In situ mapping of biomineral skeletal proteins by molecular recognition imaging with antibody-functionalized AFM tips.

Spatial localizing of skeletal proteins in biogenic minerals remains a challenge in biomineralization research. To address this goal, we developed a novel in situ mapping technique based on molecular recognition measurements via atomic force microscopy (AFM), which requires three steps: (1) the development and purification of a polyclonal antibody elicited against the target protein, (2) its covalent coupling to a silicon nitride AFM tip ('functionalization'), and (3) scanning of an appropriately prepared biomineral surface. We applied this approach to a soluble shell protein - accripin11 - recently identified as a major component of the calcitic prisms of the fan mussel Pinna nobilis [1]. Multiple tests reveal that accripin11 is evenly distributed at the surface of the prisms and also present in the organic sheaths surrounding the calcitic prisms, indicating that this protein is both intra- and inter-crystalline. We observed that the adhesion force in transverse sections is about twice higher than in longitudinal sections, suggesting that accripin11 may exhibit preferred orientation in the biomineral. To our knowledge, this is the first time that a protein is localized by molecular recognition atomic force microscopy with antibody-functionalized tips in a biogenic mineral. The 'pros' and 'cons' of this methodology are discussed in comparison with more 'classical' approaches like immunogold. This technique, which leaves the surface to analyze clean, might prove useful for clinical tests on non-pathological (bone, teeth) or pathological (kidney stone) biomineralizations. Studies using implants with protein-doped calcium phosphate coating can also benefit from this technology. STATEMENT OF SIGNIFICANCE: Our paper deals with an unconventional technical approach for localizing proteins that are occluded in biominerals. This technique relies on the use of molecular recognition atomic force microscopy with antibody-functionalized tips. Although such approach has been employed in other system, this is the very first time that it is developed for biominerals. In comparison to more classical approaches (such as immunogold), AFM microscopy with antibody-functionalized tips allows higher magnification and keeps the scanned surface clean for other biophysical characterizations. Our method has a general scope as it can be applied in human health, for non-pathological (bone, teeth) and pathological (kidney stone) biomineralizations as well as for bone implants coated with protein-doped calcium phosphate.

Biochemistry and Nanomechanical Properties of Human Colon Cells upon Simvastatin, Lovastatin, and Mevastatin Supplementations: Raman Imaging and AFM Studies.

One of the most important areas of medical science is oncology, which is responsible for both the diagnostics and treatment of cancer diseases. Over the years, there has been an intensive development of cancer diagnostics and treatment. This paper shows the comparison of normal (CCD-18Co) and cancerous (CaCo-2) cell lines of the human gastrointestinal tract on the basis of nanomechanical and biochemical properties to obtain information on cancer biomarkers useful in oncological diagnostics. The research techniques used were Raman spectroscopy and imaging and atomic force microscopy (AFM). In addition, the studies also included the effect of the statin compounds─mevastatin, lovastatin, and simvastatin─and their influence on biochemical and nanomechanical changes of cell properties using Raman imaging and AFM techniques. The cytotoxicity of statins was determined using XTT tests.

Elastic shell theory for plant cell wall stiffness reveals contributions of cell wall elasticity and turgor pressure in AFM measurement.

The stiffness of a plant cell in response to an applied force is determined not only by the elasticity of the cell wall but also by turgor pressure and cell geometry, which affect the tension of the cell wall. Although stiffness has been investigated using atomic force microscopy (AFM) and Young's modulus of the cell wall has occasionally been estimated using the contact-stress theory (Hertz theory), the existence of tension has made the study of stiffness more complex. Elastic shell theory has been proposed as an alternative method; however, the estimation of elasticity remains ambiguous. Here, we used finite element method simulations to verify the formula of the elastic shell theory for onion (Allium cepa) cells. We applied the formula and simulations to successfully quantify the turgor pressure and elasticity of a cell in the plane direction using the cell curvature and apparent stiffness measured by AFM. We conclude that tension resulting from turgor pressure regulates cell stiffness, which can be modified by a slight adjustment of turgor pressure in the order of 0.1 MPa. This theoretical analysis reveals a path for understanding forces inherent in plant cells.

Atomic Force Microscopy (AFM) Applications in Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disorder characterized by progressive replacement of cardiomyocytes by fibrofatty tissue, ventricular dilatation, cardiac dysfunction, arrhythmias, and sudden cardiac death. Interest in molecular biomechanics for these disorders is constantly growing. Atomic force microscopy (AFM) is a well-established technic to study the mechanobiology of biological samples under physiological and pathological conditions at the cellular scale. However, a review which described all the different data that can be obtained using the AFM (cell elasticity, adhesion behavior, viscoelasticity, beating force, and frequency) is still missing. In this review, we will discuss several techniques that highlight the potential of AFM to be used as a tool for assessing the biomechanics involved in ACM. Indeed, analysis of genetically mutated cells with AFM reveal abnormalities of the cytoskeleton, cell membrane structures, and defects of contractility. The higher the Young's modulus, the stiffer the cell, and it is well known that abnormal tissue stiffness is symptomatic of a range of diseases. The cell beating force and frequency provide information during the depolarization and repolarization phases, complementary to cell electrophysiology (calcium imaging, MEA, patch clamp). In addition, original data is also presented to emphasize the unique potential of AFM as a tool to assess fibrosis in cardiac tissue.

DNA building blocks for AFM tip functionalization: An easy, fast and stable strategy.

Biosensing atomic force microscopy (AFM) offers the unique feature to determine the energy landscape of a bimolecular interaction at the real single molecule level. Furthermore, simultaneous and label-free mapping of molecular recognition and the determination of sample topography at the nanoscale gets possible. A prerequisite and one of the major parts in biosensing AFM are the bio-functionalized AFM tips. In the past decades, different approaches for tip functionalization have been developed. Using these functionalization strategies, several biological highly relevant interactions at the single molecule level have been explored. For the most common approach, the use of a heterobifunctional poly(ethylenglycol) crosslinker, a broad range of linkers for different chemical coupling strategies is available. Nonetheless, the time consuming functionalization protocol as well as the broad distribution of rupture length reduces the possibility of automation and may reduce the accuracy of the results. Here we present a stable and fast forward approach based on tetra-functional DNA tetrahedra. A fast functionalization and a sharp defined distribution of rupture length gets possible with low effort and high success rate. We tested the performance on the classical avidin biotin system by using tetrahedra with three disulfide legs for stable and site directed coupling to gold coated tips and a biotinylated end at the fourth vertex. A special advantage appears when working with a DNA aptamer as sensing molecule. In this case, the fourth strand can be extended by a certain DNA sequence complementary to the linkage part of an aptamer. This AFM tip functionalization protocol was applied on thrombin using DNA aptamers directed against the fibrinogen binding side of human thrombin.

Facile isolation and analysis of sporopollenin exine from bee pollen.

We present facile methods to obtain purified sporopollenin exine capsules, and provide mass balances for classical and novel purification procedures. An ionic liquid, tetrabutyl phosphonium hydroxide turned out to be the most effective in removing the intine wall. The sporopollenin capsules were investigated by fluorescent microscopy, AFM, solid-state NMR and infrared Raman spectroscopy. The latter two methods showed that sunflower and rape exines have different proportions of O-aliphatic and aromatic constituents. Purified exine capsules were coated with functionalized fluorophores. The procedures presented in this paper could contribute to further spread of the applications of this hollow, and chemically highly resistant material.

3D mechanical characterization of single cells and small organisms using acoustic manipulation and force microscopy.

Quantitative micromechanical characterization of single cells and multicellular tissues or organisms is of fundamental importance to the study of cellular growth, morphogenesis, and cell-cell interactions. However, due to limited manipulation capabilities at the microscale, systems used for mechanical characterizations struggle to provide complete three-dimensional coverage of individual specimens. Here, we combine an acoustically driven manipulation device with a micro-force sensor to freely rotate biological samples and quantify mechanical properties at multiple regions of interest within a specimen. The versatility of this tool is demonstrated through the analysis of single Lilium longiflorum pollen grains, in combination with numerical simulations, and individual Caenorhabditis elegans nematodes. It reveals local variations in apparent stiffness for single specimens, providing previously inaccessible information and datasets on mechanical properties that serve as the basis for biophysical modelling and allow deeper insights into the biomechanics of these living systems.

Comparison of Electropolishing of Aluminum in a Deep Eutectic Medium and Acidic Electrolyte.

Research advances in electropolishing, with respect to the field of metalworking, have afforded significant improvements in the surface roughness and conductivity properties of aluminum polished surfaces in ways that machine polishing and simple chemical polishing cannot. The effects of a deep eutectic medium as an acid-free electrolyte were tested to determine the potential energy thresholds during electropolishing treatments based upon temperature, experiment duration, current, and voltage. Using voltammetry and chronoamperometry tests during electropolishing to supplement representative recordings via atomic force microscopy (AFM), surface morphology comparisons were performed regarding the electropolishing efficiency of phosphoric acid and acid-free ionic liquid treatments for aluminum. This eco-friendly solution produced polished surfaces superior to those surfaces treated with industry standard acid electrochemistry treatments of 1 M phosphoric acid. The roughness average of the as-received sample became 6.11 times smoother, improving from 159 nm to 26 nm when electropolished with the deep eutectic solvent. This result was accompanied by a mass loss of 0.039 g and a 7.2 µm change in step height along the edge of the electropolishing interface, whereas the acid treatment resulted in a slight improvement in surface roughness, becoming 1.63 times smoother with an average post-electropolishing roughness of 97.7 nm, yielding a mass loss of 0.0458 g and a step height of 8.1 µm.

Dry Two-Step Self-Assembly of Stable Supported Lipid Bilayers on Silicon Substrates.

Artificial membranes are models for biological systems and are important for applications. We introduce a dry two-step self-assembly method consisting of the high-vacuum evaporation of phospholipid molecules over silicon, followed by a subsequent annealing step in air. We evaporate dipalmitoylphosphatidylcholine (DPPC) molecules over bare silicon without the use of polymer cushions or solvents. High-resolution ellipsometry and AFM temperature-dependent measurements are performed in air to detect the characteristic phase transitions of DPPC bilayers. Complementary AFM force-spectroscopy breakthrough events are induced to detect single- and multi-bilayer formation. These combined experimental methods confirm the formation of stable non-hydrated supported lipid bilayers with phase transitions gel to ripple at 311.5 ± 0.9 K, ripple to liquid crystalline at 323.8 ± 2.5 K and liquid crystalline to fluid disordered at 330.4 ± 0.9 K, consistent with such structures reported in wet environments. We find that the AFM tip induces a restructuring or intercalation of the bilayer that is strongly related to the applied tip-force. These dry supported lipid bilayers show long-term stability. These findings are relevant for the development of functional biointerfaces, specifically for fabrication of biosensors and membrane protein platforms. The observed stability is relevant in the context of lifetimes of systems protected by bilayers in dry environments.

Preparation and characterization of grass carp collagen-chitosan-lemon essential oil composite films for application as food packaging.

Encapsulation effectively delays the volatilization of lemon essential oil (LEO). Here, various chitosan (CS)-LEO nanoparticles were prepared by emulsification using different CS and Tween-80 concentrations and CS:TPP and CS:LEO ratios. The CS-LEO nanoparticles were spherical, small in size (58 ± 9 nm), had a low polydispersity index (0.15), and were highly stable under the right conditions. FTIR spectra indicated that they were fully encapsulated in the films. The composite films are characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), scanning electron microscope (SEM) and X-ray diffraction (XRD). Relative to grass carp collagen (GCC) films, edible GCC/CS-LEO had lower oxygen permeability (OP), higher tensile strength (TS), and higher elongation at break (EB). The LEO release rate increased with decreasing GCC:CS-LEO ratio. At GCC:CS-LEO ratio = 7:3, a maximum LEO release rate of 83.6 ± 9.7% was achieved over 15 days. GCC/CS-LEO films can effectively inhibit lipid oxidation, prevent microbial proliferation, and delay the deterioration of pork at 4 °C for 21 days.

Revealing the Architecture of the Cell Wall in Living Plant Cells by Bioimaging and Enzymatic Degradation.

Plant cell walls consist mostly of crystalline cellulose fibrils embedded in a matrix of complex polysaccharides, but information on their morphological features has generally been limited to that obtained from nonliving plant specimens. Here, we characterized the primary cell wall of a living plant cell (from the tobacco BY-2 suspension culture) at nanometer resolution using high-speed atomic force microscopy and at micrometer resolution using confocal laser scanning microscopy. Our results showed aligned and disordered cellulose fibrils coexisting in the outermost layer of the cell wall. We investigated the orientation of the aligned cellulose fibrils in the outer lamellae of the cell wall of living plant cells after removing cellulose, hemicellulose, and pectin by enzymatic degradation to make the cellulose fibrils more visible and, accordingly, to reveal the structure of the nanoachitecture formed by these fibrils within the cell wall. We observed that the cellulose fibrils in the outermost layer were usually oriented close to the direction of cell growth, whereas the orientation of the cellulose fibrils in the successive lamellae further inward changed randomly. Such organization should be crucial to render the plant cell wall both rigid and flexible. This finding provides insight not only into the structure of the functional plant cell wall but also into its growth mechanism.

Biomechanical Characterization of Human Pluripotent Stem Cell-Derived Cardiomyocytes by Use of Atomic Force Microscopy.

Atomic force microscopy (AFM) is not only a high-resolution imaging technique but also a sensitive tool able to study biomechanical properties of bio-samples (biomolecules, cells) in native conditions-i.e., in buffered solutions (culturing media) and stable temperature (mostly 37 °C). Micromechanical transducers (cantilevers) are often used to map surface stiffness distribution, adhesion forces, and viscoelastic parameters of living cells; however, they can also be used to monitor time course of cardiomyocytes contraction dynamics (e.g. beating rate, relaxation time), together with other biomechanical properties. Here we describe the construction of an AFM-based biosensor setup designed to study the biomechanical properties of cardiomyocyte clusters, through the use of standard uncoated silicon nitride cantilevers. Force-time curves (mechanocardiograms, MCG) are recorded continuously in real time and in the presence of cardiomyocyte-contraction affecting drugs (e.g., isoproterenol, metoprolol) in the medium, under physiological conditions. The average value of contraction force and the beat rate, as basic biomechanical parameters, represent pharmacological indicators of different phenotype features. Robustness, low computational requirements, and optimal spatial sensitivity (detection limit 200 pN, respectively 20 nm displacement) are the main advantages of the presented method.

Efeitos do jato de plasma frio sob pressão atmosférica sobre Candida albicans / Cell effects of atmospheric pressure low temperature plasma jet on Candida albicans

As infecções causadas por Candida albicans são consideradas um desafio importante na área médica. Além dos antifúngicos convencionais apresentarem elevada toxicidade, casos de resistência de C. albicans a estes medicamentos têm sido descritos com frequência. Devido a estas limitações do uso de antifúngicos, a busca por terapias alternativas é alvo crescente de estudos. Dentre as novas terapias, o jato de plasma frio destaca-se a como agente eficaz frente a cepas de C. albicans, sendo considerada uma alternativa promissora. Seu efeito antifúngico é associado à presença de espécies reativas de oxigênio e nitrogênio liberadas, assim como de íons e fótons. Todavia até o presente momento, o exato mecanismo de ação não foi elucidado. Portanto, o objetivo deste estudo é prospectar os efeitos do jato de plasma frio sobre C. albicans. Perseguindo este objetivo, foram utilizadas duas técnicas complementares às técnicas microbiológicas clássicas, a espectroscopia no Infra-vermelho (FT-IR) e a microscopia de força atômica (AFM). Suspensões padronizadas da cepa C. albicans SC 5314 e 1 cepa clínica foram expostas ao jato de plasma. A seguir, foram analisadas por espectroscopia no infra-vermelho (FT-IR) e microscopia de força atômica (AFM) para identificar os efeitos do plasma sobre a parede celular de C. albicans. Controle negativo (sem exposição) e positivo (caspofungina) também foram analisados. Os ensaios foram realizados em triplicata em três experimentos independentes e a análise estatística foi realizada ao nível de significância de 5 %. As alterações morfológicas e topográficas nas células tratadas com plasma foram similares às do grupo tratado com caspofungina. O perfil bioquímico das células após tratamento com o plasma foi também similar ao das células tratadas com caspofungina. Conclui-se que o plasma agiu diretamente sobre as estruturas da parede celular fúngica, mais especificamente sobre os polissacarídeos, com provável influencia na redução de glucanos e aumento de mananas e quitinas. Esse remodelamento da parede celular pode ter correlação com as alterações morfológicas observadas, tais como mudança no tamanho da célula e aumento da rugosidade superficial(AU)

Infections caused by Candida albicans are considered important challenges in the medical area. Besides the high toxicity of conventional antifungals, cases of C. albicans resistance have been reported with increasing frequency. Due to these limitations of the antifungals, the search for alternative therapies is increasing. Among these therapies, cold plasma showed effectiveness against C. albicans isolates and is considered a promising alternative. Its antifungal effect is associated to the presence of oxygen and nitrogen reactive species, as well as ions. Nonetheless, the exact mechanism of action on C. albicans has not yet been elucidated. The aim of this study is to investigate the effects of plasma jet on C. albicans cell wall. For this purpose, two techniques complementing to classic the microbiological methods were used, the infrared spectroscopy (FT-IR) and atomic force microscopy (AFM). Standardized suspensions of C. albicans SC 5314 and 1 clinical isolate was exposed to plasma jet. Samples were analyzed by FT-IR and AFM to identifying the effects of plasma on C. albicans. Negative control (no exposition) and positive (caspofungin) were also analyzed. The experiments will be done in triplicate in three independent experiments and the statistical analyzes were done adopting the level of significance of 5%. The morphological and topographic alterations in cells treated with plasma were similar to the group treated with caspofungin. The biochemical profile of the cells after treatment with the plasma jet was also similar to those treated with caspofungin. It could be concluded that plasma acted directly on the structures of fungal cell wall, more specifically on polysaccharides, influencing the reduction of glucans and increased of mannans and chitins. This remodeling of cell wall can be correlated with the detected morphological alterations, such as changes in cell size and increase in superficial roughness(AU)

Developing High Performance GaP/Si Heterojunction Solar Cells.

To improve the efficiency of Si-based solar cells beyond their Shockley-Queisser limit, the optimal path is to integrate them with III-V-based solar cells. In this work, we present high performance GaP/Si heterojunction solar cells with a high Si minority-carrier lifetime and high crystal quality of epitaxial GaP layers. It is shown that by applying phosphorus (P)-diffusion layers into the Si substrate and a SiNx layer, the Si minority-carrier lifetime can be well-maintained during the GaP growth in the molecular beam epitaxy (MBE). By controlling the growth conditions, the high crystal quality of GaP was grown on the P-rich Si surface. The film quality is characterized by atomic force microscopy and high-resolution x-ray diffraction. In addition, MoOx was implemented as a hole-selective contact that led to a significant increase in the short-circuit current density. The achieved high device performance of the GaP/Si heterojunction solar cells establishes a path for further enhancement of the performance of Si-based photovoltaic devices.

Stochastic Binding Process of Blunt-End Stacking of DNA Molecules Observed by Atomic Force Microscopy.

Hydrophobic attraction is often a physical origin of nonspecific and irreversible (uncontrollable) processes observed for colloidal and biological systems, such as aggregation, precipitation, and fouling with biomolecules. On the contrary, blunt-end stacking of complementary DNA duplex chain pairs, which is also mainly driven by hydrophobic interaction, is specific and stable enough to lead to self-assemblies of DNA nanostructures. To understand the reason behind these contradicting phenomena, we measured forces operating between two self-assembled monolayers of duplexed DNA molecules with blunt ends (DNA-SAMs) and analyzed their statistics. We found the high specificity and stability of blunt-end stacking that resulted in the high resemblance between the interaction forces measured on approaching and retracting. The other finding is on the stochastic formation process of blunt-end stacking, which appeared as a significant fluctuation of the interaction forces at separations smaller than 2.5 nm. Based on these results, we discuss the underlying mechanism of the specificity and stability of blunt-end stacking.

The Sapphire (0001) Surface: A Transparent and Ultraflat Substrate for DNA Nanostructure Imaging.

Mica is the current substrate of choice for DNA nanostructure imaging, mainly due to its atomically flat surface. However, these mica substrates are often not optically clear. In this work, sapphire has been evaluated as an alternative substrate, with potential to enable parallel optical and AFM studies. Well known for its thermal and chemical properties, sapphire is a hard ionic material with excellent optical properties. Because sapphire lacks the excellent basal cleavage properties of the sheet silicate mica, a process to anneal it at high temperature in water vapor was developed to achieve near atomically smooth (average roughness = 0.141 nm) terraces. AFM imaging was used to determine the dimensions of these terraces and to characterize the morphology of the DNA nanostructures, revealing that their structures were preserved, indicating that annealed c-plane cut (0001) sapphire is a promising substitute for mica as a flat and transparent substrate for DNA nanostructure studies.

Getting into shape: the mechanics behind plant morphogenesis.

The process of shape change in cells and tissues inevitably involves the modification of structural elements, therefore it is necessary to integrate mechanics with biochemistry to develop a full understanding of morphogenesis. Here, we discuss recent findings on the role of biomechanics and biochemical processes in plant cell growth and development. In particular, we focus on how the plant cytoskeleton components, which are known to regulate morphogenesis, are influenced by biomechanical stress. We also discuss new insights into the role that pectin plays in biomechanics and morphogenesis. Using the jigsaw-shaped pavement cells of the leaf as a case study, we review new findings on the biomechanics behind the morphogenesis of these intricately-shaped cell types. Finally, we summarize important quantitative techniques that has allowed for the testing and the generation of hypotheses that link biomechanics to morphogenesis.

Infrared Nanospectroscopy Reveals the Chemical Nature of Pit Membranes in Water-Conducting Cells of the Plant Xylem.

In the xylem of angiosperm plants, microscopic pits through the secondary cell walls connect the water-conducting vessels. Cellulosic meshes originated from primary walls, and middle lamella between adjacent vessels, called the pit membrane, separates one conduit from another. The intricate structure of the nano-sized pores in pit membranes enables the passage of water under negative pressure without hydraulic failure due to obstruction by gas bubbles (i.e. embolism) under normal conditions or mild drought stress. Since the chemical composition of pit membranes affects embolism formation and bubble behavior, we directly measured pit membrane composition in Populus nigra wood. Here, we characterized the chemical composition of cell wall structures by synchrotron infrared nanospectroscopy and atomic force microscopy-infrared nanospectroscopy with high spatial resolution. Characteristic peaks of cellulose, phenolic compounds, and proteins were found in the intervessel pit membranes of P. nigra wood. In addition, the vessel to parenchyma pit membranes and developing cell walls of the vascular cambium showed clear signals of cellulose, proteins, and pectin. We did not find a distinct peak of lignin and other compounds in these structures. Our investigation of the complex chemical composition of intervessel pit membranes furthers our understanding of the flow of water and bubbles between neighboring conduits. The advances presented here pave the way for further label-free studies related to the nanochemistry of plant cell components.

Single-molecule force spectroscopy study of interactions between angiotensin II type 1 receptor and different biased ligands in living cells.

Angiotensin II type 1 receptor (AT1R), a typical G protein-coupled receptor, plays a key role in regulating many cardiovascular functions. Different ligands can bind with AT1R to selectively activate either G protein (Gq) or ß-arrestin (ß-arr) pathway, or both pathways, but the molecular mechanism is not clear yet. In this work, we used, for the first time, atomic force microscopy-based single molecule force spectroscopy (SMFS) to study the interactions of AT1R with three types of ligands, balanced ligand, Gq-biased ligand, and ß-arr-biased ligand, in living cells. The results revealed their difference in binding force and binding stability. The complex of the Gq-biased ligand-AT1R overcame two energy barriers with an intermediate state during dissociation, whereas that of ß-arr-biased ligand-AT1R complex overcame one energy barrier. This indicated that AT1R had different ligand-binding conformational substates and underwent different structural changes to activate downstream signaling pathways with variable agonist efficacies. Quantitative analysis of AT1R-ligand binding in living cells at the single-molecule level offers a new tool to study the molecular mechanism of AT1R biased activation. Graphical Abstract Single-molecule force measurement on the living cell expressing AT1R-eGFP with a ligand modified AFM tip (left), the dynamic force spectra of ß-arrestin biased ligands-AT1R (middle), and Gq-biased ligands-AT1R (right). The complexes of ß-arr-biased ligand-AT1R overcame one energy barrier, with one linear region in the spectra, whereas the Gq-biased ligand-AT1R complexes overcame two energy barriers with two linear regions.

Raman, AFM and SNOM high resolution imaging of carotene crystals in a model carrot cell system.

Three non-destructive and complementary techniques, Raman imaging, Atomic Force Microscopy and Scanning Near-field Optical Microscopy were used simultaneously to show for the first time chemical and structural differences of carotenoid crystals. Spectroscopic and microscopic scanning probe measurements were applied to the released crystals or to crystals accumulated in a unique, carotenoids rich callus tissue growing in vitro that is considered as a new model system for plant carotenoid research. Three distinct morphological crystal types of various carotenoid composition were identified, a needle-like, rhomboidal and helical. Raman imaging using 532 and 488 nm excitation lines provided evidence that the needle-like and rhomboidal crystals had similar carotenoid composition and that they were composed mainly of ß-carotene accompanied by α-carotene. However, the presence of α-carotene was not identified in the helical crystals, which had the characteristic spatial structure. AFM measurements of crystals identified by Raman imaging revealed the crystal topography and showed the needle-like and rhomboidal crystals were planar but they differed in all three dimensions. Combining SNOM and Raman imaging enabled indication of carotenoid rich structures and visualised their distribution in the cell. The morphology of identified subcellular structures was characteristic for crystalline, membraneous and tubular chromoplasts that are plant organelles responsible for carotenoid accumulation in cells.