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We present a bimodal endocytic tracer, fluorescent BSA-gold (fBSA-Au), as a fiducial marker for 2D and 3D correlative light and electron microscopy (CLEM) applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent BSA. The conjugate is efficiently endocytosed and distributed throughout the 3D endolysosomal network of cells and has an excellent visibility in both fluorescence microscopy (FM) and electron microscopy (EM). We demonstrate that fBSA-Au facilitates rapid registration in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryoelectron microscopy (cryo-EM). Endocytosed fBSA-Au benefits from a homogeneous 3D distribution throughout the endosomal system within the cell, does not obscure any cellular ultrastructure, and enables accurate (50-150 nm) correlation of fluorescence to EM data. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo)CLEM applications., Competing Interests: The endocytic fBSA-Au5 and fBSA-Au10 fiducials reported here are available in the product listing of Cell Microscopy Core, UMC Utrecht., (© 2022 The Author(s).)

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Therefore, directly linking molecular to nanoscale ultrastructural information is crucial in understanding cellular physiology. Volume or three-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. However, the application of volume-CLEM is hampered by limitations in throughput and 3D correlation efficiency. In order to address these limitations, we describe a novel pipeline for volume-CLEM that provides high-precision (<100 nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) datasets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated into a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume EM and obviates the need for post-correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between the endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Loginov, Fermie, Fokkema, Agronskaia, De Heus, Blab, Klumperman, Gerritsen and Liv.)

Multiple samples are required to monitor and optimize the quality and reliability of quantitative measurements of stimulated emission depletion (STED) and confocal microscopes. Here, we present a single sample to calibrate these microscopes, align their laser beams and measure their point spread function (PSF) in 3D. The sample is composed of a refractive index matched colloidal crystal of silica beads with fluorescent and gold cores. The microscopes can be calibrated in three dimensions using the periodicity of the crystal; the alignment of the laser beams can be checked using the reflection of the gold cores; and the PSF can be measured at multiple positions and depths using the fluorescent cores. It is demonstrated how this sample can be used to visualize and improve the quality of STED and confocal microscopy images. The sample is adjustable to meet the requirements of different NA objectives and microscopy techniques and additionally can be used to evaluate refractive index mismatches as a function of depth quantitatively., (© 2021. The Author(s).)

Colloidal copper(I) sulfide (Cu 2- x S) nanocrystals (NCs) have attracted much attention for a wide range of applications because of their unique optoelectronic properties, driving scientists to explore the potential of using Cu 2- x S NCs as seeds in the synthesis of heteronanocrystals to achieve new multifunctional materials. Herein, we developed a multistep synthesis strategy toward Cu 2- x S/ZnS heteronanorods. The Janus-type Cu 2- x S/ZnS heteronanorods are obtained by the injection of hexagonal high-chalcocite Cu 2- x S seed NCs in a hot zinc oleate solution in the presence of suitable surfactants, 20 s after the injection of sulfur precursors. The Cu 2- x S seed NCs undergo rapid aggregation and coalescence in the first few seconds after the injection, forming larger NCs that act as the effective seeds for heteronucleation and growth of ZnS. The ZnS heteronucleation occurs on a single (100) facet of the Cu 2- x S seed NCs and is followed by fast anisotropic growth along a direction that is perpendicular to the c -axis, thus leading to Cu 2- x S/ZnS Janus-type heteronanorods with a sharp heterointerface. Interestingly, the high-chalcocite crystal structure of the injected Cu 2- x S seed NCs is preserved in the Cu 2- x S segments of the heteronanorods because of the high-thermodynamic stability of this Cu 2- x S phase. The Cu 2- x S/ZnS heteronanorods are subsequently converted into single-component Cu 2- x S and CuInS 2 nanorods by postsynthetic topotactic cation exchange. This work expands the possibilities for the rational synthesis of colloidal multicomponent heteronanorods by allowing the design principles of postsynthetic heteroepitaxial seeded growth and nanoscale cation exchange to be combined, yielding access to a plethora of multicomponent heteronanorods with diameters in the quantum confinement regime., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)

The application of both fluorescence and electron microscopy results in a powerful combination of imaging modalities called "correlative light and electron microscopy" (CLEM). Whereas conventional transmission electron microscopy (TEM) tomography is only able to image sections up to a thickness of ~300nm, scanning transmission electron microscopy (STEM) tomography at 200kV allows the analysis of sections up to a thickness of 900nm in three dimensions. In the current study we have successfully integrated STEM tomography into CLEM as demonstrated for human retinal pigment epithelial 1 (RPE1) cells expressing various fluorescent fusion proteins which were high-pressure frozen and then embedded in Lowicryl HM20. Fluorescently labeled gold nanoparticles were applied onto resin sections and imaged by fluorescence and electron microscopy. STEM tomograms were recorded at regions of interest, and overlays were generated using the eC-CLEM software package. Through the nuclear staining of living cells, the use of fluorescently labeled gold fiducials for the generation of overlays, and the integration of STEM tomography we have markedly extended the application of the Kukulski protocol (Kukulski et al., 2011, 2012). Various fluorescently tagged proteins localizing to different cellular organelles could be assigned to their ultrastructural compartments. By combining STEM tomography with on-section CLEM, fluorescently tagged proteins can be localized in three-dimensional ultrastructural environments with a volume of at least 2.7×2.7×0.5μm., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In correlative light and electron microscopy (CLEM), the capabilities of fluorescence microscopy (FM) and electron microscopy (EM) are united. FM combines a large field of view with high sensitivity for detecting fluorescence, which makes it an excellent tool for identifying regions of interest. EM has a much smaller field of view but offers superb resolution that allows studying cellular ultrastructure. In CLEM, the potentials of both techniques are combined but a limiting factor is the large difference in resolution between the two imaging modalities. Adding super resolution FM to CLEM reduces the resolution gap between FM and EM; it offers the possibility of identifying multiple targets within the diffraction limit and can increase correlation accuracy. CLEM is usually carried out in two separate setups, which requires transfer of the sample. This may result in distortion and damage of the specimen, which can complicate finding back regions of interest. By integrating the two imaging modalities, such problems can be avoided. Here, an integrated super resolution correlative microscopy approach is presented based on a wide-field super resolution FM integrated in a Transmission Electron Microscope (TEM). Switching imaging modalities is accomplished by rotation of the TEM sample holder. First imaging experiments are presented on sections of Lowicryl embedded Human Umbilical Vein Endothelial Cells labeled for Caveolin both with Protein A-Gold, and Alexa Fluor®647. TEM and FM images were overlaid using fiducial markers visible in both imaging modalities with an overlay accuracy of 28 ± 11 nm. This is close to the optical resolution of ~50 nm., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Fiducial markers are used in correlated light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. Currently used fiducial markers, e.g. dye-labelled nanoparticles and quantum dots, suffer from irreversible quenching of the luminescence after electron beam exposure. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can (partially) withstand electron bombardment, are interesting because of the recent development of integrated CLEM microscopes. In addition, nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow switching back from EM to LM and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; 130 nm gold-core rhodamine B-labelled silica particles, 15 nm CdSe/CdS/ZnS core-shell-shell quantum dots (QDs) and 230 nm Y 2 O 3 :Eu 3+ particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The gold-core rhodamine B-labelled silica NPs and QDs are quenched after a single exposure to 60 ke -  nm -2 with an energy of 120 keV, while Y 2 O 3 :Eu 3+ NPs are robust and still show luminescence after five doses of 60 ke - nm -2 . In addition, the luminescence intensity of Y 2 O 3 :Eu 3+ NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that Y 2 O 3 :Eu 3+ NPs are promising as robust fiducial marker in CLEM. LAY DESCRIPTION: Luminescent particles are used as fiducial markers in correlative light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. The currently used fiducial markers, e.g. dyes and quantum dots, loose their luminescence after exposure to the electron beam of the electron microscope. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can withstand electron exposure, are interesting because of recent developments in integrated CLEM microscopes. Also nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow for switching back to fluorescence imaging after the recording of electron microscopy imaging and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; dye-labelled silica particles, quantum dots and lanthanide-doped inorganic particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The dye-labelled silica NPs and QDs are quenched after a single exposure to 60 ke - nm -2 with an energy of 120 keV, while lanthanide-doped inorganic NPs are robust and still show luminescence after five doses of 60 ke - nm -2 . In addition, the luminescence intensity of lanthanide-doped inorganic NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that lanthanide-doped NPs are promising as robust fiducial marker in CLEM., (© 2019 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.)

Insight in the structure of nanoparticle assemblies up to a single particle level is key to understand the collective properties of these assemblies, which critically depend on the individual particle positions and orientations. However, the characterization of large, micron sized assemblies containing small, 10-500 nanometer, sized colloids is highly challenging and cannot easily be done with the conventional light, electron or X-ray microscopy techniques. Here, we demonstrate that focused ion beam-scanning electron microscopy (FIB-SEM) tomography in combination with image processing enables quantitative real-space studies of ordered and disordered particle assemblies too large for conventional transmission electron tomography, containing particles too small for confocal microscopy. First, we demonstrate the high resolution structural analysis of spherical nanoparticle assemblies, containing small anisotropic gold nanoparticles. Herein, FIB-SEM tomography allows the characterization of assembly dimensions which are inaccessible to conventional transmission electron microscopy. Next, we show that FIB-SEM tomography is capable of characterizing much larger ordered and disordered assemblies containing silica colloids with a diameter close to the resolution limit of confocal microscopes. We determined both the position and the orientation of each individual (nano)particle in the assemblies by using recently developed particle tracking routines. Such high precision structural information is essential in the understanding and design of the collective properties of new nanoparticle based materials and processes.

Fluorescence microscopy (FM) and electron microscopy (EM) are complementary techniques. FM affords examination of large fields of view and identifying regions of interest but has a low resolution. EM exhibits excellent resolution over a limited field of view. The combination of these two techniques, correlative microscopy, received considerable interest in the past years and has proven its potential in biology and material science. Accurate correlation of FM and EM images is, however, challenging due to the differences in contrast mechanism, size of field of view and resolution. We report an accurate, fast and robust method to correlate FM and EM images using low densities of fiducial markers. Here, 120 nm diameter fiducial markers consisting of fluorescently labelled silica coated gold nanoparticles are used. The method relies on recording FM, low magnification EM and high magnification EM images. Two linear transformation matrices are constructed, FM to low magnification EM and low magnification EM to high magnification EM. Combination of these matrices results in a high accuracy transformation of FM to high magnification EM coordinates. The method was tested using two different transmission electron microscopes and different Tokuyasu and Lowicryl sections. The overall accuracy of the correlation method is high, 5-30 nm.

In this work, gold nanoparticles coated with a fluorescently labelled (rhodamine B) silica shell are presented as fiducial markers for correlative light and electron microscopy (CLEM). The synthesis of the particles is optimized to obtain homogeneous, spherical core-shell particles of arbitrary size. Next, particles labelled with different fluorophore densities are characterized to determine under which conditions bright and (photo)stable particles can be obtained. 2 and 3D CLEM examples are presented where optimized particles are used for correlation. In the 2D example, fiducials are added to a cryosection of cells whereas in the 3D example cells are imaged after endocytosis of the fiducials. Both examples demonstrate that the particles are clearly visible in both modalities and can be used for correlation. Additionally, the recognizable core-shell structure of the fiducials proves to be very powerful in electron microscopy: it makes it possible to irrefutably identify the particles and makes it easy to accurately determine the center of the fiducials.

The knowledge of the quantum dot (QD) concentration in a colloidal suspension and the quantitative understanding of the size-dependence of the band gap of QDs are of crucial importance from both applied and fundamental viewpoints. In this work, we investigate the size-dependence of the optical properties of nearly spherical wurtzite (wz) CuInS 2 (CIS) QDs in the 2.7 to 6.1 nm diameter range (polydispersity ≤10%). The QDs are synthesized by partial Cu + for In 3+ cation exchange in template Cu 2- x S nanocrystals, which yields CIS QDs with very small composition variations (In/Cu = 0.91 ± 0.11), regardless of their sizes. These well-defined QDs are used to investigate the size-dependence of the band gap of wz CIS QDs. A sizing curve is also constructed for chalcopyrite CIS QDs by collecting and reanalyzing literature data. We observe that both sizing curves follow primarily a 1/ d dependence. Moreover, the molar absorption coefficients and the absorption cross-section per CIS formula unit, both at 3.1 eV and at the band gap, are analyzed. The results demonstrate that the molar absorption coefficients of CIS QDs follow a power law at the first exciton transition energy (ε E 1 = 5208 d 2.45 ) and scale with the QD volume at 3.1 eV. This latter observation implies that the absorption cross-section per unit cell at 3.1 eV is size-independent and therefore can be estimated from bulk optical constants. These results also demonstrate that the molar absorption coefficients at 3.1 eV are more reliable for analytical purposes, since they are less sensitive to size and shape dispersion.

Synthesis protocols for anisotropic CuInX 2 (X = S, Se, Te)-based heteronanocrystals (HNCs) are scarce due to the difficulty in balancing the reactivities of multiple precursors and the high solid-state diffusion rates of the cations involved in the CuInX 2 lattice. In this work, we report a multistep seeded growth synthesis protocol that yields colloidal wurtzite CuInS 2 /ZnS dot core/rod shell HNCs with photoluminescence in the NIR (∼800 nm). The wurtzite CuInS 2 NCs used as seeds are obtained by topotactic partial Cu + for In 3+ cation exchange in template Cu 2- x S NCs. The seed NCs are injected in a hot solution of zinc oleate and hexadecylamine in octadecene, 20 s after the injection of sulfur in octadecene. This results in heteroepitaxial growth of wurtzite ZnS primarily on the Sulfur-terminated polar facet of the CuInS 2 seed NCs, the other facets being overcoated only by a thin (∼1 monolayer) shell. The fast (∼21 nm/min) asymmetric axial growth of the nanorod proceeds by addition of [ZnS] monomer units, so that the polarity of the terminal (002) facet is preserved throughout the growth. The delayed injection of the CuInS 2 seed NCs is crucial to allow the concentration of [ZnS] monomers to build up, thereby maximizing the anisotropic heteroepitaxial growth rates while minimizing the rates of competing processes (etching, cation exchange, alloying). Nevertheless, a mild etching still occurred, likely prior to the onset of heteroepitaxial overgrowth, shrinking the core size from 5.5 to ∼4 nm. The insights provided by this work open up new possibilities in designing multifunctional Cu-chalcogenide based colloidal heteronanocrystals.

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The localized inner 4f shell transitions of lanthanide ions are largely independent of the local surroundings. The luminescence properties of Ln 3+ ions doped into nanocrystals (NCs) are therefore similar to those in bulk crystals. Quantum size effects, responsible for the unique size-dependent luminescence of semiconductor NCs, are generally assumed not to influence the optical properties of Ln 3+ -doped insulator NCs. However, phonon confinement effects have been reported to hamper relaxation between closely spaced Stark levels in Ln 3+ -doped NCs. At cryogenic temperatures emission and excitation from higher Stark levels was observed for Ln 3+ ions in NCs only and were explained by a cutoff in the acoustic phonon spectrum. Relaxation would be inhibited as no resonant low energy (long wavelength) acoustic phonon modes can exist in nanometer sized crystals, and this prevents relaxation by direct phonon emission between closely spaced Stark levels. This phenomenon is known as a phonon bottleneck. Here, we investigate the role of phonon confinement in Ln-doped NCs. High resolution emission spectra at temperatures down to 2.2 K are reported for various Ln 3+ ions (Er 3+ , Yb 3+ , Eu 3+ ) doped into monodisperse 10 nm NaYF 4 NCs and compared with spectra for bulk (microcrystalline) material. Contrary to previous reports, we find no evidence for phonon bottleneck effects in the emission spectra. Emission from closely spaced higher Stark levels is observed only at high excitation powers and is explained by laser heating. The present results indicate that previously reported effects in NCs may not be caused by phonon confinement., Competing Interests: The authors declare no competing financial interest.

Establishing structure-activity relationships in complex, hierarchically structured nanomaterials, such as fluid catalytic cracking (FCC) catalysts, requires characterization with complementary, correlated analysis techniques. An integrated setup has been developed to perform transmission electron microscopy (TEM) and single-molecule fluorescence (SMF) microscopy on such nanostructured samples. Correlated structure-reactivity information was obtained for 100 nm thin, microtomed sections of a single FCC catalyst particle using this novel SMF-TEM high-resolution combination. High reactivity in a thiophene oligomerization probe reaction correlated well with TEM-derived zeolite locations, while matrix components, such as clay and amorphous binder material, were found not to display activity. Differences in fluorescence intensity were also observed within and between distinct zeolite aggregate domains, indicating that not all zeolite domains are equally active., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Lanthanide-doped nanocrystals (NCs) differ from their bulk counterparts due to their large surface to volume ratio. It is generally assumed that the optical properties are not affected by size effects as electronic transitions occur within the well-shielded 4f shell of the lanthanide dopant ions. However, defects and disorder in the surface layer can affect the luminescence properties. Trivalent europium is a suitable ion to investigate the subtle influence of the surface, because of its characteristic luminescence and high sensitivity to the local environment. Here, we investigate the influence of disorder in NCs on the optical properties of lanthanide dopants by studying the inhomogeneous linewidth, emission intensity ratios, and luminescence decay curves for LaPO 4 :Eu 3+ samples of different sizes (4 nm to bulk) and core-shell configurations (core, core-isocrystalline shell, and core-silica shell). We show that the emission linewidths increase strongly for NCs. The ratio of the intensities of the forced electric dipole (ED) and magnetic dipole (MD) transitions, a measure for the local symmetry distortion around Eu 3+ ions, is higher for samples with a large fraction of Eu 3+ ions close to the surface. Finally, we present luminescence decay curves revealing an increased nonradiative decay rate for Eu 3+ in NCs. The effects are strongest in core and core-silica shell NCs and can be reduced by growth of an isocrystalline LaPO 4 shell. The present systematic study provides quantitative insight into the role of surface disorder on the optical properties of lanthanide-doped NCs. These insights are important in emerging applications of lanthanide-doped nanocrystals.

Copper indium sulfide (CIS) quantum dots (QDs) are attractive as labels for biomedical imaging, since they have large absorption coefficients across a broad spectral range, size- and composition-tunable photoluminescence from the visible to the near-infrared, and low toxicity. However, the application of NIR-emitting CIS QDs is still hindered by large size and shape dispersions and low photoluminescence quantum yields (PLQYs). In this work, we develop an efficient pathway to synthesize highly luminescent NIR-emitting wurtzite CIS/ZnS QDs, starting from template Cu 2- x S nanocrystals (NCs), which are converted by topotactic partial Cu + for In 3+ exchange into CIS NCs. These NCs are subsequently used as cores for the overgrowth of ZnS shells (≤1 nm thick). The CIS/ZnS core/shell QDs exhibit PL tunability from the first to the second NIR window (750-1100 nm), with PLQYs ranging from 75% (at 820 nm) to 25% (at 1050 nm), and can be readily transferred to water upon exchange of the native ligands for mercaptoundecanoic acid. The resulting water-dispersible CIS/ZnS QDs possess good colloidal stability over at least 6 months and PLQYs ranging from 39% (at 820 nm) to 6% (at 1050 nm). These PLQYs are superior to those of commonly available water-soluble NIR-fluorophores (dyes and QDs), making the hydrophilic CIS/ZnS QDs developed in this work promising candidates for further application as NIR emitters in bioimaging. The hydrophobic CIS/ZnS QDs obtained immediately after the ZnS shelling are also attractive as fluorophores in luminescent solar concentrators.

Previous studies have established that 7,12-dimethylbenz(a)anthracene (DMBA) can initiate skin tumourigenesis in conventional furred mouse models by acting on hair follicle stem cells. However, further cancer progression depends on repeated applications of tumour promoter agents. This study evaluated the timeline involved in skin tumourigenesis and progression in immunocompetent hairless SKH1-hr mice with dysfunctional hair follicles using only DMBA with no additional tumour promoter agents. The results showed that topical application of 30 µg (117 nmol) of DMBA over the back and flank regions of the mouse once a week and 15 µg (58.5 nmol) twice a week produced skin tumours after 7-8 weeks. However, by week 14 a heavy benign tumour load required the mice to be euthanized. Lowering the DMBA dose to 15 µg (58.5 nmol) once a week produced tumours more slowly and allowed the mice to be studied for a longer period to week 23. This low-dose DMBA regimen yielded a high percentage of malignant tumours (58.8%) after 23 weekly applications. Additionally DMBA-treated skin showed an increase in mean epidermal thickness in comparison to untreated and acetone-treated skin. Despite the aberrant hair follicles in SKH1-hr mice, this chemically driven skin cancer model in hairless mice can serve as a suitable alternative to the ultraviolet-induced skin cancer models and can be reliably replicated as demonstrated by both the pilot and main experiments.

Lanthanide ions are promising for the labeling of silica nanoparticles with a specific luminescent fingerprint due to their sharp line emission at characteristic wavelengths. With the increasing use of silica nanoparticles in consumer products, it is important to label silica nanoparticles in order to trace the biodistribution, both in the environment and living organisms.In this work, we synthesized LaPO4 nanocrystals (NCs) with sizes ranging from 4 to 8 nm doped with europium or cerium and terbium. After silica growth using an inverse micelle method, monodisperse silica spheres were obtained with a single LaPO4 NC in the center. We demonstrate that the size of the silica spheres can be tuned in the 25-55 nm range by addition of small volumes of methanol during the silica growth reaction. Both the LaPO4 core and silica nanocrystal showed sharp line emission characteristic for europium and terbium providing unique optical labels in silica nanoparticles of variable sizes.

Early detection of cutaneous squamous cell carcinoma (cSCC) can enable timely therapeutic and preventive interventions for patients. In this study, in vivo nonlinear optical imaging (NLOI) based on two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG), was used to non-invasively detect microscopic changes occurring in murine skin treated topically with 7,12-dimethylbenz(a)anthracene (DMBA). The optical microscopic findings and the measured TPEF-SHG index show that NLOI was able to clearly detect early cytostructural changes in DMBA treated skin that appeared clinically normal. This suggests that in vivo NLOI could be a non-invasive tool to monitor early signs of cSCC. In vivo axial NLOI scans of normal murine skin (upper left), murine skin with preclinical hyperplasia (upper right), early clinical murine skin lesion (lower left) and late or advanced murine skin lesion (lower right)., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nonlinear optical imaging (NLOI) has emerged to be a promising tool for bio-medical imaging in recent times. Among the various applications of NLOI, its utility is the most significant in the field of pre-clinical and clinical cancer research. This review begins by briefly covering the core principles involved in NLOI, such as two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG). Subsequently, there is a short description on the various cellular components that contribute to endogenous optical fluorescence. Later on the review deals with its main theme--the challenges faced during label-free NLO imaging in translational cancer research. While this review addresses the accomplishment of various label-free NLOI based studies in cancer diagnostics, it also touches upon the limitations of the mentioned studies. In addition, areas in cancer research that need to be further investigated by label-free NLOI are discussed in a latter segment. The review eventually concludes on the note that label-free NLOI has and will continue to contribute richly in translational cancer research, to eventually provide a very reliable, yet minimally invasive cancer diagnostic tool for the patient., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Timely detection of cutaneous squamous cell carcinoma with non-invasive modalities like nonlinear spectral imaging (NLSI) can ensure efficient preventive or therapeutic measures for patients. In this study, in vivo NLSI was used to study spectral characteristics in murine skin treated with 7, 12-dimethylbenz(a)anthracene. The results show that NLSI could detect emission spectral changes during the early preclinical stages of skin carcinogenesis. Analyzing these emission spectra using simulated band-pass filters at 450-460 nm and 525-535 nm, gave parameters that were expressed as a ratio. This ratio was increased and thus suggestive of elevated metabolic activity in early stages of skin carcinogenesis.

Skin contains many autofluorescent components that can be studied using spectral imaging. We employed a spectral phasor method to analyse two photon excited autofluorescence and second harmonic generation images of in vivo human skin. This method allows segmentation of images based on spectral features. Various structures in the skin could be distinguished, including Stratum Corneum, epidermal cells and dermis. The spectral phasor analysis allowed investigation of their fluorescence composition and identification of signals from NADH, keratin, FAD, melanin, collagen and elastin. Interestingly, two populations of epidermal cells could be distinguished with different melanin content., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

High power femto-second (fs) laser pulses used for in-vivo nonlinear optical (NLO) imaging can form cyclobutane pyrimidine dimers (CPD) in DNA, which may lead to carcinogenesis via subsequent mutations. Since UV radiation from routine sun exposure is the primary source of CPD lesions, we evaluated the risk of CPD-related squamous cell carcinoma (SCC) in human skin due to NLO imaging relative to that from sun exposure. We developed a unique cancer risk model expanding previously published estimation of risk from exposure to continuous wave (CW) laser. This new model showed that the increase in CPD-related SCC in skin from NLO imaging is negligible above that due to regular sun exposure., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Optical aberrations have detrimental effects in multiphoton microscopy. These effects can be curtailed by implementing model-based wavefront sensorless adaptive optics, which only requires the addition of a wavefront shaping device, such as a deformable mirror (DM) to an existing microscope. The aberration correction is achieved by maximizing a suitable image quality metric. We implement a model-based aberration correction algorithm in a second-harmonic microscope. The tip, tilt, and defocus aberrations are removed from the basis functions used for the control of the DM, as these aberrations induce distortions in the acquired images. We compute the parameters of a quadratic polynomial that is used to model the image quality metric directly from experimental input-output measurements. Finally, we apply the aberration correction by maximizing the image quality metric using the least-squares estimate of the unknown aberration.

The combined analysis of spectral and lifetime images has the potential to provide more accurate and more detailed information about Förster resonance energy transfer (FRET). We have developed a novel FRET analysis method to analyze images recorded by multispectral lifetime imaging. The new method is based on a phasor approach and facilitates the simultaneous analysis of decay kinetics of donor and acceptor molecules. The method is applicable to both molecules that exhibit a mono-exponential decay and a bi-exponential decay. As an example we show the possibility of extracting the energy transfer efficiency and the fraction of interacting molecules even in the presence of non-interacting molecules. The reliability of the method is investigated by comparing it with conventional FRET-FLIM analyses. We show that, with the same number of detected photons, the spectrally resolved phasor approach provides higher accuracy than other analysis methods; the confidence interval is improved and the FRET efficiency is closer to the real value.

Deep imaging in turbid media such as biological tissue is challenging due to scattering and optical aberrations. Adaptive optics has the potential to compensate the tissue aberrations. We present a wavefront sensing scheme for multi-photon scanning microscopes using the pulsed, near-infrared light reflected back from the sample utilising coherence gating and a confocal pinhole to isolate the light from a layer of interest. By interfering the back-reflected light with a tilted reference beam, we create a fringe pattern with a known spatial carrier frequency in an image of the back-aperture plane of the microscope objective. The wavefront aberrations distort this fringe pattern and thereby imprint themselves at the carrier frequency, which allows us to separate the aberrations in the Fourier domain from low spatial frequency noise. A Fourier analysis of the modulated fringes combined with a virtual Shack-Hartmann sensor for smoothing yields a modal representation of the wavefront suitable for correction. We show results with this method correcting both DM-induced and sample-induced aberrations in rat tail collagen fibres as well as a Hoechst-stained MCF-7 spheroid of cancer cells.

Low-density lipoprotein (LDL) plays a critical role in cholesterol transport and is closely linked to the progression of several diseases. This motivates the development of methods to study LDL behavior from the microscopic to whole-body level. We have developed an approach to efficiently load LDL with a range of diagnostically active nanocrystals or hydrophobic agents. We performed focused experiments on LDL labeled with gold nanocrystals (Au-LDL). The labeling procedure had minimal effect on LDL size, morphology, or composition. Biological function was found to be maintained from both in vitro and in vivo experiments. Tumor-bearing mice were injected intravenously with LDL, DiR-LDL, Au-LDL, or a gold-loaded nanoemulsion. LDL accumulation in the tumors was detected with whole-body imaging methods, such as computed tomography (CT), spectral CT, and fluorescence imaging. Cellular localization was studied with transmission electron microscopy and fluorescence techniques. This LDL labeling procedure should permit the study of lipoprotein biointeractions in unprecedented detail.

EGFR signaling is attenuated by endocytosis and degradation of receptor-ligand complexes in lysosomes. Endocytosis of EGFR is known to be regulated by multiple post-translational modifications. The observation that prevention of these modifications does not block endocytosis completely, suggests the involvement of other mechanism(s). Recently, receptor clustering has been suggested to induce internalization of multiple types of membrane receptors. However, the mechanism of clustering-induced internalization remains unknown. We have used biparatopic antibody fragments from llama (VHHs) to induce EGFR clustering without stimulating tyrosine kinase activity. Using this approach, we have found an essential role for the N-terminal GG4-like dimerization motif in the transmembrane domain (TMD) for clustering-induced internalization. Moreover, conventional EGF-induced receptor internalization depends exclusively on this TMD dimerization and kinase activity. Mutations in this dimerization motif eventually lead to reduced EGFR degradation and sustained signaling. We propose a novel role for the TMD dimerization motif in the negative-feedback control of EGFR. The widely conserved nature of GG4-like dimerization motifs in transmembrane proteins suggests a general role for these motifs in clustering-induced internalization.

Initial platelet arrest at the exposed arterial vessel wall is mediated through glycoprotein Ibα binding to the A1 domain of von Willebrand factor. This interaction occurs at sites of elevated shear force, and strengthens upon increasing hydrodynamic drag. The increased interaction requires shear-dependent exposure of the von Willebrand factor A1 domain, but the contribution of glycoprotein Ibα remains ill defined. We have previously found that glycoprotein Ibα forms clusters upon platelet cooling and hypothesized that such a property enhances the interaction with von Willebrand factor under physiological conditions. We analyzed the distribution of glycoprotein Ibα with Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy. Perfusion at a shear rate of 1,600 s(-1) induced glycoprotein Ibα clusters on platelets adhered to von Willebrand factor, while clustering did not require von Willebrand factor contact at 10,000 s(-1). Shear-induced clustering was reversible, not accompanied by granule release or αIIbβ3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor. Clustering required glycoprotein Ibα translocation to lipid rafts and critically depended on arachidonic acid-mediated binding of 14-3-3ζ to its cytoplasmic tail. This newly identified mechanism emphasizes the ability of platelets to respond to mechanical force and provides new insights into how changes in hemodynamics influence arterial thrombus formation.

Label-free nonlinear spectral imaging microscopy (NLSM) records two-photon-excited fluorescence emission spectra of endogenous fluorophores within the specimen. Here, NLSM is introduced as a novel, minimally invasive method to analyze the metabolic state of fungal hyphae by monitoring the autofluorescence of NAD(P)H and flavin adenine dinucleotide (FAD). Moreover, the presence of melanin was analyzed by NLSM. NAD(P)H, FAD, and melanin were used as biomarkers for freshness of mushrooms of Agaricus bisporus (white button mushroom) that had been stored at 4°C for 0 to 17 days. During this period, the mushrooms did not show changes in morphology or color detectable by eye. In contrast, FAD/NAD(P)H and melanin/NAD(P)H ratios increased over time. For instance, these ratios increased from 0.92 to 2.02 and from 0.76 to 1.53, respectively, at the surface of mushroom caps that had been harvested by cutting the stem. These ratios were lower under the skin than at the surface of fresh mushrooms (0.78 versus 0.92 and 0.41 versus 0.76, respectively), indicative of higher metabolism and lower pigment formation within the fruiting body. Signals were different not only between tissues of the mushroom but also between neighboring hyphae. These data show that NLSM can be used to determine the freshness of mushrooms and to monitor the postharvest browning process at an early stage. Moreover, these data demonstrate the potential of NLSM to address a broad range of fundamental and applied microbiological processes.

Dynamical control of the luminescence of quantum dots is highly important for technology in the field of telecommunication, displays, and photovoltaics. In this work we use an a-Si:H solar cell structure in which CdSe quantum dots are sandwiched. By applying a positive potential over the device, charge carriers generated in the quantum dots are transported to the a-Si:H layer and transformed into electrical energy, changing the luminescence intensity with a switching time lower than 60 ms. This is a promising new step towards using quantum dots in optical switching devices.

A method, is presented for blind unmixing spectrally resolved fluorescence lifetime images. The method is based on the combined analysis of spectral and lifetime phasors and allows unmixing of up to three components without any prior knowledge. Fractional intensities, spectra and decay curves of the individual components can be extracted with this new technique. The reliability and sensitivity are investigated and the possibility of extending the method to unmix more components is discussed. The method is evaluated on mixtures of fluorescent dyes and labeled cells.

We report a spectrally resolved fluorescence lifetime imaging system based on time gated single photon detection with a fixed gate width of 200 ps and 7 spectral channels. Time gated systems can operate at high count rates but usually have large gate widths and sample only part of the fluorescence decay curve. In the system presented in this work, the fluorescence signal is sampled using a high speed transceiver. An error analysis is carried out to characterize the performance of both lifetime and spectral detection. The effect of gate width and spectral channel width on the accuracy of estimated lifetimes and spectral widths is described. The performance of the whole instrument is evaluated at count rates of up to 12 MHz. Accurate fluorescence lifetimes (error < 2%) are recorded at count rates as high as 5 MHz. This is limited by the PMT performance, not by the electronics. Analysis of the large spectral lifetime image sets is challenging and time-consuming. Here, we demonstrate the use of lifetime and spectral phasors for analyzing images of fibroblast cells with 2 different labeled components. The phasor approach provides a fast and intuitive way of analyzing the results of spectrally resolved fluorescence lifetime imaging experiments.

While cycling through a fluid catalytic cracking (FCC) unit, the structure and performance of FCC catalyst particles are severely affected. In this study, we set out to characterize the damage to commercial equilibrium catalyst particles, further denoted as ECat samples, and map the different pathways involved in their deactivation in a practical unit. The degradation was studied on a structural and a functional level. Transmission electron microscopy (TEM) of ECat samples revealed several structural features; including zeolite crystals that were partly or fully severed, mesoporous, macroporous, and/or amorphous. These defects were then correlated to structural features observed in FCC particles that were treated with different levels of hydrothermal deactivation. This allowed us not only to identify which features observed in ECat samples were a result of hydrothermal deactivation, but also to determine the severity of treatments resulting in these defects. For functional characterization of the ECat sample, the Brønsted acidity within individual FCC particles was studied by a selective fluorescent probe reaction with 4-fluorostyrene. Integrated laser and electron microscopy (iLEM) allowed correlating this Brønsted acidity to structural features by combining a fluorescence and a transmission electron microscope in a single set-up. Together, these analyses allowed us to postulate a plausible model for the degradation of zeolite crystals in FCC particles in the ECat sample. Furthermore, the distribution of the various deactivation processes within particles of different ages was studied. A rim of completely deactivated zeolites surrounding each particle in the ECat sample was identified by using iLEM. These zeolites, which were never observed in fresh or steam-deactivated samples, contained clots of dense structures. The structures are proposed to be carbonaceous deposits formed during the cracking process, and seem resistant towards burning off during catalyst regeneration., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

One of the major challenges for correlative microscopy is the preparation of the sample; the protocols for transmission electron microscopy (TEM) and fluorescence microscopy (FM) often prove to be incompatible. Here, we introduce 2+Staining: an improved contrasting procedure for Tokuyasu sections that yields both excellent positive membrane contrast in the TEM and bright fluorescence of the probe labeled on the section. 2+Staining involves the contrasting of the immunolabeled sections with 1% osmium tetroxide, 2% uranyl acetate and lead citrate in sequential steps, followed by embedding in 1.8% methyl cellulose. In addition, we demonstrate an amplification of the fluorescent signal by introducing additional antibody incubation steps to the immunolabeling procedure. The methods were validated using the integrated laser and electron microscope (iLEM), a novel tool for correlative microscopy combining FM and TEM in a single setup. The approaches were tested on HL-60 cells labeled for lysosomal-associated membrane protein 2 (LAMP-2) and on sections of muscle from a facioscapulohumeral dystrophy mouse model. Yielding excellent results and greatly expediting the workflow, the methods are of great value for those working in the field of correlative microscopy and indispensible for future users of integrated correlative microscopy.

In neurons, the distinct molecular composition of axons and dendrites is established through polarized targeting mechanisms, but it is currently unclear how nonpolarized cargoes, such as mitochondria, become uniformly distributed over these specialized neuronal compartments. Here, we show that TRAK family adaptor proteins, TRAK1 and TRAK2, which link mitochondria to microtubule-based motors, are required for axonal and dendritic mitochondrial motility and utilize different transport machineries to steer mitochondria into axons and dendrites. TRAK1 binds to both kinesin-1 and dynein/dynactin, is prominently localized in axons, and is needed for normal axon outgrowth, whereas TRAK2 predominantly interacts with dynein/dynactin, is more abundantly present in dendrites, and is required for dendritic development. These functional differences follow from their distinct conformations: TRAK2 preferentially adopts a head-to-tail interaction, which interferes with kinesin-1 binding and axonal transport. Our study demonstrates how the molecular interplay between bidirectional adaptor proteins and distinct microtubule-based motors drives polarized mitochondrial transport., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Growth factor receptors are present in the plasma membrane of resting cells as monomers or (pre)dimers. Ligand binding results in higher-order oligomerization of ligand-receptor complexes. To study the regulation of receptor clustering, several experimental techniques have been developed in the last decades. However, many involve invasive approaches that are likely to disturb the integrity of the membrane, thereby affecting receptor interactions. In this chapter, we describe the use of a noninvasive approach to study receptor dimerization and oligomerization. This method is based upon the Förster energy transfer between identical adjacent fluorescent proteins (homo-FRET) and is determined by analyzing the change in fluorescence anisotropy. Homo-FRET takes place within a distance of 10nm, making this an excellent approach for studying receptor-receptor interactions in intact cells. After excitation of monomeric GFP (mGFP) with polarized light, limiting anisotropy values (r(inf)) of the emitted light are determined, where proteins with known cluster sizes are used as references. Dimerization and oligomerization of the epidermal growth factor receptor (EGFR) in response to ligand binding is determined by using receptors that have been fused with mGFP at their C-terminus. In this chapter, we describe the involved technology and discuss the feasibility of homo-FRET experiments for the determination of cluster sizes of growth factor receptors like EGFR., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Storing platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor., Design and Methods: We examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy., Results: Cold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-D-glucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions., Conclusions: We conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches., (Copyright © 2012 Elsevier Inc. All rights reserved.)