Synthesis of nanomaterials with multi-imaging modality is of great importance in clinical molecular imaging and diagnostics. This work reports novel synthetic strategy to create ultrasmall and hexagonal upconversion nanoparticles (UCNPs), -NaGdF 4 : Yb 3+ , Er 3+ , and -NaGdF 4 : Yb 3+ , Tm 3+ , with inherent magnetic and efficient upconversion properties. The use of new combination of lanthanide chloride and sodium TFA as the precursors for UCNPs gave the best results in terms of size (10-40 nm), crystallinity and morphology, and proved to be cost-and time-saving. Water solubilization of both NaGdF 4 : Yb 3+ , Er 3+ , and -NaGdF 4 : Yb 3+ , Tm 3+ UCNPs was achieved by homogeneous polymer coating using amphiphilic poly(acrylic acid) derivatives. The strong upconversion and magnetic properties were maintained after extensive polymer coating process. To see the potential of the UCNPs for biological applications, the surface of NaGdF 4 : Yb 3+ , Er 3+ UCNPs were functionalized with Ni-nitrilotriacetate (NiNTA) moiety. The remarkable specificity of these NiNTAUCNPs for the oligohistidine peptide was clearly shown by both magnetic resonance and optical imaging. Finally, the cellular uptake of these UCNPs was investigated by fluorescence microscope using spectral imaging technique.

Multimodal nanoparticles have been extensively studied for target-specific imaging and therapy of various diseases, including cancer. In this study, radiolabeled arginine-glycine-aspartic acid (RGD)-functionalized Er 31 /Yb 31 co-doped NaGdF 4 upconversion nanophosphors (UCNPs) were synthesized and evaluated as a multimodal PET/MR/optical probe with tumor angiogenesisspecific targeting properties. Methods: A dimeric cyclic RGDyk ((cRGDyk) 2 ) peptide was conjugated to polyacrylic acid-coated NaGdF 4 :Yb 31 /Er 31 UCNPs along with polyethylene glycol molecules and was consecutively radiolabeled with 124 I. In vitro cytotoxicity testing was performed for 3 d. Upconversion luminescence imaging of (cRGDyk) 2 -UCNP was performed on U87MG cells with a laboratory-made confocal microscope. In vivo small-animal PET and clinical 3-T T1-weighted MR imaging of 124 I-labeled RGD-functionalized UCNPs was acquired with or without blocking of cyclic RGD peptide in a U87MG tumor model. Inductively coupled plasma mass spectrometry and biologic transmission electron microscopy were done to evaluate gadolinium concentration and UCNP localization, respectively. Results: Polymer-coated UCNPs and dimeric RGD-conjugated UCNPs were monodispersely synthesized, and those of hydrodynamic size were 30 6 8 nm and 32 6 9 nm, respectively. (cRGDyk) 2 -UCNPs have a low cytotoxic effect on cells. Upconversion luminescence signals of (cRGDyk) 2 -UCNP were specifically localized on the surface of U87MG cells. 124 I-c(RGDyk) 2 -UCNPs specifically accumulated in U87MG tumors (2.8 6 0.8 vs. 1.3 6 0.4 percentage injected dose per gram in the blocking experiment), and T1-weighted MR images showed significant positive contrast enhancement in U87MG tumors. Tumor localization of 124 I-c(RGDyk) 2 -UCNPs was confirmed by inductively coupled plasma mass spectrometry and biologic transmission electron microscopy analysis. Conclusion: These results suggest that 124 Ilabeled RGD-functionalized UCNPs have high specificity for a v b 3 integrin-expressing U87MG tumor cells and xenografted tumor models. Multimodal UCNPs can be used as a platform nanoparticle with multimodal imaging for cancer-specific diagnoses.

The exon-exon junction complex (EJC) forms via association of proteins during splicing of mRNA in a defined manner. Its organization provides a link between biogenesis, nuclear export, and translation of the transcripts. The EJC proteins accumulate in nuclear speckles alongside most other splicing-related factors. We followed the establishment of the EJC on mRNA by investigating the mobility and interactions of a representative set of EJC factors in vivo using a complementary analysis with different fluorescence fluctuation microscopy techniques. Our observations are compatible with cotranscriptional binding of the EJC protein UAP56 confirming that it is involved in the initial phase of EJC formation. RNPS1, REF/Aly, Y14/Magoh, and NXF1 showed a reduction in their nuclear mobility when complexed with RNA. They interacted with nuclear speckles, in which both transiently and long-term immobilized factors were identified. The location-and RNA-dependent differences in the mobility between factors of the so-called outer shell and inner core of the EJC suggest a hypothetical model, in which mRNA is retained in speckles when EJC outer-shell factors are missing.

We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope. We have replaced the conventional detection scheme by a prism-based spectrometer and an electron-multiplying charge-coupled device camera used to record the photons. This allows us to read out more than 80,000 full spectra per second with a signal-to-noise ratio and a quantum efficiency high enough to allow single photon counting. We can identify up to four spectrally different quantum dots in vitro and demonstrate that spectrally resolved detection can be used to characterize photophysical properties of fluorophores by measuring the spectral dependence of quantum dot fluorescence emission intermittence. Moreover, we can confirm intracellular cross-correlation results as acquired with a conventional setup and show that spectral flexibility can help to optimize the choice of the detection windows.

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/ mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells. ' International Society for Advancement of CytometryKey terms fluorescence resonance energy transfer; two-photon spectral imaging; unmixing; protein-protein interaction; YFP; mCherry THE current advances in light microscopy and engineering of fluorescent proteins (FPs) with improved properties and altered colors have provided the cellular biologist with the ability to investigate fine molecular events in living cells (1,2). Properly combined, fluorescent molecules allow the study of protein conformational changes or inter protein interaction by means of fluorescence resonance energy transfer (FRET) (3,4). When an excited donor fluorophore is proximate (=10 nm) and suitably oriented to an acceptor molecule, FRET occurs through a nonradiative dipoledipole interaction. Currently, the most popular FP pairs for FRET analysis are cyan with yellow fluorescent protein (CFP/YFP) and green with red fluorescent protein (GFP/RFP) (5,6).Among the methodologies based on nonradiative resonance energy transfer, two approaches prevail, the fluorescence lifetime imaging microscopy (FLIM), and the fluorescence intensity-based FRET imaging. The FLIM is known to be cumbersome to require highly specialized equipment and as well to involve long acquisition time (7). To the contrary, fluorescence intensity-based FRET estimation provides