Jun-ichi Hotta

Transfection of living HeLa cells with fluorescent poly-cytosine encapsulated Ag nanoclusters

The fluorescence of silver clusters encapsulated by single stranded oligo-DNA (24 cytosine base pairs, C(24):Ag(n)) was used to monitor the transfection of this new silver/DNA fluorophore inside living HeLa cells. For this, the C(24):Ag(n) molecules were complexed with a commercially available transfection reagent Lipofectamine and the internalization of C(24):Ag(n) was followed with confocal fluorescence microscopy. Bright near-infrared fluorescence was observed from inside the transfected HeLa cells, when exciting with 633 nm excitation, opening up the possibility for the use of these C(24):Ag(n) clusters for biological labelling and imaging of living cells and for monitoring the transfection process with limited harm to the living cells.

Optical Encoding of Silver Zeolite Microcarriers

and,thus,onlyalimited number of unique codes is available. More recentencoding strategies rely on photobleaching of microcarriersduringthemicroscopicassay,alsocalledactiveencodingopposedto the previous fixed encoding but the technique suffers fromlimited pattern readability due to the negative contrast and fastphotobleaching.

Spectroscopic Rationale for Efficient Stimulated-Emission Depletion Microscopy Fluorophores

We report a rationale for identifying superior dyes for stimulated-emission depletion (STED) microscopy. We compared the dyes pPDI and pTDI, which displayed excellent photostability in single-molecule spectroscopy. Surprisingly, their photostability and performance in STED microscopy differed significantly. While single pTDI molecules could be visualized with excellent resolution (35 nm), pPDI molecules bleached rapidly under similar conditions. Femtosecond transient absorption measurements proved that the overlap between the stimulated-emission band and the excited-state absorption band is the main reason for the observed difference. Thus, assessment of the excited-state absorption band provides a rational means of dye selection and determination of the optimal wavelength for STED.

DNA fluorocode: A single molecule, optical map of DNA with nanometre resolution

We present a new method for single-molecule optical DNA mapping using an exceptionally dense, yet sequence-specific coverage of DNA with a fluorescent probe. The method employs a DNA methyltransferase enzyme to direct the DNA labelling, followed by molecular combing of the DNA onto a polymer-coated surface and subsequent sub-diffraction limit localization of the fluorophores. The result is a ‘DNA fluorocode’; a simple description of the DNA sequence, with a maximum achievable resolution of less than 20 bases, which can be read and analyzed like a barcode. We demonstrate the generation of a fluorocode for genomic DNA from the lambda bacteriophage using a DNA methyltransferase, M.HhaI, to direct fluorescent labels to four-base sequences reading 5′-GCGC-3′. A consensus fluorocode that allows the study of the DNA sequence at the level of an individual labelling site can be generated from a handful of molecules.

In situ measurements of ion-exchange processes in single polymer particles : laser trapping microspectroscopy and confocal fluorescence microspectroscopy

Ion-exchange processes of a cationic dye (Rhodamine B; RhB) were studied for individual polymer particles (diameter of 16-20 μm) by laser trapping microspectroscopy and confocal fluorescence laser microspectroscopy. The absorbance of RhB at 565 nm adsorbed on a cation-exchange particle increased linearly with the concentration of RhB in the aqueous phase, while it was independent of the particle diameter. Fluorescence intensity profile measurements of RhB along the particle diameter by confocal fluorescence microspectroscopy directly proved that ion exchange took place in the surface layer (∼2-μm thickness) of the particle in the initial stage (1 h). Diffusion of RhB in the particle was very slow, and ion exchange proceeded gradually to the inner volume in the order of days. The ion-exchange processes were analyzed on the basis of simulation of the time course of the concentration profile of RhB in the particle, and the diffusion coefficient of RhB was determined to be (2-4) × 10(-11) cm(2)·s(-1).

Fano resonance in a multimode tapered fiber coupled with a microspherical cavity

Fano resonance in a tapered optical fiber in contact with a high-Q microsphere is demonstrated. Multimode waves propagating in a 2.3μm diameter taper were coupled with a single whispering gallery mode of a 220μm sphere, and their coherent interaction resulted in Fano resonance. The asymmetric line shapes of the transmission spectra changed periodically with scanning of the coupling position along the taper. The observed 24μm period was due to modal dispersion in the tapered fiber.

The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering

Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein–protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting.

Data storage based on photochromic and photoconvertible fluorescent proteins

The recent discovery of photoconvertible and photoswitchable fluorescent proteins (PCFPs and RSFPs, respectively) that can undergo photoinduced changes of their absorption/emission spectra opened new research possibilities in subdiffraction microscopy and optical data storage. Here we demonstrate the proof-of-principle for read only and rewritable data storage both in 2D and 3D, using PCFPs and RSFPs. The irreversible burning of information was achieved by photoconverting from green to red defined areas in a layer of the PCFP Kaede. Data were also written and erased several times in layers of the photochromic fluorescent protein Dronpa. Using IrisFP, which combines the properties of PCFPs and RSFPs, we performed the first encoding of data in four colours using only one type of fluorescent protein. Finally, three-dimensional optical data storage was demonstrated using three mutants of EosFP (d1EosFP, mEosFP and IrisFP) in their crystalline form. Two-photon excitation allowed the precise addressing of regions of interest (ROIs) within the three-dimensional crystalline matrix without excitation of out-of-focus optical planes. Hence, this contribution highlights several data storage schemes based on the remarkable properties of PCFPs/RSFPs.

Excitation energy migration processes in cyclic porphyrin arrays probed by single molecule spectroscopy.

By using single molecule fluorescence spectroscopy we have investigated the excitation energy migration processes occurring in a series of cyclic porphyrin arrays bearing a close proximity in overall architectures to the LH2 complexes in purple bacterial photosynthetic systems. We have revealed that the conformational heterogeneity induced by the structural flexibility in large cyclic porphyrin arrays, which provides the nonradiative deactivation channels as an energy sink or trap, reduces significantly the energy migration efficiency. Our study provides detailed information on the energy migration efficiency of the artificial light-harvesting arrays at the single molecule level, which will be a guideline for future applications in single molecular photonic devices in the solid state.

Resonant Frequency Control of a Microspherical Cavity by Temperature Adjustment

We demonstrate frequency tuning of whispering-gallery mode resonances in a fused-silica microsphere by means of temperature control. Scattering spectra of the microsphere are measured using a tunable narrow-linewidth diode laser, and the frequency scale is precisely calibrated from a simultaneously observed spectrum of rubidium vapor. The resonant frequencies of whispering-gallery modes can be varied linearly by temperature elevation and lowering, at a rate of -2.6 GHz/K for a 190 µm sphere.