Postdoctoral researcher, University of Copenhagen/Nano-Science center & Department of chemistry
Fluorescence microscopy relies on the separation of a specific fluorescence signal from the background signal. However, the background rarely is truly dark and high concentration of organic dyes, strong illumination and image analysis is needed to enhance the specific fluorescence signal of the dye. In contrast, lanthanide centered emission enables background-free imaging through, e.g. time-gating or by using near infrared (NIR) wavelengths for excitation and/or emission. This is due to the unique optical properties of lanthanides: very narrow absorption and emission peaks in the wavelength range all the way from UV to NIR with long luminescent lifetimes (up to milliseconds). Therefore, the long-lived lanthanide luminescence can be read long after the short-lived background luminescence has died out. Furthermore, NIR wavelengths are poorly absorbed by biological material, so by using the NIR-emitting lanthanides or upconverting nanophosphors with NIR-excitation and anti-Stokes emission, the background can be reduced or completely removed. We have explored the use of spectral imaging with lanthanides. The narrow emission peaks of lanthanides can readily be identified even from under a strong background luminescence or wide peaks of organic fluorophores. Therefore, a high contrast fluorescence microscopy image can be obtained merely by forming the image using only the photons arising from the narrow lanthanide peaks while excluding the photons arising from any broad spectral feature. Thus, the origin of the photons can be assigned with absolute certainty.
Abstract: The narrow, near infrared (NIR) emission from lanthanide ions has attracted great interest, particularly with regard to developing tools for bioimaging, where the long lifetimes of lanthanide excited states can be exploited to address problems arising from autofluorescence and sample transparency. Despite the promise of lanthanide-based probes for near-IR imaging, few reports on their use are present in the literature. Here, we demonstrate that images can be recorded by monitoring NIR emission from lanthanide complexes using detectors, optical elements and a microscope that were primarily designed for the visible part of the spectrum.
Pub.: 08 Jan '15, Pinned: 27 Jun '17
Abstract: Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process and also, acidification of vesicles destined for exocytosis. To monitor these events the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low energy 980 nm laser excitation, that facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and in in vivo applications, e.g. for the bioimaging of small animal models.
Pub.: 16 Dec '16, Pinned: 27 Jun '17
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