Quantum dots (QDs) are semiconductor nanocrystals with extensive imaging and diagnostic capabilities, including the
potential for single molecule tracking. Commercially available QDs offer distinct advantages over organic fluorophores,
such as increased photostability and tunable emission spectra, but their cadmium selenide (CdSe) core raises toxicity
concerns. For this reason, replacements for CdSe-based QDs have been sought that can offer equivalent optical
properties. The spectral range, brightness and stability of InP QDs may comprise such a solution. To this end,
LANL/CINT personnel fabricated moderately thick-shell novel InP QDs that retain brightness and emission over time in
an aqueous environment. We are interested in evaluating how the composition and surface properties of these novel QDs
affect their entry and sequestration within the cell. Here we use epifluorescence and transmission electron microscopy
(TEM) to evaluate the structural properties of cultured Xenopus kidney cells (A6; ATCC) that were exposed either to
commercially available CdSe QDs (Qtracker® 565, Invitrogen) or to heterostructured InP QDs (LANL). Epifluorescence
imaging permitted assessment of the general morphology of cells labeled with fluorescent molecular probes (Alexa
Fluor® ® phalloidin; Hoechst 33342), and the prevalence of QD association with cells. In contrast, TEM offered unique
advantages for viewing electron dense QDs at higher resolution with regard to subcellular sequestration and
compartmentalization. Preliminary results show that in the absence of targeting moieties, InP QDs (200 nM) can
passively enter cells and sequester nonspecifically in cytosolic regions whereas commercially available targeted QDs
principally associate with membranous structures within the cell. Supported by: NIH 5R01GM084702.
Quantum dots (QDs) could offer significant advantages in clinical settings due to their high photostability and quantum yield. We are investigating the uptake and compartmentalization of QDs by cells because these processes are not fully characterized and there is potential for heavy metal toxicity when semiconductor nanocrystals are sequestered. Here we demonstrate the intracellular accumulation of QDs in human embryonic kidney cells (HEK-293; ATCC) exposed to nontargeted (Qtracker 565nm; QDot Corp.) or targeted (Qtracker 565 Cell Labeling Kit; QDot Corp.) QDs. As expected, 10 nM targeted QDs (Lagerholm et al., 2004, Nano Letters, 4:2019-2022) accumulated in HEK-293 cells and normal human astrocytes (NHA; Cambrex Biosciences) after 1 hr, while nontargeted QDs (200 nM) could be detected after 24 hr in HEK-293 but not NHA. The uptake of 10 nM targeted QDs was greater than the uptake of 200 nM nontargeted QDs as confirmed by the number and intensity of puncta visible in HEK-293 cells imaged with confocal microscopy. QD uptake was not detected in two <i>Xenopus</i> kidney cell lines (XLK-WG and A6; ATCC) exposed to nontargeted QDs (10-500 nM) for 18 hours. Co-labeling of HEK-293 cultures with CellTracker Red CMTPX (Invitrogen) following QD uptake verified that QD accumulation does not affect cell viability. Differences in QD uptake between cell lines could be species-specific or due to different growth conditions. The unexpected accumulation of nontargeted QDs raises questions about the uptake mechanism and the intracellular location that are being investigated with TEM. Supported by NIH-NIDCD (DC003292) and NMSU-ADVANCE (NSF0123690) to EES.
Quantum dot bioconjugates offer unprecedented opportunities for monitoring biological processes and molecular interactions in cells, tissues, and organs. We are interested in developing applications that permit investigation of physiological processes and cytoskeletal organization in live cells, and allow imaging of complex organs, such as the auditory and vestibular sensory structures of the inner ear. Multiphoton microscopy is a powerful technique for acquiring images from deep within a sample while reducing phototoxic effects of laser light exposure on cells. Previous studies have established that a solid-state Nd:YLF laser can be used to acquire two-photon and three-photon images from live cells while minimizing phototoxic side effects (Wokosin et al., 1996, <i>Bioimaging</i>, 4:208-214; Squirrell et al., 1999, <i>Nature Biotechnology</i>, 8:763-767). We present here the results of experiments using an all-solid-state Nd:YLF 1047 nm femtosecond laser (Microlase DPM1000) source to excite quantum dot bioconjugates. Cells were labeled with Qdot (Quantum Dot Corporation) bioconjugates or with Alexa Fluor (Molecular Probes) bioconjugates and then imaged with a BioRad 1024 confocal microscope configured for multiphoton imaging using internal or external (non-descanned) detectors. Results demonstrate that the Nd:YLF laser can be used to stimulate fluorescence emission of quantum dots and Alexa Fluor bioconjugates in cultured amphibian (<i>Xenopus</i>) and mammalian (rat, chinese hamster) cells. We conclude that the Nd:YLF laser is a viable excitation source that extends the applicability of quantum dots for investigation of biological processes using multiphoton microscopy.