We present a new strategy for the fabrication of magneto-fluorescent nanoparticles designed for bimodal imaging. These hybrid nanostructures comprise an optically active semiconductor nanoparticle quantum dot core with tunable fluorescence, encapsulated within a hollow paramagnetic iron oxide shell that serves as an MRI contrast agent. The yolk–shell morphology enables incorporation of the semiconductor and magnetic domains into a single structure, while avoiding direct contact between them, which typically results in quenching of the desired optical fluorescence. We successfully demonstrate utilization of the ultrasmall (15 nm hydrodynamic size) magneto-fluorescent CdSe@CdS@Hollow-Fe2O3 nanoparticles for multimodal imaging of cells at the intracellular level.

The development of imaging methodologies for single cell measurements over extended timescales of up to weeks, in the intact animal, will depend on signal strength, stability, validity and specificity of labeling. Whereas light-microscopy can achieve these with genetically-encoded probes or dyes, this modality does not allow mesoscale imaging of entire intact tissues. Non-invasive imaging techniques, such as magnetic resonance imaging (MRI), outperform light microscopy in field of view and depth of imaging, but do not offer cellular resolution and specificity, suffer from low signal-to-noise ratio and, in some instances, low temporal resolution. In addition, the origins of the signals measured by MRI are either indirect to the process of interest or hard to validate. It is therefore highly warranted to find means to enhance MRI signals to allow increases in resolution and cellular-specificity. To this end, cell-selective bi-functional magneto-fluorescent contrast agents can provide an elegant solution. Fluorescence provides means for identification of labeled cells and particles location after MRI acquisition, and it can be used to facilitate the design of cell-selective labeling of defined targets. Here we briefly review recent available designs of magneto-fluorescent markers and elaborate on key differences between them with respect to durability and relevant cellular highlighting approaches. We further focus on the potential of intracellular labeling and basic functional sensing MRI, with assays that enable imaging cells at microscopic and mesoscopic scales. Finally, we illustrate the qualities and limitations of the available imaging markers and discuss prospects for in vivo neural imaging and large-scale brain mapping.

Background Site-directed mutagenesis (SDM) is a key method in molecular biology; allowing to modify DNA sequences at single base pair resolution. Although many SDM methods have been developed, methods that increase efficiency and versatility of this process remain highly desired. Method We present a versatile and simple method to efficiently introduce a variety of mutation schemes using Gibson-assembly but without the need to design uniquely designated Gibson primers. Instead, we explore the re-use of standard SDM primers (completely overlapping in sequence) in combination with regular primers (~ 25 bps long) for amplification of fragments flanking the site of mutagenesis. We further introduce a rapid amplification step of the Gibson-assembled product for analysis and quality control, as well as for ligation, or re-ligation at instances the process fails (avoiding expenditure of added Gibson reaction mixtures). Results We first demonstrate that standard SDM primers can be used with the Gibson assembly method and, despite the need for extensive digestion of the DNA past the entire primer sequence, the reaction is attainable within as short as 15 min. We also find that the amount of the assembled Gibson product is too low to be visualized on standard agarose gel. Our added amplification step (by use of the same short primers initially employed) remedies this limitation and allows to resolve whether the desired Gibson-assembled product has been obtained on agarose gel or by sequencing of amplicons. It also provides large amounts of amplicons for subsequent ligations, bypassing the need to re-employ Gibson mixtures. Lastly, we find that our method can easily accommodate SDM primers with degenerate sequences. Conclusion We employ our alternative approach to delete, replace, insert, and degenerate sequences within target DNA sequences, specifically DNA sequences that proved very resistant to mutagenesis by multiple other SDM methods (standard and commercial). Importantly, our approach involves the re-use of SDM primers from our primer-inventory. Our scheme thereby reduces the need (and time and money) to design and order new custom Gibson-primers. Together, we provide a simple and versatile protocol that spans only 4 days (including the added amplification step), requires minimal primer sets and provides very high yields and success rates (> 98%).

Multimodal imaging of optically-cleared brains, ex vivo, by Magnetic Resonance Imaging (MRI) and light microscopy (LM) presents unique opportunities for studying the brain at various scales and resolutions. However, CLARITY -cleared brains lack MRI contrast, implicating lipids as the major source of MRI contrast. We explored the ex vivo MRI compatibility of uDISCO, ECi and Scale -cleared brains. Surprisingly, uDISCO and ECi -cleared brains retain MRI contrast, whereas Scale-cleared brains show a severe loss of MRI contrast, as CLARITY. Determination of lipid-content in cleared samples shows that CLARITY, uDISCO and ECi are strongly delipidating, whereas Scale preserves most lipids. We conclude that MRI contrast can be associated with tissue expansion (and hyperhydration) rather than with lipid-content. Thus, we present two clearing methods compatible with ex vivo MRI.

We assessed the feasibility of using stop codons as means to obtain polycistronic expression in eukaryotic cells. We show robust bicistronic expression of different open reading frames (ORFs), when these are cloned in sequence and simply separated by stop codons (in or out of frame), in heterologous expression systems and primary neurons. We further find this method to support polycistronic expression of three stop codon separated ORFs in vivo, which guided us to develop a technicolor Genetically Encoded Functional Rainbow Indicators (GEFRIs) for monitoring cellular morphology and neuronal firing, concomitantly. These findings guided us to develop a new technique we denote SPLIT: Stop codon mediated Polycistronic Induction in HeTerologous expression systems, for rapid and easy development of fragmented proteins by the sole use of stop codons. We validated the SPLIT method by generating several new split-GFP variants, then engineer a palette of functional split-GCaMP6s variants and, lastly, generate a split ca2+-probe localized at ER and mitochondria junctions, denoted split-MEGIC. With the use of the probe, we show presence and activity of mito-ER contact sites within individual dendritic spines. Split MEGIC can thereby be imaged by two photon excitation in vivo in mice brains and, by standard confocal microscope in transgenic zebrafish larvae. Together, we explore non canonical translation mechanisms and show these to be highly pervasive in various cell types in vitro and in vivo. We harness translation reinitiation to express multiple ORFs, to engineer rainbow indicators and to swiftly produce functional split-proteins and probes.

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Background: Site-directed mutagenesis (SDM) is a key method in molecular biology; allowing to modify DNA sequences at single base pair resolution. Although many SDM methods have been developed, methods that increase efficiency and versatility of this process remain highly desired. Method: We present a versatile and simple method to efficiently introduce a variety of mutation schemes using the Gibson-assembly without the need for unique Gibson primers. The method entails use of standard SDM primers (shorter and completely overlapping in sequences in contrast to Gibson primers) that are separately employed with common primer (~25 bps long) for amplification of fragments flanking the site of mutagenesis, followed by rapid amplification of the Gibson-assembled product for added visualization and sequencing steps for ensuring high success rates.Results: We find that assembly of the fragments via the Gibson reaction mixture is attainable within as short as 15 minutes, despite the need for extensive digestion of the DNA (by exonuclease) past the entire SDM primer sequence (to expose non-clashing overlap between the fragments). We also find that the amount of the assembled Gibson product is too low to be visualized and assessed on standard agarose gel. We thereby introduce a short amplification step (by use of the same short primers initially employed) to 1) easily resolve whether the product (only the correct size can yield a product) has been obtained, and 2) for isolation of product for DNA-sequencing (to assess whether mutation(s) have been introduced). No other SDM method enables assessment of mutagenesis prior completion of the process. Conclusion: We employ our approach to delete, replace, insert, and degenerate sequences within target DNA sequences, specifically in DNA sequences that proved very resistant to mutagenesis by multiple other SDM methods (standard and commercial). The entire protocol spans only four days, requires minimal primers sets (as well as can be used with most in-house primers) and provides very high yields and success rates (>98%).