Yan-Kai Tzeng

Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds

Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost lung tissue in patients. Several optical imaging methods and probes have been used to track how stem cells incorporate and regenerate themselves in vivo over time. However, these approaches are limited by photobleaching, toxicity and interference from background tissue autofluorescence. Here we show that fluorescent nanodiamonds, in combination with fluorescence-activated cell sorting, fluorescence lifetime imaging microscopy and immunostaining, can identify transplanted CD45–CD54+CD157+ lung stem/progenitor cells in vivo, and track their engraftment and regenerative capabilities with single-cell resolution. Fluorescent nanodiamond labelling did not eliminate the cells’ properties of self-renewal and differentiation into type I and type II pneumocytes. Time-gated fluorescence imaging of tissue sections of naphthalene-injured mice indicates that the fluorescent nanodiamond-labelled lung stem/progenitor cells preferentially reside at terminal bronchioles of the lungs for 7 days after intravenous transplantation. Supplementary information The online version of this article (doi:10.1038/nnano.2013.147) contains supplementary material, which is available to authorized users.

Sub-20-nm fluorescent nanodiamonds as photostable biolabels and fluorescence resonance energy transfer donors.

Adv. Mater. 2010, 22, 843–847 2010 WILEY-VCH Verlag Gm A current trend in fluorescent probe technology is to expand the role of fluorophores that emit light in the far red and near infrared region for biomedical use. The negatively charged nitrogenvacancy defect center, (N-V) , in type Ib diamond is one of such fluorophores and has drawn much attention in recent years. The center exhibits several distinct features such as extended red emission at 700 nm, near-unity fluorescence quantum yield, and ultrahigh photostability (neither photobleaching nor photoblinking). These characteristics, along with the diverse surface functionalizability and non-toxic nature of the material, have made fluorescent nanodiamond (FND) a promising candidate among other conventional markers and labels, for example, organic dyes, fluorescent proteins, and quantum dots, for bioimaging applications. Despite the unique photophysical and biochemical properties of FND, the size is an important parameter to characterize. Decreasing the size, clearly, will increase the mobility of the fluorescent nanoprobe inside a cell, avoid altering the properties of targeted molecules, and enhance its translocation into cell nuclei. Additionally, particles of size in the range of 10 nm are more suitable for biolabeling and fluorescence resonance energy transfer (FRET) applications. However, so far the sizecontrolled production of FNDs has not yet been so well developed as that of other probes and scale-up synthesis of smaller and brighter FNDs remains a challenging task. In this communication, we report procedures of production and isolation of sub-20-nm FND particles utilizing He ion irradiation and differential centrifugation methods, and experiments demonstrating that they are useful as far-red fluorescent biolabels as well as FRET donors are presented. Table 1 compares the optical properties between (N-V) and two representative red and far-red fluorescent proteins. The in vivo structure of the latter protein is in form of tetramer and, therefore, has a size close to 10 nm. A simple calculation based on the molar extinction coefficient and the fluorescence quantum yield indicates that the brightness of a single (N-V) center is 2-fold as high as that of this far-red fluorescent protein, owing to the superb quantum yield of the atom-like fluorophore. Moreover, the (N-V) center fluoresces in the farther red region, where both autofluorescence and light scattering from the active biological environment are reduced to a greater extent. While compared to the infrared dye (such as IRDye-800CW in Table 1), the (N-V) center is a factor of 5 lower in brightness, this deficit can be readily compensated if each FND particle contains multiple (N-V) centers. It is the aim of this work to produce FNDs with a size comparable to those of far-red fluorescent proteins and concurrently containing as many (N-V) centers as possible. A previous experiment has shown that FNDs can be mass-produced with 40-keV Heþ irradiation of synthetic type Ib diamond nanocrystallites, followed by thermal annealing at 800 8C. For diamond powders with amedian size of 35 nm from a commercial source (MSY; Microdiamant), they usually have a broad size distribution and 10% of the particles are smaller than 15 nm. By optimizing the experimental parameters in differential centrifugation, we extracted sub-20-nm FNDs from the 35-nm ensembles after theHeþ irradiation andmulti-step centrifugation procedures. The sizes of these particles were finally characterized with dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). As revealed by DLS (Fig. 1a), the average diameter of the smallest FNDs that we have been able to extract with use of a standard centrifuge (Model 3700; Kubota) is 11.5 nm, accompanied with a narrow size deviation from 10 to 14 nm. TEM and AFM (Figs. S1 and S2 in Supporting Information) also confirmed independently the size distribution centered in the range of 10 nm at the single particle level. To make a direct comparison of the optical properties between the 11-nm FNDs and monomeric red fluorescent proteins (recombinant DsRed; BioVision), we characterized both specimens in solution with fluorescence correlation spectroscopy (FCS).

Time-Resolved Luminescence Nanothermometry with Nitrogen-Vacancy Centers in Nanodiamonds.

Measuring temperature in nanoscale spatial resolution either at or far from equilibrium is of importance in many scientific and technological applications. Although negatively charged nitrogen-vacancy (NV(-)) centers in diamond have recently emerged as a promising nanometric temperature sensor, the technique has been applied only under steady state conditions so far. Here, we present a three-point sampling method that allows real-time monitoring of the temperature changes over ±100 K and a pump-probe-type experiment that enables the study of nanoscale heat transfer with a temporal resolution of better than 10 μs. The utility of the time-resolved luminescence nanothermometry was demonstrated with 100 nm fluorescent nanodiamonds spin-coated on a glass substrate and submerged in gold nanorod solution heated by a near-infrared laser, and the validity of the measurements was verified with finite-element numerical simulations. The combined theoretical and experimental approaches will be useful to implement time-resolved temperature sensing in laser processing of materials and even for devices in operation at the nanometer scale.

Facile MALDI-MS analysis of neutral glycans in NaOH-doped matrixes: microwave-assisted deglycosylation and one-step purification with diamond nanoparticles.

A streamlined protocol has been developed to accelerate, simplify, and enhance matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) of neutral underivatized glycans released from glycoproteins. It involved microwave-assisted enzymatic digestion and release of glycans, followed by rapid removal of proteins and peptides with carboxylated/oxidized diamond nanoparticles, and finally treating the analytes with NaOH before mixing them with acidic matrix (such as 2,5-dihydroxybenzoic acid) to suppress the formation of both peptide and potassiated oligosaccharide ions in MS analysis. The advantages of this protocol were demonstrated with MALDI-TOF-MS of N-linked glycans released from ovalbumin and ribonuclease B.