Dhermendra Tiwari

Fluorescent Platinum Nanoclusters: Synthesis, Purification, Characterization, and Application to Bioimaging†

Noble-metal nanoclusters consisting of several atoms have been gaining much attention as novel fluorescent markers owing to their optical properties, which include size-dependent emission wavelength and discrete electronic state, features that are similar to semiconductor quantum dots. However, their smaller size and lower cytotoxicity in some ways make metal nanoclusters superior. Dickson and coworkers reported the synthesis and characterization of gold and silver 7] nanoclusters and successful bioimaging of actin filament labeled with these nanoclusters, while Lin et al. used peptide-conjugated gold nanoclusters as nuclear targeting and intracellular imaging probes. However, among the noble-metal clusters, platinum clusters have not yet been used as bioimaging probes except as a reducing catalyst. Herein, we describe water-soluble platinum nanoclusters that are less cytotoxic and emit a brighter fluorescence at 470 nm than other fluorescent nanomaterials, such as gold clusters. The synthesis of such platinum nanoclusters is achieved by a simple chemical reduction; they are purified by size-exclusion high performance liquid chromatography (HPLC). The size of the noble-metal nanoclusters is crucial because it affects the photoluminescence wavelength. 2, 3a, 4a,5a] This parameter can be regulated by a macromolecule-based template such as a dendrimer. One example is the fourth-generation polyamidoamine dendrimer (PAMAM (G4-OH)), which has been used as a molecular template in the synthesis of fluorescent gold nanoclusters. 8a,b] This dendrimer has nanometer-scale size and a uniform structure comprising an internal core and an external shell. Since the internal core contains tertiary amines that can form coordination bonds with the metallic ions, PAMAM dendrimers can trap these metal ions to form metal nanoclusters. Herein, we apply PAMAM (G4-OH) to synthesize Pt nanoclusters. Electronspray ionization (ESI) mass spectrometry confirmed that the synthesized nanoclusters were atomically monodispersed Pt5. Platinum nanoclusters were prepared by reducing H2PtCl6 (6.0 mL, 0.5m) with NaBH4 (3.0 mmol) in the presence of PAMAM (G4-OH) (0.5 mmol, 71.4 mg; Scheme 1). NaBH4

Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells

The early detection of HER2 (human epidermal growth factor receptor 2) status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs) have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs) using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC). As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs) with fluorescence quantum yields of 0.23∼0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line) was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission) on the fluorescence image of KPL-4 cells.

A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy

Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm2) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy.

Smart fluorescent proteins: Innovation for barrier-free superresolution imaging in living cells

During the past decade, several novel fluorescence microscopy techniques have emerged that achieve incredible spatial and temporal resolution beyond the diffraction limit. These microscopy techniques depend on altered optical setups, unique fluorescent probes, or post‐imaging analysis. Many of these techniques also depend strictly on the use of unique fluorescent proteins (FPs) with special photoswitching properties. These photoswitchable FPs are capable of switching between two states in response to light. All localization precision and patterned illumination techniques—such as photo‐activation localization microscopy, stochastic optical reconstruction microscopy, reversible saturable optically linear transitions, and saturated structured illumination microscopy—take advantage of these inherent switching properties to achieve superior spatial resolution. This review provides extensive analysis of the positive and negative aspects of photoswitchable FPs, highlighting their application in diffraction‐unlimited imaging and suggesting the most suitable fluorescent proteins for superresolution imaging.