A cost-effective approach to measurements of fluorophore temperature sensitivity and temperature change with reasonable accuracy.

The demand for measuring fluorophore temperature sensitivity and temperature change in chemical or biological samples has spurred the search for effective methods. While infrared (IR) light-based thermal devices are popular, they are limited to surface temperature measurement. Fluorescence-based thermometry, which utilizes intensity, lifetime, polarization, and spectrum change, provides the temperature information directly from the samples and can have high temporal and spatial resolution. However, measuring fluorescence can be tricky and expensive. A cost-effective approach to achieving reasonable accuracy is highly desired. This study introduces such an approach, employing a light-emitting diode (LED) for fluorophore excitation and a laser diode (LD) for sample heating, with a phone camera recording fluorescence changes. A data processing method converts the video into digital data, processed through digital filters. Utilizing a small-volume cuvette enhances heating efficiency. This study serves as a practical guide for inexperienced individuals, including students, instructors, and researchers, facilitating entry into the field and navigating the complexities of fluorescence-based thermometry.

Mapping human tissues with highly multiplexed RNA in situ hybridization.

In situ transcriptomic techniques promise a holistic view of tissue organization and cell-cell interactions. There has been a surge of multiplexed RNA in situ mapping techniques but their application to human tissues has been limited due to their large size, general lower tissue quality and high autofluorescence. Here we report DART-FISH, a padlock probe-based technology capable of profiling hundreds to thousands of genes in centimeter-sized human tissue sections. We introduce an omni-cell type cytoplasmic stain that substantially improves the segmentation of cell bodies. Our enzyme-free isothermal decoding procedure allows us to image 121 genes in large sections from the human neocortex in <10 h. We successfully recapitulated the cytoarchitecture of 20 neuronal and non-neuronal subclasses. We further performed in situ mapping of 300 genes on a diseased human kidney, profiled >20 healthy and pathological cell states, and identified diseased niches enriched in transcriptionally altered epithelial cells and myofibroblasts.

Mapping Human Tissues with Highly Multiplexed RNA in situ Hybridization.

In situ transcriptomic techniques promise a holistic view of tissue organization and cell-cell interactions. Recently there has been a surge of multiplexed RNA in situ techniques but their application to human tissues and clinical biopsies has been limited due to their large size, general lower tissue quality and high background autofluorescence. Here we report DART-FISH, a versatile padlock probe-based technology capable of profiling hundreds to thousands of genes in centimeter-sized human tissue sections at cellular resolution. We introduced an omni-cell type cytoplasmic stain, dubbed RiboSoma that substantially improves the segmentation of cell bodies. We developed a computational decoding-by-deconvolution workflow to extract gene spots even in the presence of optical crowding. Our enzyme-free isothermal decoding procedure allowed us to image 121 genes in a large section from the human neocortex in less than 10 hours, where we successfully recapitulated the cytoarchitecture of 20 neuronal and non-neuronal subclasses. Additionally, we demonstrated the detection of transcripts as short as 461 nucleotides, including neuropeptides and discovered new cortical layer markers. We further performed in situ mapping of 300 genes on a diseased human kidney, profiled >20 healthy and pathological cell states, and identified diseased niches enriched in transcriptionally altered epithelial cells and myofibroblasts.

Effects of equine joint injury on boundary lubrication of articular cartilage by synovial fluid: role of hyaluronan.

OBJECTIVE: To compare equine synovial fluid (SF) from injured and control joints for cartilage boundary lubrication function; concentrations of the putative boundary lubricant molecules hyaluronan (HA), proteoglycan 4 (PRG4), and surface-active phospholipids (SAPLs); relationships between lubrication function and composition; and lubrication restoration by addition of HA. METHODS: Equine SF from normal joints, joints with acute injury, and joints with chronic injury were analyzed for boundary lubrication of normal articular cartilage (kinetic friction coefficient [µ(kinetic) ]). Equine SF samples were analyzed for HA, PRG4, and SAPL concentrations and HA molecular weight distribution. The effect of the addition of HA, of different concentrations and molecular weight, on the µ(kinetic) of equine SF samples from normal joints and joints with acute injury was determined. RESULTS: The µ(kinetic) of equine SF from joints with acute injury (0.036) was higher (+39%) than that of equine SF from normal joints (0.026). Compared to normal equine SF, SF from joints with acute injury had a lower HA concentration (-30%) of lower molecular weight forms, higher PRG4 concentration (+83%), and higher SAPL concentration (+144%). Equine SF from joints with chronic injury had µ(kinetic) , PRG4, and SAPL characteristics intermediate to those of equine SF from joints with acute injury and normal equine SF. Regression analysis revealed that the µ(kinetic) value decreased with increasing HA concentration in equine SF. The friction-reducing properties of HA alone improved with increasing concentration and molecular weight. The addition of high molecular weight HA (4,000 kd) to equine SF from joints with acute injury reduced the µ(kinetic) to a value near that of normal equine SF. CONCLUSION: In the acute postinjury stage, equine SF exhibits poor boundary lubrication properties, as indicated by a high µ(kinetic) . HA of diminished concentration and molecular weight may be the basis for this, and adding HA to deficient equine SF restored lubrication function.