ABSTRACT
When performing multiphoton fluorescence lifetime imaging in multiple spectral emission channels, an instrument response function must be acquired in each channel if accurate measurements of complex fluorescence decays are to be performed. Although this can be achieved using the reference reconvolution technique, it is difficult to identify suitable fluorophores with a mono-exponential fluorescence decay across a broad emission spectrum. We present a solution to this problem by measuring the IRF using the ultrafast luminescence from gold nanorods. We show that ultrafast gold nanorod luminescence allows the IRF to be directly obtained in multiple spectral channels simultaneously across a wide spectral range. We validate this approach by presenting an analysis of multispectral autofluorescence FLIM data obtained from human skin ex vivo.
Subject(s)
Gold/chemistry , Imaging, Three-Dimensional/instrumentation , Imaging, Three-Dimensional/methods , Luminescence , Microscopy, Fluorescence, Multiphoton/instrumentation , Microscopy, Fluorescence, Multiphoton/methods , Humans , In Vitro Techniques , Nanotubes , Spectrometry, Fluorescence , Time FactorsABSTRACT
Multiphoton tomography for in vivo high-resolution multidimensional imaging has been used in clinical investigations and small animal studies. The novel femtosecond laser tomographs have been employed to detect cosmetics and pharmaceutical components in situ as well as to study the interaction of drugs with intratissue cells and the extracellular matrix under physiological conditions. Applications include the intra-tissue accumulation of sunscreen nanoparticles in humans, the monitoring the metabolic status of patients with dermatitis, the biosynthesis of collagen after administration of anti-aging products, and the detection of porphyrins after application of 5-aminolevulinic acid. More than 2000 patients and volunteers in Europe, Australia, and Asia have been investigated with these unique tomographs. In addition, femtosecond laser nanoprocessing microscopes have been employed for targeted delivery and deposition in body organs, optical transfection and optical cleaning of stem cells, as well as for the optical transfer of molecular beacons to track microRNAs. These diverse applications highlight the capacity for multiphoton tomography and femtosecond laser nanoprocessing tools to advance drug delivery research.
Subject(s)
Drug Delivery Systems , Microscopy, Confocal/methods , Tomography/methods , Animals , Humans , Microscopy, Fluorescence, Multiphoton/methods , Tomography, Optical Coherence/methodsABSTRACT
The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA-122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMB-probes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells. In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus.
Subject(s)
Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Hepatocytes/metabolism , Hepatocytes/ultrastructure , MicroRNAs/pharmacokinetics , Microscopy, Fluorescence, Multiphoton/methods , Biological Transport, Active/physiology , Cell Line , HumansABSTRACT
Telling the difference quickly: Femtosecond laser pulses are not only suitable to distinguish structural isomers. They also provide access to the distinction of enantiomers by combination of circular dichroism and mass spectrometry [picture: see text].
