- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Customized MFM probes with high lateral resolution
摘要： Magnetic force microscopy (MFM) is a widely used technique for magnetic imaging. Besides its advantages such as the high spatial resolution and the easy use in the characterization of relevant applied materials, the main handicaps of the technique are the lack of control over the tip stray field and poor lateral resolution when working under standard conditions. In this work, we present a convenient route to prepare high-performance MFM probes with sub-10 nm (sub-25 nm) topographic (magnetic) lateral resolution by following an easy and quick low-cost approach. This allows one to not only customize the tip stray field, avoiding tip-induced changes in the sample magnetization, but also to optimize MFM imaging in vacuum (or liquid media) by choosing tips mounted on hard (or soft) cantilevers, a technology that is currently not available on the market.
关键词： magnetic materials,AFM probes,high-resolution microscopy,atomic force microscopy (AFM),magnetic force microscopy (MFM)
更新于2025-09-04 15:30:14
-
Super-resolution microscopy and empirically validated autocorrelation image analysis discriminates microstructures of dairy derived gels
摘要： The food industry must capitalise on advancing technologies in order to optimise the potential from emerging ingredient technologies. These can aid in product optimisation and provide quantitative empirical data to which there is a fundamental physical understanding. Super-resolution microscopy provides a tool to characterise the microstructure of complex colloidal materials under near native conditions. Coherent Anti-Stokes Raman Scattering (CARS) microscopy was used to show the presence of fluorescent dye required for imaging does not affect gel microstructure and super-resolution Stimulated Emission Depletion (STED) microscopy is used to image four dairy derived gels. Image analysis has been developed based on 2D spatial autocorrelation, and a model that extracts parameters corresponding to a typical length of the protein domains and the inter pore distance. The model has been empirically validated through the use of generated images to show the fitting parameters relate to precise physical features. The fractal dimension is extracted from Fourier space analysis. The combination of STED microscopy and image analysis is sensitive enough to significantly differentiate samples based on whether gels were made from fresh or reconstituted milk, and whether gelation was induced through acidification or rennet addition. Rheometry shows that the samples exhibit different macroscopic behaviours, and these differences become increasingly significant with time. Samples can be differentiated earlier in the gelation process with imaging as compared to rheometry. This highlights the potential of STED imaging and image analysis to characterise the size of protein domains, pore spacing and the fractal dimensions of microstructures to aid product optimisation.
关键词： Stimulated Emission Depletion (STED) microscopy,Super-resolution microscopy,Fractal dimension,Coherent Anti-stokes Raman Scattering (CARS) microscopy,2D spatial autocorrelation analysis
更新于2025-09-04 15:30:14
-
Optoacoustic microscopy at multiple discrete frequencies
摘要： Optoacoustic (photoacoustic) sensing employs illumination of transient energy and is typically implemented in the time domain using nanosecond photon pulses. However, the generation of high-energy short photon pulses requires complex laser technology that imposes a low pulse repetition frequency (PRF) and limits the number of wavelengths that are concurrently available for spectral imaging. To avoid the limitations of working in the time domain, we have developed frequency-domain optoacoustic microscopy (FDOM), in which light intensity is modulated at multiple discrete frequencies. We integrated FDOM into a hybrid system with multiphoton microscopy, and we examine the relationship between image formation and modulation frequency, showcase high-fidelity images with increasing numbers of modulation frequencies from phantoms and in vivo, and identify a redundancy in optoacoustic measurements performed at multiple frequencies. We demonstrate that due to high repetition rates, FDOM achieves signal-to-noise ratios similar to those obtained by time-domain methods, using commonly available laser diodes. Moreover, we experimentally confirm various advantages of the frequency-domain implementation at discrete modulation frequencies, including concurrent illumination at two wavelengths that are carried out at different modulation frequencies as well as flow measurements in microfluidic chips and in vivo based on the optoacoustic Doppler effect. Furthermore, we discuss how FDOM redefines possibilities for optoacoustic imaging by capitalizing on the advantages of working in the frequency domain.
关键词： Doppler effect,Frequency-domain,Multiphoton microscopy,Optoacoustic microscopy,Microcirculatory blood flow
更新于2025-09-04 15:30:14
-
Ex Vivo Confocal Microscopy Of Basal Cell Carcinoma On A 3-Color Scale
摘要： Ex vivo confocal microscopy (CM) is capable of visualizing freshly excised tissue in real-time with cellular resolution without routine processing. Depending on the laser wavelength, either reflectance (RCM) or fluorescence (FCM) is utilized. Ex vivo CM is useful for the rapid evaluation of tumor margins during Mohs micrographic surgery (MMS). Initially, ex vivo RCM studies used acetic acid as contrast agent to enhance basal cell carcinoma (BCC) cell nuclei. However, thin strands of BCC were frequently missed. The use of fluorophores improves contrast, so that even small strands of BCC can be spotted in FCM mosaics. Acridine orange (AO) is the dye most widely used. AO binds specifically to DNA and emits fluorescence, so images of living cell nuclei can be enhanced and displayed as bright structures in FCM mosaics. Even though good resolution and morphological correlation are achieved with this standard technique, nowadays confocal mosaics are displayed in a grey scale format. However, dermato-pathologists are often neither familiar with nor comfortable assessing these black-and-white images. We herein report a new technique for obtaining 3-color scale confocal mosaics (3CS-FCM) with the simultaneous use of AO and ethidium bromide (EB) as fluorescent dyes. In this technique, the excised skin sample is first soaked with liquid nitrogen. The sample is then sectioned into 20-30 μm-thick slices using a cryostat and stained with the dye mixture (AO 0.1 mM + EB 0.25 mM) for about one minute. The sample is then placed in the confocal microscope plate for imaging (Nikon A1R+, NIKON CORPORATION?, Japan). The tissue is scanned simultaneously with two different wavelength lasers (405 and 488 nm) and the collected fluorescence displayed on the screen as a 3-color-scale mosaic. Around 10 to 15 minutes are required for completion of the tissue processing and for final mosaics to be developed. Unlike AO, EB binds specifically to the DNA of BCC cells that are damaged due to freezing. As a result, BCC nests are stained by EB and emit red fluorescence after laser stimulation; in contrast, the epidermis and dermis are stained by AO and emit green fluorescence. Blue color corresponds to the background tissue autofluorescence. All fluorescence is collected by the microscope displaying the final images in a 3-color scale format. AO and EB staining do not affect additional fixation or staining of the sample. Figure 1 shows completed BCC mosaics displayed with this new technique. Each color represents a different skin structure, making the mosaics easier to read. In this way, 3CS confocal mosaics are more user-friendly and can be interpreted by healthcare professionals without previous experience with FCM. Moreover, with frozen sample processing, the tissue is completely flattened and the entire sample can be displayed on the screen. These developments represent important advantages over previously described images obtained with CM. In conclusion, 3CS-FCM is an innovative technique that provides colored images, expanding significantly the applicability of FCM. Larger studies are nevertheless required to validate the technique for MMS and other applications.
关键词： fluorescence confocal microscopy,3-color scale mosaics,basal cell carcinoma,ethidium bromide,acridine orange,ex vivo confocal microscopy,Mohs micrographic surgery
更新于2025-09-04 15:30:14
-
Characterization of neurite dystrophy after trauma by high speed structured illumination microscopy and lattice light sheet microscopy
摘要： Background: Unbiased screening studies have repeatedly identified actin-related proteins as one of the families of proteins most influenced by neurotrauma. Nevertheless, the status quo model of cytoskeletal reorganization after neurotrauma excludes actin and incorporates only changes in microtubules and intermediate filaments. Actin is excluded in part because it is difficult to image with conventional techniques. However, recent innovations in fluorescent microscopy provide an opportunity to image the actin cytoskeleton at super-resolution resolution in living cells. This study applied these innovations to an in vitro model of neurotrauma. New method: New methods are introduced for traumatizing neurons before imaging them with high speed structured illumination microscopy or lattice light sheet microscopy. Also, methods for analyzing structured illumination microscopy images to quantify post-traumatic neurite dystrophy are presented. Results: Human induced pluripotent stem cell-derived neurons exhibited actin organization typical of immature neurons. Neurite dystrophy increased after trauma but was not influenced by jasplakinolide treatment. The F-actin content of dystrophies varied greatly from one dystrophy to another. Comparison with existing methods: In contrast to fixation dependent methods, these methods capture the evolution of the actin cytoskeleton over time in a living cell. In contrast to prior methods based on counting dystrophies, this quantification scheme parameterizes the severity of a given dystrophy as it evolves from a local swelling to an almost-perfect spheroid that threatens to transect the neurite. Conclusions: These methods can be used to investigate genetic factors and therapeutic interventions that modulate the course of neurite dystrophy after trauma.
关键词： Traumatic brain injury,Dystrophy,Structured illumination microscopy,Human induced pluripotent stem cell derived neurons,Lattice light sheet microscopy
更新于2025-09-04 15:30:14
-
Whole-cell, 3D and multi-color STED imaging with exchangeable fluorophores
摘要： We demonstrate STED microscopy of whole bacterial and eukaryotic cells using fluorogenic labels that reversibly bind to their target structure. A constant exchange of labels guarantees the removal of photobleached fluorophores and their replacement by intact fluorophores, thereby circumventing bleaching-related limitations of STED super-resolution imaging. We achieve a constant labeling density and demonstrate a fluorescence signal for long and theoretically unlimited acquisition times. Using this concept, we demonstrate whole-cell, 3D, multi-color and live cell STED microscopy.
关键词： PAINT,fluorogenic labels,multicolor imaging,live-cell STED microscopy,volumetric imaging,exchange-based STED microscopy
更新于2025-09-04 15:30:14
-
Deep learning in imaging
摘要： Machine learning approaches that include deep learning are moving beyond image classification to change the way images are made. Computers are powerful tools for carrying out tasks such as image classification or identification as well as or better than human experts. Conventional machine learning approaches are widely used for segmentation and phenotyping in fluorescence microscopy. These tools are now being largely outperformed by their deep-learning-based counterparts, some of which are available as user-friendly tools. But a perhaps more astonishing wave of developments has recently come about through the use of deep learning not for image analysis but for image transformation. In these cases, deep convolutional networks are trained to transform one type of image into another. For example, two studies have shown the power of deep learning for the creation of fluorescence micrographs of cells directly from bright-field or phase images, to facilitate multiplexed and longitudinal imaging. Researchers have also used deep learning to go from low signal-to-noise images to high-quality images, which opens the door to extended imaging of even very light-sensitive living organisms. Deep learning can similarly overcome obstacles associated with super-resolution microscopy. Two approaches, ANNA-PALM and DeepSTORM, were developed to improve the speed of localization microscopy, which is one of the major hurdles of the technique. Deep learning can also enable cross-modality imaging, where applications such as a shift from confocal images to stimulated-emission-depletion-microscopy-resolution images could democratize super-resolution imaging. As with any method, the caveats associated with deep learning in such applications, such as the potential for artifacts, must be carefully considered and analyzed. Nevertheless, we think we have seen only the tip of the iceberg, and that deep learning stands to improve all aspects of imaging, from acquisition to analysis.
关键词： image transformation,machine learning,fluorescence microscopy,deep learning,super-resolution microscopy,imaging
更新于2025-09-04 15:30:14
-
DNA-Based Super-Resolution Microscopy: DNA-PAINT
摘要： Super-resolution microscopies, such as single molecule localization microscopy (SMLM), allow the visualization of biomolecules at the nanoscale. The requirement to observe molecules multiple times during an acquisition has pushed the field to explore methods that allow the binding of a fluorophore to a target. This binding is then used to build an image via points accumulation for imaging nanoscale topography (PAINT), which relies on the stochastic binding of a fluorescent ligand instead of the stochastic photo-activation of a permanently bound fluorophore. Recently, systems that use DNA to achieve repeated, transient binding for PAINT imaging have become the cutting edge in SMLM. Here, we review the history of PAINT imaging, with a particular focus on the development of DNA-PAINT. We outline the different variations of DNA-PAINT and their applications for imaging of both DNA origamis and cellular proteins via SMLM. Finally, we reflect on the current challenges for DNA-PAINT imaging going forward.
关键词： DNA PAINT,SMLM,DNA origami,DNA,fluorescence microscopy,super-resolution microscopy
更新于2025-09-04 15:30:14
-
Confocal Microscopy and Lentigo Maligna: An in vivo Pilot Study for the Assessment of Response to Imiquimod Therapy
摘要： Background: Reflectance confocal microscopy (RCM) is a noninvasive technique that provides real-time in vivo images of the epidermal layer. Imiquimod has been recommended as an alternative treatment in lentigo maligna (LM) when surgical excision is not the treatment of choice. In the present study we compare the results of in vivo RCM to the histopathological examination before and after treatment of LM with topical imiquimod. Methods: Thirty-four patients with confirmed LM were included. Imiquimod 5% was applied until a weeping erosion appeared in the LM-affected skin. Evaluation was performed by clinical examination, dermatoscopy, histopathology and RCM. Results: During the follow-up, 27 of 34 patients (79.42%) demonstrated a total tumor clearance by imiquimod treatment. In the treated area, a significant decrease of atypical cells was detected using RCM (p < 0.0001). Furthermore, a significant positive correlation in the detected atypical cells was shown using confocal microscopy and histology (p = 0.0001, r = 0.7335, respectively). Conclusion: In patients not suitable for surgical intervention imiquimod treatment is an appropriate treatment alternative. Thereby, in vivo RCM was demonstrated to be an excellent examining device, which not only allows diagnosis of LM, but also therapy and follow-up examinations. An important benefit of RCM, in contrast to conventional histopathology, is the simple handling with in vivo examination of epidermal skin without any pain for the patient.
关键词： Imiquimod therapy,Lentigo maligna,Noninvasive follow-up,Reflectance confocal microscopy
更新于2025-09-04 15:30:14
-
Building two-dimensional materials one row at a time: Avoiding the nucleation barrier
摘要： Assembly of two-dimensional (2D) molecular arrays on surfaces produces a wide range of architectural motifs exhibiting unique properties, but little attention has been given to the mechanism by which they nucleate. Using peptides selected for their binding affinity to molybdenum disulfide, we investigated nucleation of 2D arrays by molecularly resolved in situ atomic force microscopy and compared our results to molecular dynamics simulations. The arrays assembled one row at a time, and the nuclei were ordered from the earliest stages and formed without a free energy barrier or a critical size. The results verify long-standing but unproven predictions of classical nucleation theory in one dimension while revealing key interactions underlying 2D assembly.
关键词： two-dimensional materials,atomic force microscopy,nucleation,peptides,molecular dynamics simulations
更新于2025-09-04 15:30:14