Miniaturized integration of a fluorescence microscope

The diagnosis of parasitic worm (helminth) infections requires specialized laboratory settings, but most affected individuals reside in locations without access to such facilities. We tested two portable microscopic devices for the diagnosis of helminth infections in a cross-sectional survey in rural Côte d'Ivoire. We examined 164 stool samples under a light microscope and then re-examined with a commercial portable light microscope and an experimental mobile phone microscope for the diagnosis of Schistosoma mansoni and soil-transmitted helminths. Additionally, 180 filtered urine samples were examined by standard microscopy and compared with the portable light microscope for detection of Schistosoma haematobium eggs. Conventional microscopy was considered the diagnostic reference standard. For S. mansoni, S. haematobium and Trichuris trichiura, the portable light microscope showed sensitivities of 84.8%, 78.6% and 81.5%, respectively, and specificities of 85.7%, 91.0% and 93.0%, respectively. For S. mansoni and T. trichiura, we found sensitivities for the mobile phone microscope of 68.2% and 30.8%, respectively, and specificities of 64.3% and 71.0%, respectively. We conclude that the portable light microscope has sufficient diagnostic yield for Schistosoma and T. trichiura infections, while the mobile phone microscope has only modest sensitivity in its current experimental set-up. Development of portable diagnostic technologies that can be used at point-of-sample collection will enhance diagnostic coverage in clinical and epidemiological settings.

Functional imaging in behaving animals is essential to understanding brain function. However, artifacts resulting from animal motion, including locomotion, can severely corrupt functional measurements. To dampen tissue motion, we designed a new optical window with minimal optical aberrations. Using the newly developed high-speed continuous volumetric imaging system based on an optical phase-locked ultrasound lens, we quantified motion of the cerebral cortex and hippocampal surface during two-photon functional imaging in behaving mice. We find that the out-of-plane motion is generally greater than the axial dimension of the point-spread-function during mouse locomotion, which indicates that high-speed continuous volumetric imaging is necessary to minimize motion artifacts.

To analyze various aspects of complex tissue, there is increasing demand to study each sample at different length scales in biology. Correlative light and electron microscopy (CLEM) is the latest technique to correlate two different types of information on the exact same histological area of interest: histology (from light microscopy) and ultrastructure (from electron microscopy). The three-dimensional fine structures of the maculae flavae (MFe) of the human vocal fold were investigated using CLEM. Five normal human adult vocal folds as specimens embedded in paraffin, sectioned, and mounted on glass slides with/without a chemical digestion method (modified sodium hydroxide maceration method) were investigated. Observations using CLEM were performed. The fine structures of cells and extracellular matrices in the MFe and their peripheral regions were able to be observed on the exact same histological area of interest with the light microscope and field emission-scanning electron microscope. Cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells were intermingled in the MFe of the human vocal fold. The extracellular matrices surrounding each three types of cell in the MFe differed, suggesting the cells were different in functional property. CLEM is a useful technique to observe the three-dimensional fine structures of the human vocal fold mucosa. The results of the present study are consistent with the hypothesis that the cells in the MFe of the human vocal fold have heterogeneity and each three types of cell have different properties.

Velocity characteristics in boundary layer flow caused by solitary wave traveling over horizontal bottom

ABSTRACTA portable, lightweight, and compact microscope was developed for rapid on‐site identification of Cryptosporidium spp. oocysts, a major cause of diarrhoea in neonatal ruminants. Cryptosporidiosis is an infectious parasitic disease that causes diarrhoea, and rapid diagnosis plays a critical role in initiating timely treatment. A method for diagnosing cryptosporidiosis involves detecting oocysts excreted in faeces using a light microscope with an appropriate staining method. However, transporting samples to a laboratory often takes time, during which the animal's health may deteriorate. The developed portable microscope provides up to 1000x magnification, enabling the diagnosis of cryptosporidiosis using the carbol fuchsin staining method. It is compatible with almost any smartphone and does not require additional digital or optical magnification. Through an integrated mobile telemedicine application, veterinarians can instantly consult specialists by transmitting patient information and images. Calibration tests have demonstrated that the system can generate high‐resolution images of oocysts. This innovative microscope is a crucial tool for veterinarians working in the field, allowing for rapid detection of Cryptosporidium spp. oocysts, reducing neonatal calf mortality, and preventing the spread of infectious diseases.

Peering into Batteries: Electrochemical Insight Through In Situ and Operando Methods over Multiple Length Scales

<p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="first" dir="auto" id="d1087e127">The butterfly wing scales are biocomposites with impressive structural and optical properties, owing to their features spanning from hundreds of microns to a few nanometers. Though they demonstrate efficient color production and structural rigidity, the mechanisms to achieve them are well-concealed across different length scales. As a result, understanding these biocomposites' composition, structure, and corresponding function remains challenging. No single technique can provide a high-resolution three-dimensional (3D) volume with an accurate color map. Traditionally, approaches such as light microscopy (LM) and spectrophotometry for color responses, and scanning electron microscopes (SEM) and transmission electron microscopes (TEM) for structural imaging provide key insights [ <a class="xref-link" href="#r1">1</a>]. However, these methods offer either limited resolution (LM) or limited field of view (TEM), falling short of capturing the full complexity of the scales [ <a class="xref-link" href="#r2">2</a>]. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d1087e135">Here, we used ptychographic X-ray computed tomography (PXCT), which provides 3D density maps at an intermediate resolution (66.5nm) over hundreds of microns. This allowed us to combine the inferences from the high-resolution (&lt;1 nm), limited view structural insights from TEM/SEM with the low-resolution (&gt;1 μm) color response from LM/spectrophotometry [ <a class="xref-link" href="#r3">3</a>, <a class="xref-link" href="#r4">4</a>]. In <a class="xref-link" href="#fg001">Figure 1</a>, the images obtained from various modalities are shown along with their corresponding length scales. Notably, PXCT provides both 3D structural measurements and density values. The 3D structural measurements enable us to estimate the tilt map of the continuous lower lamina structure of the scale and the thickness map of the intricate upper lamina structure. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d1087e146">The known physics behind the reflectance and absorbance spectra enables us to combine insights from a wide range of imaging modalities synergistically. <div class="fig panel" id="fg001"> <a class="named-anchor" id="fg001"> <!-- named anchor --> </a> <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/d9324456-0185-45c5-85af-242f85581685/ScienceOpen/image/285_6725df8b9ab10-DeepanBalakrishnanAPMC2025_fig1.jpg"/> </div> <div class="panel-content"> <div class="label">Fig. 1.</div> <div class="caption" id="d1087e151"> <p class="first" dir="auto" id="d1087e152">Butterfly wing scales have features spanning from 100s of microns to a few nanometers captured using different imaging modalities. From left to right: 1, light microscope (LM), 2, ptychographic X-ray computed tomography (PXCT), scanning electron microscope (SEM), focused ion beam SEM (FIB-SEM), transmission electron microscope (TEM), resin embedding TEM (RE-TEM). The scale bar represents the corresponding length shown below each image. </div> </div> </div>

Correlative light and electron microscopy (CLEM) combines the strengths of both light and electron imaging modalities and enables linking of biological spatiotemporal information from live-cell fluorescence light microscopy (fLM) to high-resolution cellular ultra-structures from cryo-electron microscopy and tomography (cryoEM/ET). This has been previously achieved by using fLM signals to localize the regions of interest under cryogenic conditions. The correlation process, however, is often tedious and time-consuming with low throughput and limited accuracy, because multiple correlation steps at different length scales are largely carried out manually. Here, we present an experimental workflow, AutoCLEM, which overcomes the existing limitations and improves the performance and throughput of CLEM methods, and associated software. The AutoCLEM system encompasses a high-speed confocal live-cell imaging module to acquire an automated fLM grid atlas that is linked to the cryoEM grid atlas, followed by cryofLM imaging after freezing. The fLM coordinates of the targeted areas are automatically converted to cryoEM/ET and refined using fluorescent fiducial beads. This AutoCLEM workflow significantly accelerates the correlation efficiency between live-cell fluorescence imaging and cryoEM/ET structural analysis, as demonstrated by visualizing human immunodeficiency virus type 1 (HIV-1) interacting with host cells.

An experimental study of energy dissipation in two-dimensional unsteady plunging breakers and an eddy viscosity model to simulate the dissipation due to wave breaking are reported in this paper. Measured wave surface elevations are used to examine the characteristic time and length scales associated with wave groups and local breaking waves, and to estimate and parameterize the energy dissipation and dissipation rate due to wave breaking. Numerical tests using the eddy viscosity model are performed and we find that the numerical results well capture the measured energy loss. In our experiments, three sets of characteristic time and length scales are defined and obtained: global scales associated with the wave groups, local scales immediately prior to breaking onset and post-breaking scales. Correlations among these time and length scales are demonstrated. In addition, for our wave groups, wave breaking onset predictions using the global and local wave steepnesses are found based on experimental results. Breaking time and breaking horizontal length scales are determined with high-speed imaging, and are found to depend approximately linearly on the local wave steepness. The two scales are then used to determine the energy dissipation rate, which is the ratio of the energy loss to the breaking time scale. Our experimental results show that the local wave steepness is highly correlated with the measured dissipation rate, indicating that the local wave steepness may serve as a good wave-breaking-strength indicator. To simulate the energy dissipation due to wave breaking, a simple eddy viscosity model is proposed and validated with our experimental measurements. Under the small viscosity assumption, the leading-order viscous effect is incorporated into the free-surface boundary conditions. Then, the kinematic viscosity is replaced with an eddy viscosity to account for energy loss. The breaking time and length scales, which depend weakly on wave breaking strength, are applied to evaluate the magnitude of the eddy viscosity using dimensional analysis. The estimated eddy viscosity is of the order of 10−3 m2s−1 and demonstrates a strong dependence on wave breaking strength. Numerical simulations with the eddy viscosity estimation are performed to compare to the experimental results. Good agreement as regards energy dissipation due to wave breaking and surface profiles after wave breaking is achieved, which illustrates that the simple eddy viscosity model functions effectively.

Introduction:Foldscope is an origami-based portable paper microscope, developed in 2014, that can provide up to 2000 times magnification. It has the potential use as a low-cost diagnostic alternative in areas with limited resources. Oral cancer screening camps rely on visual examination of the patients’ oral cavity. In-field diagnosis of cytological smears can be very beneficial, although it requires more resources. Therefore, we designed a study to compare the accuracy of Foldscope in the identification of features in cytology smears, when compared with light microscope.Methodology:Two examiners with adequate experience evaluated 35 oral cytology smears. The Foldscope was used twice for evaluation by each examiner, with a gap of 15 days between the two evaluations. Both examiners also evaluated the same set of slides using a light microscope. Accuracy of identification of cells using Foldscope, when compared with the light microscope was evaluated. Interobserver agreement in the identification of various features was also determined using kappa statistics, for light microscope examination, and two Foldscope examinations.Results:We found that both examiners could identify epithelial cells accurately using the Foldscope. Mucous cells, inflammatory cells, and microorganisms could also be identified, with slightly lesser accuracy. Interobserver agreement was good in the light microscope. However, with the Foldscope, especially after the second sitting, the interobserver agreement was found to be comparably good.Conclusion:With adequate training and practice, Foldscopes could be valuable tools for rapid and immediate diagnosis of exfoliative cytology smears in mass oral cancer screening camps.

Simultaneous Correlative Light and Electron Microscopy of Samples in Liquid

Recently, Shy and his co-workers developed a new turbulent flow system that used a pair of counter-rotating fans and perforated plates to generate stationary near-isotropic turbulence, as verified by LDV measurements, for the study of premixed turbulent combustion processes. This paper evaluates for the first time the correlations between spatial and temporal properties of small-scale intermittency in such a fan-stirred near-isotropic turbulence. These spatiotemporal properties are obtained simultaneously via high-speed digital particle image velocimetry together with wavelet analyses. It is found that the wavelet energy spectra in the inertial range of near-isotropic region all exhibit a slope of nearly −5/3 which spans at least from 3Hz to 100Hz. Characteristic scales, including the integral time and length scales, Taylor microscales, and viscous dissipation scales, are identified without the use of Taylor hypothesis. Thus, a direct evaluation of Taylor hypothesis in near-isotropic turbulence with zero mean velocity can be made. From variations of the flatness factor, equivalent to the 4th order velocity structure function, in the spatial and time domains, it is found that the characteristic spatial and temporal intermittent scales of intense vorticity structures in the dissipation range of the fan-stirred near-isotropic turbulence occur around 5 ∼ 8η and τk, respectively, where η and τk are the Kolmogorov length and time scales. These results are useful for further study of particle settling in turbulence, a problem of both engineering and geophysical interest.

Cellular processes span a huge range of length and time scales from the molecular to the near macroscopic. Understanding how effects on one scale influence, and are themselves influenced by, those on lower and higher scales is a critical issue for the construction of models in systems biology. Advances in computing hardware and software now allow explicit simulation of some aspects of cellular dynamics close to the molecular scale. Vesicle fusion is one example of such a process. Experiments, however, typically probe cellular behaviour from the molecular scale up to microns. Standard particle-based simulation techniques cannot capture such a broad range. Consequently, at long length scales, models have often been of the mass action variety, in which molecular constituents are represented by density fields that vary continuously in space and time, rather than involving discrete molecules. But these models struggle to represent processes that are localized in space and time or involve the transport of material through a crowded environment. A novel class of mesoscopic simulation techniques are now able to span length and time scales from nanometres to microns for hundreds of microseconds and may soon be coupled to mass action models allowing the parameters in such models to be continuously tuned according to the finer resolution simulation. This will help realize the goal of a computational cellular simulation that is able to capture the dynamics of membrane-associated processes such as endo- and exocytosis.

Fourier light field microscopy (FLFM) shows great potential in high-speed volumetric imaging of biodynamics. However, due to the inherent disadvantage of wide-field illumination, it suffers from intense background, arising from out of the depth-of-field signal and tissue scattered noise. The background will not only deteriorate the image contrast, making quantitative measurement difficult, but also introduce artifacts, especially in functional imaging of the neuronal network activity in vivo. Here, we propose the robust Fourier light field microscopy (RFLFM), which suppresses the background in FLFM by introducing structured illumination and computational reconstruction based on HiLo. The superior performance of RFLFM is verified by volumetric imaging of biological dynamics in larval zebrafish and mouse in vivo, at a volumetric imaging rate up to 33.3 Hz. The statistical results show that the fluorescence background can be significantly depressed, with the signal-to-background ratio improved by orders of magnitude and the whole image contrast improved by as much as ∼ 10.4 times. Moreover, we stress that, in functional imaging of neuronal network activity in turbid brain tissues, our system can avoid artifacts resulting from background fluctuations, while conventional light field microscopy fails. As a simple but powerful tool, we anticipate our technique to be widely adopted in robust, high-contrast, high-speed volumetric imaging.

In vivo imaging of zebrafish embryogenesis