Interactions between colloidal-scale structures govern the physical properties of soft and biological materials, and knowledge of the forces associated with these interactions is critical for understanding and controlling these materials. A common approach to quantify colloidal interactions is to measure the interaction forces between colloids and a fixed surface. The centrifuge force microscope (CFM), a miniaturized microscope inside a centrifuge, is capable of performing hundreds of force measurements in parallel over a wide force range (10 −2 to 10 4 pN), but CFM instruments are not widely used to measure colloid–surface interaction forces. In addition, current CFM instruments rely on brightfield illumination and are not capable of fluorescence microscopy. Here we present a fluorescence CFM (F-CFM) that combines both fluorescence and brightfield microscopy and demonstrate its use for measuring microscale colloidal-surface interaction forces. The F-CFM operates at speeds up to 5000 RPM, 2.5× faster than those previously reported, yielding a 6.25× greater maximum force than previous instruments. A battery-powered GoPro video camera enables real-time viewing of the microscopy video on a mobile device, and frequency analysis of the audio signal correlates centrifuge rotational speed with the video signal. To demonstrate the capability of the F-CFM, we measure the force required to detach hundreds of electrostatically stabilized colloidal microspheres attached to a charged glass surface as a function of ionic strength and compare the resulting force distributions with an approximated DLVO theory. The F-CFM will enable microscale force measurements to be correlated with fluorescence imaging in soft and biological systems.
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In situ high-speed brightfield imaging for studies of aquatic organisms
Behavioral measurements of fragile aquatic organisms require specialized in situ techniques.We developed an in situ brightfield camera set-up for use during SCUBA diving in aquatic ecosystems.The system uses brightfield illumination with collimated light and an underwater camera to highlight morphological details, body motion and interactions between organisms with high spatial (4K: 3840x2160 pixels) and temporal resolution (up to 120 fps).This technique is particularly useful for gelatinous organisms because of their large (centimeters in length), transparent bodies.Further, the measurements are not subject to experimental artifacts produced in laboratory studies.This method is useful for anyone seeking detailed brightfield images of organisms or nonliving material (e.g. marine snow) in the natural environment.
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- PAR ID:
- 10354278
- Date Published:
- Journal Name:
- Protocolsio
- ISSN:
- 2473-1838
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Two-photon polymerization (TPP) is an advanced 3D fabrication technique capable of creating features with submicron precision. A primary challenge in TPP lies in the facile and accurate characterization of fabrication quality, particularly for structures possessing complex internal features. In this study, we introduce an automated brightfield layerwise evaluation technique that enables a simple-to-implement approach for
in situ monitoring and quality assessment of TPP-fabricated structures. Our approach relies on sequentially acquired brightfield images during the TPP writing process and using background subtraction and image processing to extract layered spatial features. We experimentally validate our method by printing a fibrous tissue scaffold and successfully achieve an overall system-adjusted fidelity of 87.5%in situ . Our method is readily adaptable in most TPP systems and can potentially facilitate high-quality TPP manufacturing of sophisticated microstructures. -
Quantifying myelin content in brain tissue using color Spatial Light Interference Microscopy (cSLIM)Garini, Yuval (Ed.)Deficient myelination of the brain is associated with neurodevelopmental delays, particularly in high-risk infants, such as those born small in relation to their gestational age (SGA). New methods are needed to further study this condition. Here, we employ Color Spatial Light Interference Microscopy (cSLIM), which uses a brightfield objective and RGB camera to generate pathlength-maps with nanoscale sensitivity in conjunction with a regular brightfield image. Using tissue sections stained with Luxol Fast Blue, the myelin structures were segmented from a brightfield image. Using a binary mask, those portions were quantitatively analyzed in the corresponding phase maps. We first used the CLARITY method to remove tissue lipids and validate the sensitivity of cSLIM to lipid content. We then applied cSLIM to brain histology slices. These specimens are from a previous MRI study, which demonstrated that appropriate for gestational age (AGA) piglets have increased internal capsule myelination (ICM) compared to small for gestational age (SGA) piglets and that a hydrolyzed fat diet improved ICM in both. The identity of samples was blinded until after statistical analyses.more » « less
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null (Ed.)The characterization of particle and plankton populations, as well as microscale biophysical interactions, is critical to several important research areas in oceanography and limnology. A growing number of aquatic researchers are turning to holography as a tool of choice to quantify particle fields in diverse environments, including but not limited to, studies on particle orientation, thin layers, phytoplankton blooms, and zooplankton distributions and behavior. Holography provides a non-intrusive, free-stream approach to imaging and characterizing aquatic particles, organisms, and behavior in situ at high resolution through a 3-D sampling volume. Compared to other imaging techniques, e.g., flow cytometry, much larger volumes of water can be processed over the same duration, resolving particle sizes ranging from a few microns to a few centimeters. Modern holographic imaging systems are compact enough to be deployed through various modes, including profiling/towed platforms, buoys, gliders, long-term observatories, or benthic landers. Limitations of the technique include the data-intensive hologram acquisition process, computationally expensive image reconstruction, and coherent noise associated with the holograms that can make post-processing challenging. However, continued processing refinements, rapid advancements in computing power, and development of powerful machine learning algorithms for particle/organism classification are paving the way for holography to be used ubiquitously across different disciplines in the aquatic sciences. This review aims to provide a comprehensive overview of holography in the context of aquatic studies, including historical developments, prior research applications, as well as advantages and limitations of the technique. Ongoing technological developments that can facilitate larger employment of this technique toward in situ measurements in the future, as well as potential applications in emerging research areas in the aquatic sciences are also discussed.more » « less
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Abstract In shallow coastal systems, sediments are exposed to dramatic and complex variability in environmental conditions that influences sediment processes on short timescales. Sediment oxygen demand (SOD), or consumption of oxygen by sediment‐dwelling organisms and chemical reactions within sediments, is one such process and an important metric of aquatic ecosystem functioning and health. The most common instruments used to measure SOD in situ are batch‐style benthic chambers, which generally require long measurement periods to resolve fluxes and thus do not capture the high temporal variability in SOD that can be driven by dynamic coastal processes. These techniques also preclude linking changes in SOD through time to specific features of the sediment, for example, shifts in sediment faunal activities which can vary on short time scales and can also be affected by ambient oxygen concentrations. Here we present an in situ semi‐flow through instrument to repeatedly measure SOD in discrete areas of sediment. The system isolates patches of sediment in replicate benthic chambers, and measures and records oxygen decrease for a short time before refreshing the overlying water in the chamber with water from the external environment. This results in a sawtooth pattern in which each tooth is an incubation, providing an automated method to produce direct measurements of in situ SOD that can be directly linked to an area of sediment and related to rapid shifts in environmental conditions.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.17504/protocols.io.kxygxz4ykv8j/v2
