomics and transcriptomics and discuss current challenges, which need to be overcome on the way to spatial proteomics using high-resolution fluorescence microscopy
Neuronal correlates of serial position performance in amnestic mild cognitive impairment.
Objectives:
Delayed recall of the first words of a list - the primacy position â is thought to be particularly dependent on intact memory consolidation. Hippocampal volume has been suggested as the primary neuronal correlate of delayed primacy recall in cognitively normal elderly individuals. Here, we studied the association of hippocampal volume with primacy recall in individuals with amnestic mild cognitive impairment (aMCI).
Methods:
We investigated serial position performance in 88 subjects with aMCI using a 16-word list (CVLT). Primacy and recency performance were measured during learning and delayed recall. Hippocampal volumes were automatically determined from structural MRI scans. We conducted regression analyses with bilateral hippocampal volumes as predictors and serial position indices as outcomes.
Results:
After controlling for age, gender, and total intracranial volume, bilateral hippocampal volume was not associated with primacy recall either during learning or delayed recall. Primacy performance during learning was associated with the right inferior and middle temporal gyrus as well as the right inferior parietal cortex and supramerginal gyrus. During delayed recall, primacy performance was related to the bilateral supramarginal gyri.
Conclusions:
Our findings suggest a reduced primacy effect in aMCI already during learning, contrasting previous findings in normal cognitive aging. This might indicate impaired encoding and consolidation processes at an early stage of episodic memory acquisition. Furthermore, our data indicates that hippocampal volume may not be a relevant determinant of residual primacy performance in the stage of aMCI, which may rather depend on temporal and parietal neocortical networks
Calcium stabilizes the strongest protein fold
Staphylococcal pathogens adhere to their human targets using adhesins, which can withstand extremely high forces. Here, authors use single-molecule force spectroscopy to determine the similarly high unfolding forces of B domains that link the adhesin to the bacterium
Mapping the intracellular distribution of carbon nanotubes after targeted delivery to carcinoma cells using confocal Raman imaging as a label-free technique
Mapping the intracellular distribution of carbon nanotubes after targeted delivery to carcinoma cells using confocal Raman imaging as a label-free technique
A single-molecule approach to explore binding, uptake and transport of cancer cell targeting nanotubes
Linking of Sensor Molecules with Amino Groups to Amino-Functionalized AFM Tips
The measuring tip of an atomic force microscope (AFM) can be upgraded to a specific biosensor by attaching one or a few biomolecules to the apex of the tip. The biofunctionalized tip is then used to map cognate target molecules on a sample surface or to study biophysical parameters of interaction with the target molecules. The functionality of tip-bound sensor molecules is greatly enhanced if they are linked via a thin, flexible polymer chain. In a typical scheme of tip functionalization, reactive groups are first generated on the tip surface, a bifunctional cross-linker is then attached with one of its two reactive ends, and finally the probe molecule of interest is coupled to the free end of the cross-linker. Unfortunately, the most popular functional group generated on the tip surface is the amino group, while at the same time, the only useful coupling functions of many biomolecules (such as antibodies) are also NH<sub>2</sub> groups. In the past, various tricks or detours were applied to minimize the undesired bivalent reaction of bifunctional linkers with adjacent NH<sub>2</sub> groups on the tip surface. In the present study, an uncompromising solution to this problem was found with the help of a new cross-linker (âacetal-PEG-NHSâ) which possesses one activated carboxyl group and one acetal-protected benzaldehyde function. The activated carboxyl ensures rapid unilateral attachment to the amino-functionalized tip, and only then is the terminal acetal group converted into the amino-reactive benzaldehyde function by mild treatment (1% citric acid, 1â10 min) which does not harm the AFM tip. As an exception, AFM tips with magnetic coating become demagnetized in 1% citric acid. This problem was solved by deprotecting the acetal group before coupling the PEG linker to the AFM tip. Bivalent binding of the corresponding linker (âaldehyde-PEG-NHSâ) to adjacent NH<sub>2</sub> groups on the tip was largely suppressed by high linker concentrations. In this way, magnetic AFM tips could be functionalized with an ethylene diamine derivative of ATP which showed specific interaction with mitochondrial uncoupling protein 1 (UCP1) that had been purified and reconstituted in a mica-supported planar lipid bilayer