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Deep learning enables cross-modality super-resolution in fluorescence microscopy
摘要： We present deep-learning-enabled super-resolution across different fluorescence microscopy modalities. This data-driven approach does not require numerical modeling of the imaging process or the estimation of a point-spread-function, and is based on training a generative adversarial network (GAN) to transform diffraction-limited input images into super-resolved ones. Using this framework, we improve the resolution of wide-field images acquired with low-numerical-aperture objectives, matching the resolution that is acquired using high-numerical-aperture objectives. We also demonstrate cross-modality super-resolution, transforming confocal microscopy images to match the resolution acquired with a stimulated emission depletion (STED) microscope. We further demonstrate that total internal reflection fluorescence (TIRF) microscopy images of subcellular structures within cells and tissues can be transformed to match the results obtained with a TIRF-based structured illumination microscope. The deep network rapidly outputs these super-resolved images, without any iterations or parameter search, and could serve to democratize super-resolution imaging.
关键词： GAN,cross-modality,super-resolution,fluorescence microscopy,deep learning
更新于2025-11-21 11:24:58
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A Double-Hybridization Approach for the Transcription- and Amplification-Free Detection of Specific mRNA on a Microarray
摘要： A double-hybridization approach was developed for the enzyme-free detection of specific mRNA of a housekeeping gene. Targeted mRNA was immobilized by hybridization to complementary DNA capture probes spotted onto a microarray. A second hybridization step of Cy5-conjugated label DNA to another section of the mRNA enabled specific labeling of the target. Thus, enzymatic artifacts could be avoided by omitting transcription and amplification steps. This manuscript describes the development of capture probe molecules used in the transcription- and amplification-free analysis of RPLP0 mRNA in isolated total RNA. An increase in specific signal was found with increasing length of the target-specific section of capture probes. Unspecific signal comprising spot autofluorescence and unspecific label binding did not correlate with the capture length. An additional spacer between the specific part of the capture probe and the substrate attachment site increased the signal significantly only on a short capture probe of approximately 30 nt length.
关键词： gene expression,enzyme-free,fluorescence microscopy,mRNA detection,microarray
更新于2025-11-21 11:24:58
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Three-Dimensional Segmentation and Quantitative Measurement of the Aqueous Outflow System of Intact Mouse Eyes Based on Spectral Two-Photon Microscopy Techniques
摘要： PURPOSE. To visualize and quantify the three-dimensional (3D) spatial relationships of the structures of the aqueous outflow system (AOS) within intact enucleated mouse eyes using spectral two-photon microscopy (TPM) techniques. METHODS. Spectral TPM, including two-photon autofluorescence (TPAF) and second-harmonic generation (SHG), were used to image the small structures of the AOS within the limbal region of freshly enucleated mouse eyes. Long infrared excitation wavelengths (930 nm) were used to reduce optical scattering and autofluorescent background. Image stacks were collected for 3D image rendering and structural segmentation. For anatomical reference, vascular perfusion with fluorescent-conjugated dextran (150 KDa) and trans-corneal perfusion with 0.1 lm fluorescent polystyrene beads were separately performed to identify the episcleral veins (EV) and the trabecular meshwork (TM) and Schlemm's canal (SC), respectively. RESULTS. Three-dimensional rendering and segmentation of spectral two-photon images revealed detailed structures of the AOS, including SC, collector channels (CC), and aqueous veins (AV). The collagen of the TM was detected proximal to SC. The long and short axes of the SC were 82.2 ± 22.2 μm and 6.7 ± 1.4 μm. The diameters of the CC averaged 25.6 ± 7.9 μm where they originated from the SC (ostia), enlarged to 34.1 ± 13.1 μm at the midpoint, and narrowed to 18.3 ± 4.8 μm at the junction of the AV. The diameter of the AV averaged 12.5 ± 3.4 μm. CONCLUSIONS. Spectral TPM can be used to reconstruct and measure the spatial relationships of both large and small AOS structures, which will allow for better understanding of distal aqueous outflow dynamics.
关键词： second-harmonic generation,aqueous outflow,nonlinear microscopy,glaucoma,two-photon fluorescence microscopy
更新于2025-11-21 11:08:12
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Successful optimization of reconstruction parameters in structured illumination microscopy – A practical guide
摘要： The impact of the different reconstruction parameters in super-resolution structured illumination microscopy (SIM) on image artifacts is carefully analyzed. These parameters comprise the Wiener filter parameter, an apodization function, zero-frequency suppression and modifications of the optical transfer function. A detailed investigation of the reconstructed image spectrum is concluded to be suitable for identifying artifacts. For this purpose, two samples, an artificial test slide and a more realistic biological system, were used to characterize the artifact classes and their correlation with the image spectra as well as the reconstruction parameters. In addition, a guideline for efficient parameter optimization is suggested and the implementation of the parameters in selected up-to-date processing packages (proprietary and open-source) is depicted.
关键词： Super resolution,Fluorescence microscopy,Structured illumination microscopy,Parameter estimation,Image reconstruction
更新于2025-09-23 15:23:52
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Suppressed Triplet Exciton Diffusion Due to Small Orbital Overlap as a Key Design Factor for Ultralong-Lived Room-Temperature Phosphorescence in Molecular Crystals
摘要： Persistent room-temperature phosphorescence (RTP) under ambient conditions is attracting attention due to its strong potential for applications in bioimaging, sensing, or optical recording. Molecular packing leading to a rigid crystalline structure that minimizes nonradiative pathways from triplet state is often investigated for efficient RTP. However, for complex conjugated systems a key strategy to suppress the nonradiative deactivation is not found yet. Here, the origin of small rates of a nonradiative decay process from triplet states of conjugated molecular crystals showing RTP is reported. Optical microscopy analysis showed that, despite a favorable molecular stacking, an aromatic crystal with strong RTP is characterized by small diffusion length and small values of the diffusion coefficient of triplet excitons. Quantum chemical calculations reveal a large overlap between the lowest unoccupied molecular orbitals but very small overlap between the highest occupied molecular orbitals (HOMOs). Inefficient electron exchange caused by the small overlap of HOMOs prevents triplet excitons from diffusing over long distances and consequently from quenching at defect sites inside the crystal or at the crystal surface. These results will allow design of comprehensive molecular structures to obtain molecular solids with more efficient RTP.
关键词： suppressed nonradiative rate,persistent room-temperature phosphorescence,triplet exciton diffusion,molecular orbital overlap,fluorescence microscopy
更新于2025-09-23 15:23:52
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Preliminary Clinical Microneurosurgical Experience With the 4K3-Dimensional Microvideoscope (ORBEYE) System for Microneurological Surgery: Observation Study
摘要： BACKGROUND: The exoscope has been reported as a novel neurosurgical instrumentation in clinical practice. OBJECTIVE: To investigate the possibility that ORBEYE (OE), a novel instrument that excludes eyepiece lenses and allows for microsurgery by observation of the 4K3D monitor, could replace microscopes. METHODS: We report 22 clinical cases by 5 experienced neurosurgeons and the comparative results of training 10 residents. An observation study with questionnaire survey was conducted on usability. Twelve items including image quality, eyestrain, and function of the arm were evaluated. RESULTS: The following 22 clinical procedures were conducted: surgery for intracranial hemorrhage (n = 2) and brain tumor (n = 8), laminectomy (n = 3), aneurysm clipping (n = 3), vascular anastomosis (n = 2), carotid endarterectomy (n = 2), and nerve decompression (n = 1). No complications were observed. The fluorescent study, including indocyanine-green and 5-aminolevunic acid, allowed for clear depiction on the 4K monitor. The surgeon could operate in a comfortable posture. Similar to the microscope, it was possible to change the optical and viewing axes with the OE, but the OE was switched to the microscope or endoscope in hematoma removal and pituitary surgery. Residents judged that eyestrain was strong (P = .0096). Experienced neurosurgeons acting as assistants judged that the scope arm’s range of movement was narrow (P = .0204). Sixty percent of residents judged that the OE was superior to the microscope. CONCLUSION: Although based on limited experience, it was not possible to substitute the microscope with the OE in all operations; however, the OE surpasses the microscope in terms of ergonomic features.
关键词： Microsurgery,Fluorescence,Microscopy,Endoscope
更新于2025-09-23 15:23:52
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Fluorescence microscopy as a tool for determining self-incompatibility in apricot cultivars
摘要： Fluorescence microscopy is a relatively rapid and reliable method to determine self-incompatibility in fruit-tree species. It is based on observation of pollen-tube growth in the pistils. Pollen tubes stained with fluorochromes show fluorescence when exposed to ultraviolet light. Testing of the self-compatibility trait was carried out in 123 apricot cultivars using fluorescence microscopy. In self-compatible cultivars, in the majority of pistils (60-100%), the pollen tubes reached the ovary. In contrast, in self-incompatible cultivars, pollen tubes growth ceased in the style, with plugs forming at their tips. In these cultivars, pollen tubes rarely (0-30%) reached the base of the style. Although apricot cultivars of the European eco-geographical group are traditionally considered self-compatible, we identified many self-incompatible cultivars, especially among those originating from new North American and West European breeding programs. About half (62) of the studied cultivars were self-incompatible. Given that self-incompatibility occurs frequently among new apricot cultivars, special care should be taken when considering cultivar composition in new orchard plantings.
关键词： fluorescence microscopy,self-incompatibility,pollen tube growth,Prunus armeniaca,Rosaceae
更新于2025-09-23 15:23:52
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Yellow-emissive carbon dots with a large Stokes shift are viable fluorescent probes for detection and cellular imaging of?silver ions and glutathione
摘要： Yellow-emissive carbon dots (Y-CDs) were prepared by a solvothermal method using anhydrous citric acid and 2,3-phenazinediamine as the starting materials. The Y-CDs display a 24% fluorescence quantum yield, a 188-nm Stokes’ shift and excellent stability. They are shown here to be excellent fluorescent probes for the determination of Ag(I) ion and glutathione (GSH). If exposed to Ag(I) ions, they are bound by the carboxy groups of the Y-CDs, and this causes quenching of fluorescence (with excitation/emission maxima at 380/568 nm) via a static quenching mechanism. This effect was used to design a fluorometric assay for Ag(I). The quenched fluorescence of the Y-CDs can be restored by adding GSH due to the high affinity of GSH for Ag(I). The calibration plot for Ag(I) is linear in the 1–4 μM Ag(I) concentration range, and the limit of detection is 31 nM. The respective values for GSH are 5–32 μM, and 76 nM, respectively. The method was applied to the detection of Ag(I) in spiked environmental water samples and gave recoveries ranging from 93 to 107%. It was also applied to the determination of GSH in tomatoes and purple grapes and gave satisfactory recoveries. The Y-CDs display low cytotoxicity and were successfully used to image Ag(I) and GSH in H1299 cells.
关键词： Fluorescence microscopy,Applications,Stern-Volmer plot,Fluorescence imaging,Fluorescence detection
更新于2025-09-23 15:22:29
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Accelerated FRET-PAINT microscopy
摘要： Recent development of FRET-PAINT microscopy significantly improved the imaging speed of DNA-PAINT, the previously reported super-resolution fluorescence microscopy with no photobleaching problem. Here we try to achieve the ultimate speed limit of FRET-PAINT by optimizing the camera speed, dissociation rate of DNA probes, and bleed-through of the donor signal to the acceptor channel, and further increase the imaging speed of FRET-PAINT by 8-fold. Super-resolution imaging of COS-7 microtubules shows that high-quality 40-nm resolution images can be obtained in just tens of seconds.
关键词： FRET-PAINT,Super-resolution fluorescence microscopy,FRET,Single-molecule localization microscopy
更新于2025-09-23 15:22:29
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Detection of Circulating Tumor Cells in Fluorescence Microscopy Images Based on ANN Classifier
摘要： Circulating tumor cells (CTCs) is a clinical biomarker for cancer metastasis. CTCs are cells circulating in the body of patients by being separated from primary cancer and entering into blood vessel. CTCs spread every positions in the body, and this is one of the cause of cancer metastasis. To analyze them, pathologists get information about metastasis without invasive test. CTCs test is conducted by analyzing the blood sample from patient. The fluorescence microscope generates a large number of images per each sample, and images contain a lot of cells. There are only a few CTCs in images and cells often have blurry boundaries. So CTCs identification is not an easy work for pathologists. In this paper, we develop an automatic CTCs identification method in fluorescence microscopy images. This proposed method has three section. In the first approach, we conduct the cell segmentation in images by using filtering methods. Next, we compute feature values from each CTC candidate region. Finally, we identify CTCs using artificial neural network algorithm. We apply the proposed method to 5895 microscopy images (7 samplesas), and evaluate the effectiveness of our proposed method by using leave-one-out cross validation. We achieve the result of performance tests, a true positive rate is 92.57% and false positive rate is 9.156%.
关键词： Fluorescence microscopy image,Artificial neural network,Feature analysis,Computer aided diagnosis,Circulating tumor cells
更新于2025-09-23 15:22:29