Label-Free Chemical Nano-Imaging of Intracellular Drug Binding Sites
William S. HartHemmel AmraniaAlice BeckleyJochen R. BrandtSandeep SundriyalAinoa Rueda‐ZubiaurreAlexandra E. PorterEric O. AboagyeMatthew J. FuchterChris C. Phillips
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Optical microscopy has a diffraction limited resolution of about 250 nm. Fluorescence methods (e.g. PALM, STORM, STED) beat this, but they are still limited to 10 s of nm, and the images are an indirect pointillist representation of only part of the original object. Here we describe a way of combining a sample preparation technique taken from histopathology, with a probe-based nano-imaging technique, (s SNOM) from the world of Solid State Physics. This allows us to image subcellular structures optically, and at a nanoscale resolution that is about 100 x better than normal microscopes. By adding a tuneable laser source, we also demonstrate mid-infrared chemical nano-imaging (MICHNI) in human myeloma cells and we use it to map the binding sites of the anti cancer drug bortezomib to less than 10 zL sized intracellular components. MICHNI is label free and can be used with any biological material and drugs with specific functional chemistry. We believe that its combination of speed, cheapness, simplicity, safety and chemical contrast promises a transformative impact across the life sciences.Keywords:
STED microscopy
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This chapter covers the basic principles of super-resolution fluorescence microscopy but recognises that essential operational details can only be learned within an experienced imaging facility of a research group where super-resolution microscopes are the central tool. The first practical implementation of super-resolution imaging in far-field microscopy with lenses was by STED, or stimulated emission depletion, in 1994. RESOLFT is an acronym that stands for reversible saturable optical fluorescence transitions. STED microscopy differs from RESOLFT in that it uses nonlinear excitation saturation to switch fluorophores into the dark, ground, state by emission depletion. While STED microscopy was being developed by Stefan Hell's research group, Heintzmann and Gustafsson were independently developing structured illumination microscopy (SIM). Normally SIM is limited only by the readout time of the camera to about 100 ms per image. Most super-resolution methods exhibit a common drawback: with respect to the total number of emitted photons, they are less efficient than standard widefield microscopy.
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Abstract Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultra-structure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides sub-diffraction resolution by physically enlarging the sample before microscopy. Expansion of fixed cells by crosslinking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complimentary approaches, we here combined ExM with STED (ExSTED) and demonstrate an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straight forward, we found that high fidelity labelling via multi-epitopes is required to obtain emitter densities that allow to resolve ultra-structural details with ExSTED. Our work provides a robust template for super resolution microscopy of entire cells in the ten nanometer range.
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The concept of stimulated emission depletion (STED) microscopy demonstrated that the diffraction barrier could, in fact, be surpassed. In STED microscopy, the diffraction limit is overcome by exploiting the inherent photophysical properties of the fluorescent molecules to reduce the size of the effective focal volume of a laser scanning microscope (LSM). The two major classes of fluorophores available for STED microscopy are fluorescent proteins (FPs) and small-molecule organic dyes. In recent years, STED microscopy has been extended to three-dimensional, multicolor, and live-cell imaging. These innovations are already proving to be invaluable tools for cell biology. Reversible saturable optical fluorescence transitions (RESOLFT) microscopy using reversibly switching FPs or other fluorophores with on/off transitions that require low light intensities is a compelling alternative to STED microscopy. Given recent advances in imaging speed for this super-resolution modality, the ability to image subcellular dynamics with reduced phototoxicity should be of broad appeal.
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Abstract We present localization with stimulated emission depletion (LocSTED) microscopy, a combination of STED and single-molecule localization microscopy (SMLM). We use the simplest form of a STED microscope that is cost effective and synchronization free, comprising continuous wave (CW) lasers for both excitation and depletion. By utilizing the reversible blinking of fluorophores, single molecules of Alexa 555 are localized down to ~5 nm. Imaging fluorescently labeled proteins attached to nanoanchors structured by STED lithography shows that LocSTED microscopy can resolve molecules with a resolution of at least 15 nm, substantially improving the classical resolution of a CW STED microscope of about 60 nm. LocSTED microscopy also allows estimating the total number of proteins attached on a single nanoanchor.
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Abstract Optical fluorescence microscopy provides molecular specificity and high contrast, which are powerful aspects in biomedical researches. Moreover, super-resolution microscopy techniques have broken through the diffraction-limited resolution, which had hindered optical microscopy. Among various techniques, stimulated emission depletion (STED) microscopy quasi-instantaneously reduces the size of the effective focal spot by suppressing the peripheral fluorescence of the excited spot with an additional depletion laser, while also providing optical sectioning. With these advantages, the usage of STED microscopy is increasing in the various field of research. Nevertheless, STED microscopy has been continuously improved to answer more biological questions. This review summarises the recent advancements and new techniques implemented for STED microscopy, including microscopy architectures, multicolour ability, deep-tissue imaging, aberration correction, three-dimensional super-resolution, fast measurement, photostability, and multimodality. It is expected that STED microscopy will further evolve and become an more widely useful tool for life sciences.
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Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultrastructure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides subdiffraction resolution by physically enlarging the sample before microscopy. The expansion of the fixed cells by cross-linking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complementary approaches, we combined ExM with STED (ExSTED) and demonstrated an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straightforward, we found that high-fidelity labeling via multi-epitopes is required to obtain emitter densities that allow ultrastructural details with ExSTED to be resolved. Our work provides a robust template for super-resolution microscopy of entire cells in the ten nanometer range.
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