An official website of the United States government
Photoactivated localization microscopy (PALM) relies on fluorescence photoactivation and single-molecule localization to overcome optical diffraction and reconstruct images of biological samples with spatial resolution at the nanoscale. The implementation of this subdiffraction imaging method, however, requires fluorescent probes with photochemical and photophysical properties specifically engineered to enable the localization of single photoactivated molecules with nanometer precision. The synthetic versatility and outstanding photophysical properties of the borondipyrromethene (BODIPY) chromophore are ideally suited to satisfy these stringent requirements. Specifically, synthetic manipulations of the BODIPY scaffold can be invoked to install photolabile functional groups and photoactivate fluorescence under photochemical control. Additionally, targeting ligands can be incorporated in the resulting photoactivatable fluorophores (PAFs) to label selected subcellular components in live cells. Indeed, photoactivatable BODIPYs have already allowed the sub-diffraction imaging of diverse cellular substructures in live cells using PALM and can evolve into invaluable analytical probes for bioimaging applications.
more »
« less
Oxime as a general photocage for the design of visible light photo-activatable fluorophores
Photoactivatable fluorophores have been widely used for tracking molecular and cellular dynamics with subdiffraction resolution. In this work, we have prepared a series of photoactivatable probes using the oxime moiety as a new class of photolabile caging group in which the photoactivation process is mediated by a highly efficient photodeoximation reaction. Incorporation of the oxime caging group into fluorophores results in loss of fluorescence. Upon light irradiation in the presence of air, the oxime-caged fluorophores are oxidized to their carbonyl derivatives, restoring strong fluorophore fluorescence. To demonstrate the utility of these oxime-caged fluorophores, we have created probes that target different organelles for live-cell confocal imaging. We also carried out photoactivated localization microscopy (PALM) imaging under physiological conditions using low-power light activation in the absence of cytotoxic additives. Our studies show that oximes represent a new class of visible-light photocages that can be widely used for cellular imaging, sensing, and photo-controlled molecular release.
more »
« less
- Award ID(s):
- 1803066
- PAR ID:
- 10357357
- Date Published:
- Journal Name:
- Chemical Science
- Volume:
- 12
- Issue:
- 47
- ISSN:
- 2041-6520
- Page Range / eLocation ID:
- 15572 to 15580
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract The borondipyrromethene (BODIPY) chromophore is a versatile platform for the construction of photoresponsive dyes with unique properties. Specifically, its covalent connection to a photocleavable group can be exploited to engineer compounds with photoswitchable fluorescence. The resulting photoactivatable fluorophores can increase their emission intensity or shift their emission wavelengths in response to switching. Such changes permit the spatiotemporal control of fluorescence with optical stimulations and the implementation of imaging strategies that would be impossible to replicate with conventional fluorophores. Indeed, BODIPYs with photoactivatable fluorescence enable the selective highlighting of intracellular targets, the nanoscaled visualization of sub‐cellular components, the real‐time monitoring of dynamic events and the photochemical writing of optical barcodes.more » « less
-
Osiński, Marek; Kanaras, Antonios G. (Ed.)Single-molecule localization microscopy (SMLM) strategies based on fluorescence photoactivation permit the imaging of live cells with subdiffraction resolution and the high-throughput tracking of individual biomolecules in their interior. They rely predominantly on genetically-encoded fluorescent proteins to label live cells selectively and allow the sequential single-molecule localization of sparse populations of photoactivated fluorophores. Synthetic counterparts to these photoresponsive proteins are limited to a few remarkable examples at the present stage, mostly because of the daunting challenges in engineering biocompatible molecular constructs with appropriate photochemical and photophysical properties for live-cell SMLM. Our laboratory developed a new family of synthetic photoactivatable fluorophores specifically designed for these imaging applications. They combine a borondipyrromethene (BODIPY) fluorophore and an ortho-nitrobenzyl (ONB) photocage in a single molecular skeleton. The photoinduced ONB cleavage extends electronic delocalization to shift bathochromically the BODIPY absorption and emission bands. As a result, these photochemical transformations can be exploited to switch fluorescence on in a spectral region compatible with bioimaging applications and allow the localization of the photochemical product at the single-molecule level. Furthermore, our compounds can be delivered and operated in the interior of live cells to enable the visualization of organelles with nanometer resolution and the intracellular tracking of single photoactivated molecules.more » « less
-
Carbon dots (CDs) are emerging as the material of choice in a range of applications due to their excellent photoluminescence properties, ease of preparation from inexpensive precursors, and low toxicity. However, the precise nature of the mechanism for the fluorescence is still under debate, and several molecular fluorophores have been reported. In this work, a new blue fluorophore, 5-oxopyrrolidine-3-carboxylic acid, was discovered in carbon dots synthesized from the most commonly used precursors: citric acid and urea. The molecular product alone has demonstrated interesting aggregation-enhanced emission (AEE), making it unique compared to other fluorophores known to be generated in CDs. We propose that this molecular fluorophore is associated with a polymer backbone within the CDs, and its fluorescence behavior is largely dependent on intermolecular interactions with the polymers or other fluorophores. Thus, a new class of non-traditional fluorophores is now relevant to the consideration of the CD fluorescence mechanism, providing both an additional challenge to the community in resolving the mechanism and an opportunity for a greater range of CD design schemes and applications.more » « less
-
Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/d1sc05351e
