EZ Clear for simple, rapid, and robust mouse whole organ clearing

Tissue clearing for whole organ cell profiling has revolutionized biology and imaging for exploration of organs in three-dimensional space without compromising tissue architecture. But complicated, laborious procedures, or expensive equipment, as well as the use of hazardous, organic solvents prevents the widespread adoption of these methods. Here we report a simple and rapid tissue clearing method, EZ Clear, that can clear whole adult mouse organs in 48 hours in just three simple steps. Samples stay at room temperature and remain hydrated throughout the clearing process, preserving endogenous and synthetic fluorescence, without altering sample size. After wholemount clearing and imaging, EZ Cleared samples can be further processed for downstream embedding and cryosectioning followed by standard histology or immunostaining, without loss of endogenous or synthetic fluorescence signal. Overall, the simplicity, speed, and flexibility of EZ Clear make it easy to adopt and apply to diverse approaches in biomedical research.

Digital E. coli Counter: A Microfluidics and Computer Vision-Based DNAzyme Method for the Isolation and Specific Detection of E. coli from Water Samples

Biological water contamination detection-based assays are essential to test water quality; however, these assays are prone to false-positive results and inaccuracies, are time-consuming, and use complicated procedures to test large water samples. Herein, we show a simple detection and counting method for E. coli in the water samples involving a combination of DNAzyme sensor, microfluidics, and computer vision strategies. We first isolated E. coli into individual droplets containing a DNAzyme mixture using droplet microfluidics. Upon bacterial cell lysis by heating, the DNAzyme mixture reacted with a particular substrate present in the crude intracellular material (CIM) of E. coli. This event triggers the dissociation of the fluorophore-quencher pair present in the DNAzyme mixture leading to a fluorescence signal, indicating the presence of E. coli in the droplets. We developed an algorithm using computer vision to analyze the fluorescent droplets containing E. coli in the presence of non-fluorescent droplets. The algorithm can detect and count fluorescent droplets representing the number of E. coli present in the sample. Finally, we show that the developed method is highly specific to detect and count E. coli in the presence of other bacteria present in the water sample.

Measurement of scattered rays from different materials using an inorganic scintillator based optical fiber sensor and its application in radiotherapy

Measurements using an Optical Fiber OFS including an inorganic scintillator placed on the surface of a phantom show that the particle energy distribution inside the phantom remains unchanged. The backscattered intensity measured using an Optical Fiber Sensor (OFS) exhibits a linear relationship with the total radiation dose delivered to the phantom, and this relationship shows that the OFS can be used for indirect dose measurement when located on the surface of the phantom i.e. that arising from the energetic backscattered electrons and photons. Such a device can therefore be used as a clinical in-vivo dosimeter, being located on the patient’s body surface. In addition, the measurement results for the same OFS located inside and outside the radiation field of a compound water based phantom are analyzed. The differences in measurement of the fluorescence signal in response to various tissue materials representing bone or tumor tissue in the irradiation field are strongly related to the material's ability to block the scattered rays from the water phantom, as well as the scattered X-rays generated by the material located within the phantom.

High-quantum yield alloy-typed core/shell CdSeZnS/ZnS quantum dots for bio-applications

Background Quantum dots (QDs) have been used as fluorophores in various imaging fields owing to their strong fluorescent intensity, high quantum yield (QY), and narrow emission bandwidth. However, the application of QDs to bio-imaging is limited because the QY of QDs decreases substantially during the surface modification step for bio-application. Results In this study, we fabricated alloy-typed core/shell CdSeZnS/ZnS quantum dots (alloy QDs) that showed higher quantum yield and stability during the surface modification for hydrophilization compared with conventional CdSe/CdS/ZnS multilayer quantum dots (MQDs). The structure of the alloy QDs was confirmed using time-of-flight medium-energy ion scattering spectroscopy. The alloy QDs exhibited strong fluorescence and a high QY of 98.0%. After hydrophilic surface modification, the alloy QDs exhibited a QY of 84.7%, which is 1.5 times higher than that of MQDs. The QY was 77.8% after the alloy QDs were conjugated with folic acid (FA). Alloy QDs and MQDs, after conjugation with FA, were successfully used for targeting human KB cells. The alloy QDs exhibited a stronger fluorescence signal than MQD; these signals were retained in the popliteal lymph node area for 24 h. Conclusion The alloy QDs maintained a higher QY in hydrophilization for biological applications than MQDs. And also, alloy QDs showed the potential as nanoprobes for highly sensitive bioimaging analysis. Graphical Abstract

A Ratiometric Fluorescent Aptamer Homogeneous Biosensor Based on Hairpin Structure Aptamer for AFB1 Detection

On the basis of aptamer (Apt) with hairpin structure and fluorescence resonance energy transfer (FRET), a ratio fluorescent aptamer homogeneous sensor was prepared for the determination of Aflatoxin B1 (AFB1). Initially, the Apt labeled simultaneously with Cy5, BHQ2, and cDNA labeled with Cy3 were formed a double-stranded DNA through complementary base pairing. The fluorescent aptamer sensor demonstrates a weak fluorescence emission of Cy3 and a high fluorescence emission of Cy5 due to the quenching effect of BHQ2. The double-stranded DNA structure will be disintegrated in the presence of AFB1, resulting the removal of Cy3 and the close of Cy5 with BHQ2. The fluorescence signal of Cy3 and Cy5 were restored and quenched respectively. Thus, the ratio change of FCy3 to FCy5 was used to realized the detection of AFB1 with wider detection range and lower limit of detection (LOD). The response of the optimized protocol for AFB1 detection was wider linear range from 0.05 ng/mL to 100 ng/mL and the LOD was 12.6 pg/mL. The sensor designed in this strategy has the advantages of simple preparation and fast signal response. It has been used for the detection of AFB1 in labeled corn and wine, indicating it had good application potential in practical samples.

Detection and localization of calcium oxalate in kidney using synchrotron deep ultraviolet fluorescence microscopy

Renal oxalosis is a rare cause of renal failure whose diagnosis can be challenging. Synchrotron deep ultraviolet (UV) fluorescence was assayed to improve oxalosis detection on kidney biopsies spatial resolution and sensitivity compared with the Fourier transform infrared microspectroscopy gold standard. The fluorescence spectrum of synthetic mono-, di- and tri-hydrated calcium oxalate was investigated using a microspectrometer coupled to the synchrotron UV beamline DISCO, Synchrotron SOLEIL, France. The obtained spectra were used to detect oxalocalcic crystals in a case control study of 42 human kidney biopsies including 19 renal oxalosis due to primary (PHO, n = 11) and secondary hyperoxaluria (SHO, n = 8), seven samples from PHO patients who received combined kidney and liver transplants, and 16 controls. For all oxalocalcic hydrates samples, a fluorescence signal is detected at 420 nm. These spectra were used to identify standard oxalocalcic crystals in patients with PHO or SHO. They also revealed micrometric crystallites as well as non-aggregated oxalate accumulation in tubular cells. A nine-points histological score was established for the diagnosis of renal oxalosis with 100% specificity (76–100) and a 73% sensitivity (43–90). Oxalate tubular accumulation and higher histological score were correlated to lower estimated glomerular filtration rate and higher urinary oxalate over creatinine ratio.

Turn-on signal fluorescence sensor based on DNA derived bio-dots/polydopamine nanoparticles for the detection of glutathione

The turn-on fluorescence signal mechanism for detection of GSH.

Investigation of 6S 1/2–8S 1/2 two-photon transition of cesium atoms by a single 822 nm laser

We experimentally investigate the 6S 1/2–8S 1/2 two-photon transition in cesium vapor by a single laser. A blue (455.5 and 459.3 nm) fluorescence signal is observed as a result of 822.5 nm laser beams illuminating the Cs vapor with a counter-propagating configuration. The dependences of the fluorescence intensity on the polarization combinations of the laser beams, laser power and vapor temperature are studied to obtain optimal experimental parameters. The frequency difference between the two hyperfine components of 4158 (7) MHz is measured with a Fabry–Perot interferometer as a frequency reference. Such a large spectral isolation and the insensitivity to the Earth’s magnetic field enable the 6S 1/2–8S 1/2 transition to be a stable frequency standard candidate for a frequency-doubled 1644 nm laser in the U-band window for quantum telecommunication.

Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.