Quantitative analysis of vitamin D using m/MALDI-TOF mass spectrometry based on a parylene matrix chip

Vitamin D deficiency is associated with various disorders and is diagnosed based on the concentration of 25-hydroxy vitamin D3 (25(OH)D3) in serum. The parylene matrix chip was fabricated to reduce the matrix background noise, and the homogenous distribution of the matrix was retained for the quantitative analysis of 25(OH)D3. The Amplex Red assay was performed to confirm that the sample-matrix mixing zone of the parylene matrix chip was formed below the surface of the parylene-N film. The homogeneous distribution of the matrix was verified from the fluorescence image. For effective analysis using a parylene matrix chip, 25(OH)D3 was modified through the nucleophilic addition of betaine aldehyde (BA) to form a hemiacetal salt. Such modified 25(OH)D3 with a positive charge from BA could be effectively analyzed using MALDI-TOF mass spectrometry. Serum 25(OH)D3 was extracted by liquid–liquid extraction (LLE) and quantified using MALDI-TOF mass spectrometry based on the parylene matrix chip. The intensity of the mass peak of 25(OH)D3 was linearly correlated (r2 = 0.992) with the concentration of 25(OH)D3 spiked in serum, and the LOD was 0.0056 pmol/μL. Energy drinks and vitamin D3 tablets were also employed for the real sample analysis. Finally, the results of the chemiluminescence binding assay and MALDI-TOF mass spectrometry were statistically analyzed to determine the applicability of the method using the Bland–Altman test and Passing–Bablok regression.

Verification of an algorithm for actin tail detection of subviral particles by comparison with expert validated data

The locomotion of subviral particles of Marburg virus has been shown to be primarily actin based. For this work, a virologist selected 14 subviral particles that show actin tails in fluorescence image sequences. Using the tracked coordinates, examination areas around these subviral particles are defined. The brightness of within the examination area behind the subviral particle is analysed. In addition, the speed of the particle in each frame is calculated to investigate potential correlations between actin activity and particle speed. The results show that actin tracks can be automatically detected and analysed. First hints of a correlation between subviral particle movement and actin activity could be gathered with the presented actin tail quantifier.

Stable Fluorescence of Eu3+ Complex Nanostructures Beneath a Protein Skin for Potential Biometric Recognition

We designed and realized highly fluorescent nanostructures composed of Eu3+ complexes under a protein coating. The nanostructured material, confirmed by photo-induced force microscopy (PiFM), includes a bottom fluorescent layer and an upper protein layer. The bottom fluorescent layer includes Eu3+ that is coordinated by 1,10-phenanthroline (Phen) and oleic acid (O). The complete complexes (OEu3+Phen) formed higher-order structures with diameter 40–150 nm. Distinctive nanoscale striations reminiscent of fingerprints were observed with a high-resolution transmission electron microscope (HRTEM). Stable fluorescence was increased by the addition of Eu3+ coordinated by Phen and 2-thenoyltrifluoroacetone (TTA), and confirmed by fluorescence spectroscopy. A satisfactory result was the observation of red Eu3+ complex emission through a protein coating layer with a fluorescence microscope. Lanthanide nanostructures of these types might ultimately prove useful for biometric applications in the context of human and non-human tissues. The significant innovations of this work include: (1) the structural set-up of the fluorescence image embedded under protein “skin”; and (2) dual confirmations of nanotopography and unique nanofingerprints under PiFM and under TEM, respectively.

Design of a Long-Pass Filter with Effects on Fluorescence Image Observation for Surgical Fluorescence Microscope Applications

The goal of oncological surgery is to completely remove the tumor. Tumors are often difficult to observe with the naked eye because of the presence of numerous blood vessels and the fact the colors of the tumor and blood vessels are similar. Therefore, a fluorescent contrast medium using a surgical microscope is used to observe the removal status of the tumor. To observe the tumor removal status using a fluorescent contrast agent, fluorescence is expressed in the tumor by irradiating with an external light source, and the expressed tumor can be confirmed through a surgical microscope. However, not only fluorescence-expressed tumors are observed under a surgical microscope, but images from an external light source are also mixed and observed. Therefore, since the surgical microscope is connected to a filter, the quality of the diagnostic image is not uniform, and it is difficult to achieve a clear observation. As a result, an asymmetric image quality phenomenon occurs in the diagnostic images. In this paper, a filter with high clarity that provides a symmetrical observation of diagnostic images is developed and manufactured.

Fluorescence Imaging Using Enzyme-Activatable Probes for Real-Time Identification of Pancreatic Cancer

IntroductionRadical resection is the only curative treatment for pancreatic cancer, which is a life-threatening disease. However, it is often not easy to accurately identify the extent of the tumor before and during surgery. Here we describe the development of a novel method to detect pancreatic tumors using a tumor-specific enzyme-activatable fluorescence probe.MethodsTumor and non-tumor lysate or small specimen collected from the resected specimen were selected to serve as the most appropriate fluorescence probe to distinguish cancer tissues from noncancerous tissues. The selected probe was sprayed onto the cut surface of the resected specimen of cancer tissue to acquire a fluorescence image. Next, we evaluated the ability of the probe to detect the tumor and calculated the tumor-to-background ratio (TBR) by comparing the fluorescence image with the pathological extent of the tumor. Finally, we searched for a tumor-specific enzyme that optimally activates the selected probe.ResultsUsing a library comprising 309 unique fluorescence probes, we selected GP-HMRG as the most appropriate activatable fluorescence probe. We obtained eight fluorescence images of resected specimens, among which four approximated the pathological findings of the tumor, which achieved the highest TBR. Finally, dipeptidyl-peptidase IV (DPP-IV) or a DPP-IV-like enzyme was identified as the target enzyme.ConclusionThis novel method may enable rapid and real-time visualization of pancreatic cancer through the enzymatic activities of cancer tissues.