Synthesis of Rhodamines from Fluoresceins Using Pd-Catalyzed C–N Cross-Coupling

A unified, convenient, and efficient strategy for the preparation of rhodamines and <i>N,N</i>′-diacylated rhodamines has been developed. Fluorescein ditriflates were found to undergo palladium-catalyzed C–N cross-coupling with amines, amides, carbamates, and other nitrogen nucleophiles to provide direct access to known and novel rhodamine derivatives, including fluorescent dyes, quenchers, and latent fluorophores

Synthesis of Rhodamines from Fluoresceins Using Pd-Catalyzed C–N Cross-Coupling

A unified, convenient, and efficient strategy for the preparation of rhodamines and <i>N,N</i>′-diacylated rhodamines has been developed. Fluorescein ditriflates were found to undergo palladium-catalyzed C–N cross-coupling with amines, amides, carbamates, and other nitrogen nucleophiles to provide direct access to known and novel rhodamine derivatives, including fluorescent dyes, quenchers, and latent fluorophores

Correlated images of lamin B1-mEos2 PALM data with electron micrographs.

<p>(<b>A</b>) Correlated image of Lamin B1-mEos2 signal with electron micrograph. White arrows point to Au beads used for alignment. (<b>B</b>) Higher magnification view of an area from <b>A</b> showing a possible nuclear pore (red arrowhead) with corresponding gap in Lamin B1-mEos2 signal. (<b>C</b>) SEM only image of panel <b>B</b> to show cellular ultrastructure. M, mitochondrion. G, Golgi apparatus.</p

Correlative Photoactivated Localization and Scanning Electron Microscopy

<div><p>The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.</p></div

Overview of correlative PALM and SEM procedure and the PALM imaging system.

<p>(<b>A</b>) Flow diagram showing the steps involved in correlating PALM and SEM images. (<b>B</b>) PALM imaging set-up showing the 100 nm thick cryosection on an ITO coated coverslip with 80 nm Au beads that are used for image registration and alignment.</p

Caged dye-phalloidin labeling of actin and correlated PALM/SEM.

<p>(<b>A</b>) Maximum intensity projection image of confocal z-stack of a cell with actin labeled using NVOC₂-carborhodamine110–PEG₈–phalloidin (magenta) and the nuclear stain 4',6-diamidino-2-phenylindole (blue). (<b>B</b>) PALM image of caged dye labeled actin. (<b>C</b>) SEM of area imaged by PALM in panel <b>B</b>. (<b>D</b>) Correlated PALM and SEM of caged dye labeled actin.</p

Correlated images of peroxisome-localized mEos2-SKL PALM data with electron micrographs.

<p>(<b>A</b>, <b>C</b>) SEM image of cells showing mitochondria (M) and other membranous organelles. Red arrowheads indicate peroxisomes as seen by overlaying PALM data of mEos2-SKL on electron micrographs (<b>B</b>, <b>D</b>).</p

PALM imaging acquisition parameters.

<p>Power densities, lasers, filters, and the durations of activation and exposure pulses for different wavelength channels during PALM acquisition.</p

Correlated images of TFAM-mEos2 PALM data with electron micrographs.

<p>(<b>A</b>) PALM image of TFAM-mEos2. (<b>B</b>) SEM image of mitochondria (M) in the same area imaged in <b>A</b>. (<b>C</b>) Correlated image of TFAM-mEos2 PALM data with SEM data. TFAM-mEos2 resides in the mitochondrial matrix surrounded by boundary and cristae membranes.</p

Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

<p>The utility of<b> </b>small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2‑nitro-<i>N</i>-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p