Technical Reference #1655

1655.

Non-blinking and Photostable Upconverted Luminescence from Single Lanthanide Doped Nanocrystals Shiwei Wua; Gang Hana; Delia J. Millirona; Shaul Alonia; Virginia Altoea; Dmitri V. Talapinb; Bruce E. Cohena;; and P. James Schucka;, University of Chicago, PNAS, 106(1655), (2009)
Link To Paper

Abstract:
The development of probes for single-molecule imaging has dramatically facilitated the study of individual molecules in cells andother complex environments. Single-molecule probes ideally exhibitgood brightness uninterrupted emission resistance to p

Keywords:
bio-imaging; fluorescence; nanoparticle; single molecule phosphorescence

Materials & Methods:
Sample Preparation and Electron Microscopic Characterizations. A sample of oleic acid-coatedUCNPsfor single-particle characterizationwaspreparedbydrop casting a dilute solution of UCNPs in hexane on a 20-nm thick silicon nitride membrane window (4107SN-BA SPI Supplies) which allowed both optical and electron microscopic characterization. The window size of 100 m permitted us to inspect thesameregion of interest inaSEMat30kV(FESEM-Ultra 55 Zeiss)and in aTEMat 200 kV (FETEM-2100F Jeol) equipped with an energy dispersive X-ray spectrometer (INCAEnergyTEM250 Oxford) after optical characterization. The region of interest was mapped using a transmission mode in the SEM (TM-SEM) as well as the TEM. The morphology structure and composition of individual nanoparticles were obtained by HR-TEM and EDS. The samples of individual amphiphilic polymer-coated UCNPs and citric acid-coated UCNPs were prepared on a separate polyD-Lysine pretreated coverglass by drop casting. Optical Characterization. Upconverted luminescence of individual UCNPs was characterized in a modified sample-scanning confocal optical microscope (TE- 2000U Nikon). A 980-nm single-modeCWdiode laser (L980P300J Thorlabs) was tightly focused on the sample through a 0.95 NA 100X air objective (Plan Apo Nikon). The sample was raster scanned by a piezo-actuated three-dimensional nanopositioning stage (Nano-PDQ375 Madcity). At each pixel the upconverted visible luminescence was collected through the same objective and passed through a dichroic beamsplitter (750DCSPXR Chroma) and 2 short-pass filters (700SP-2P and 750SP-2P Chroma) while the 980-nm excitation light was completely filtered out. After passing through a confocal pinhole (150 m) the upconvertedluminescencewaseither detectedbyaSPAD(SPCM-AQR-15Perkin- Elmer) orbyaspectrograph (SP-2356Acton)equippedwithaCCDdetector (iXon Andor).TheupconvertedphotonsdetectedbytheSPADcanalsoberecordedand time-tagged with a TCSPC (PicoHarp 300 PicoQuant). Cell Culture and Imaging. NIH 3T3 mouse embryonic fibroblasts (ATCC) were grown to 70% confluence on polyD-lysine coated glass-bottom dishes (MatTek) in modified high-glucoseDMEMwith10%FBS. Cells were incubated for 3 h with 10pMamphiphilic polymer-coatedUCNPsinDMEMwith FBShumidifiedat37 °C under 5% CO2. Cells were washed 3 times with PBS and then resuspended in DMEMfor imaging. Images were acquired with a 1.4NA100 oil objective (Plan Apo Nikon) on the same confocal optical microscope as described above. Upconverted luminescence was excited with a 980-nm CW laser at a power of approximately 10 mW and cellular autofluorescence with a 532-nm CW laser at apowerof approximately 85 W.AZ532RDCdichroic beamsplitter (Chroma)and 3RD540LP emission filter (Omega Optical) were used for autofluorescent images and optics for upconverted images were the same as described above.

Microscopic Technique
Confocal Microscopy, Optical

Cell Type(s)
3T3