Abnormal accumulation of amyloid-β (Aβ) plaques in the brain is one of the major characteristics of Alzheimer’s disease and there is a possibility that the degree of plaque toxicity is related to the distribution of nanoscale oligomeric aggregates in plaques. Here, by super-resolution fluorescence imaging, we visualized that anti-Aβ oligomer antibodies localized differently to plaques compared to anti-Aβ monomer antibodies and characteristic plaque shapes in brain tissues were classified. These differences in nanoscale distribution were hard to be discerned by conventional fluorescence imaging, implying that super-resolution imaging has the potential to reveal the detailed features of oligomeric aggregates in plaques.
KEYWORDS: Signal detection, Super resolution, Brain, Luminescence, Visualization, Spatial resolution, Signal attenuation, Proteins, Molecules, Microscopy
Abnormal deposition of amyloid-β (Aβ) causes the formation of senile plaques, which is one of the main pathological features of Alzheimer disease. To visualize the plaques at nanometer resolution, single-molecule localization microscopy (SMLM) is promising. It is also essential to develop a method for suppressing autofluorescence especially under high-magnifications used for detecting single molecules. Here, we report a novel method to reduce autofluorescence in mouse brains which is applicable to visualize the structure of Aβ plaques by SMLM. The super-resolution images of Aβ plaques showed fibrous structures that were not able to be discerned by conventional fluorescence imaging.
Amino acids and peptides are basic components of proteins and have vital importance in various biological functions and diseases. In this research, we have attempted to detect and distinguish 20 kinds of amino acids and 39 kinds of peptides without any labeling. By using Raman microscopy, more than two thousand Raman spectra were obtained within five minutes from each analyte, at femtomolecular levels. Furthermore, deep learning analyses of the spectra yielded accuracies greater than 96 percent in discriminating between the amino acids and the peptides.
Single molecule localization microscopy (SMLM) relies on the detection of fluorescence emission from a single fluorophore molecule. Recently, several fluorescent dyes with spontaneous blinking have been reported. Last year, we reported a near-infrared dye with spontaneous blinking and high photostability. Here, we present a new orangefluorescent dye that exhibits spontaneous blinking based on equilibrium between the fluorescent and non-fluorescent forms. We have also ascertained that the new dye can be used to create a super-resolution image of cytoskeletal microtubules. In combination with our near-infrared dye reported last year, the new dye is expected to allow dual-color SMLM.
We have attempted to image three-dimensional distribution of fluorescence beads embedded within gels in a glass capillary using light-sheet illumination, as a primary step towards developing a three-dimensional molecular imaging flow cytometer. An illumination and a detection path were arranged orthogonal to the longer axis of the capillary. The light-sheet illumination was tilted with respect to the illumination axis to image a projection of a section of the sample by a CCD. Different sections of the sample were imaged through scanning the capillary itself, along its length. By stacking the images after inverse transformation, the three-dimensional distribution of the fluorescence beads was imaged.
KEYWORDS: Luminescence, Breast, Super resolution, Microscopy, Point spread functions, Spatial resolution, Tissues, 3D image processing, Tissue optics, Breast cancer
HER2 positive breast cancer is currently examined by counting HER2 genes using fluorescence in situ hybridization (FISH)-stained breast carcinoma samples. In this research, two-dimensional super resolution fluorescence microscopy based on stochastic optical reconstruction microscopy (STORM), with a spatial resolution of approximately 20 nm in the lateral direction, was used to more precisely distinguish and count HER2 genes in a FISH-stained tissue section. Furthermore, by introducing double-helix point spread function (DH-PSF), an optical phase modulation technique, to super resolution microscopy, three-dimensional images were obtained of HER2 in a breast carcinoma sample approximately 4 μm thick.
Electrochemical biosensors have been developed due to its potential to be a compact medical
diagnostic devise with high sensitivity. So far we have developed a photoelectrochemical DNA sensor
using transparent semiconductor films such as tin-doped indium oxide (ITO), in which probe DNAs that
captures fluorescence-labeled target DNAs were immobilized on semiconductor via silane coupling reagent such as aminopropyl triethoxy silane (APTES). Here we aimed to provide an effective DNA immobilization technique using gold thin layer in order to obtain higher photocurrents to noise ratio. Gold thin film (1nm thickness) deposited over semiconductor electrode serves as a substrate to immobilize a thiol-modified DNA (24bases) at its end that can capture fluorescence-labeled target DNA by hybridization. The sensitivity in this method was approximately 4times higher than that in APTES.
Here we show that femtosecond laser irradiation can be used to evoke dynamic calcium concentration changes in living cells. The relatively localized interaction that results from the two-photon absorption process allows the release of calcium from intracellular stores in cells in vitro. The self-catalytic response to calcium elevation in a cell can increase the initial release of calcium further so that the entire cell undergoes a rise in cytosolic calcium concentration (i.e. a calcium wave). The calcium stimulation was observed in HeLa (non-excitable) and PC12 (excitable) cells, and could be seen to occur inside a range of power levels between approximately 20 to 80mW. The observation of direct calcium release by femtosecond laser which leads to a calcium wave in the cell has implications for photolytic calcium uncaging experiments since it could be a competing, or even dominant factor in some experiments using caged calcium for the generation of calcium waves.
We have demonstrated that intracellular Ca2+ waves in a living HeLa cell can be induced by femtosecond near-infrared laser pulses. In this paper, we present the results of investigation on the process of the Ca2+ wave generation using pharmacological methods to determine generation mechanisms. A mode-locked Ti:Sapphire laser (780 nm, 80 fs, 82 MHz) was used as a wave-triggering light source. The laser beam was focused into HeLa cells by using a water immersion objective lens (NA 0.9). Ca2+ waves were visualized by using a fluorescent Ca2+ indicator (Fluo-4) and monitored by a fluorescence microscope. Three mechanisms for the Ca2+ wave generation were considered; (1) Ca2+ flow into cells by destruction of the cell membrane, (2) mechanical stress by shock waves associated with the laser absorption, and (3) the leaking of Ca2+ through the destruction of intracellular Ca2+ stores. To investigate the mechanisms, we have performed experiments to determine the dependence of the probability of Ca2+ wave generation with two kinds of extracellular solutions; (a) a Ca2+ free extracellular solution (by use of EGTA), and (b) a solution containing U-73122 to inhibit the response to shockwave-based mechanical effects. From these experimental results, we can conclude the main mechanism of Ca2+ wave generation by laser irradiation is due to the leaking of Ca2+ through the destruction of intracellular Ca2+ stores.
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