Improving the Signal Intensity of Cryosections Using a Conductive Adhesive Film in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique was used to obtain the molecular images of cryosections without labeling. Although MALDI-MSI has been widely used to detect small molecules from biological tissues, issues remain due to the technical process of cryosectioning and limited mass spectrometry parameters. The use of a conductive adhesive film is a unique method to obtain high-quality sections from cutting tissue, such as bone, muscle, adipose tissue, and whole body of mice or fish, and we have reported the utilization of the film for MALDI-MSI in previous. However, some signal of the small molecules using the conductive adhesive films was still lower than on the indium tin oxide (ITO) glass slide. Here, the sample preparation and analytical conditions for MALDI-MSI using an advanced conductive adhesive film were optimized to obtain strong signals from whole mice heads. The effects of tissue thickness and laser ionization power on signal intensity were verified using MALDI-MSI. The phospholipid signal intensity was measured for samples with three tissue thicknesses (5, 10, and 20 µm); compared to the signals from the samples on the ITO glass slides, the signals with conductive adhesive films exhibited significantly higher intensities when a laser with a higher range of power was used to ionize the small molecules. Thus, the technique using the advanced conductive adhesive film showed an improvement in MALDI-MSI analysis.

Raman spectroscopic insight into osteoarthritic cartilage regeneration by mRNA therapeutics encoding cartilage-anabolic transcription factor Runx1.

While joint arthroplasty remains nowadays the most popular option available to repair chronically degenerated osteoarthritic joints, possibilities are recently emerging for regeneration of damaged cartilage rather than its replacement with artificial biomaterials. This latter strategy could allow avoiding the quite intrusive surgical procedures associated with total joint replacement. Building upon this notion, we first apply Raman spectroscopy to characterize diseased cartilage in a mice model of instability-induced knee osteoarthritis (OA) upon medial collateral ligament (MCL) and medial meniscus (MM) transections. Then, we examine the same OA model after cartilage regeneration by means of messenger RNA (mRNA) delivery of a cartilage-anabolic runt-related transcription factor 1 (RUNX1). Raman spectroscopy is shown to substantiate at the molecular scale the therapeutic effect of the Runx1 mRNA cartilage regeneration approach. This study demonstrates how the Raman spectroscopic method could support and accelerate the development of new therapies for cartilage diseases.

Preparation of Thin Frozen Sections from Nonfixed and Undecalcified Hard Tissues Using Kawamoto's Film Method (2020).

A method for preparing frozen sections with an adhesive film is described. In order to observe fine structures and weak fluorescence of samples, new types of adhesive films [Cryofilm type 3C(16UF) and 4D(16UF)] are used. The adhesive film is made with very clear and very low autofluorescence. For gene analysis, a very thin adhesive film (LMD film) is used to cut by means of the laser microdissection (LMD). For MALDI mass spectrometry imaging (MALDI-MSI), a conductive adhesive film (Cryofilm type MS) is used to avoid electric charge of the sample. A biological sample is frozen quickly and freeze-embedded. The frozen sample is cut with a very sharp disposable blade made from fine tungsten carbide. The combination of the adhesive films and the blade can generate 3 micrometer thick sections from samples including bone, while it is also possible to generate 1 µm thick sections. The morphology of bone and soft tissues are preserved using this method. Cells such as osteoblasts, fibroblasts, and osteoclasts are clearly observed with an oil immersion lens at high magnification. Sections generated using the Cryofilm type 3C(16UF) shows weak fluorescent signals more clearly than sections generated with the previously reported adhesive films [Cryofilm type 2C(9) and 2C(10)]. Furthermore fluorescence of the fine structures in cells is clearly shown using a super-high-resolution microscope. Several staining and experimental methods such as histology, histochemistry, enzyme histochemistry, immunohistochemistry, and in situ hybridization can be performed on these sections. This method is also useful for preparing frozen sections of large sample such as a whole-body mouse and rat. In gene analysis, gene quality of sample collected from the section made with the LMD film is superior to that of sample made by a conventional method. The Cryofilm type MS makes almost complete section from tissues including hard tissues and large samples. The satisfactory signals are detected from the section with MALDI-MSI.

Conductive Adhesive Film Expands the Utility of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique is a promising approach for detecting the distribution of small molecules in a section of biological tissue. However, when a cryosection is created from fragile, hard, or whole-body samples, obtaining a high-quality section that maintains the distribution of the various components has been difficult. Since adhesive films have the potential to obtain high-quality cryosections, we attempted to utilize a conductive adhesive film for MALDI-MSI. To this end, cryosections of the whole body of a 9-day-old mouse were directly prepared on indium tin oxide (ITO) glass slides, nonconductive adhesive films, or conductive adhesive films, and the signal intensities from each section were measured by MALDI-MSI. We measured the differences in the ion intensity among these three slides/films by means of multivariate analyses and found that both the nonconductive and conductive adhesive films gave rise to high-quality sections in comparison with the ITO glass slide. The conductive adhesive film gave higher signals that were comparable to those of the ITO glass slide in comparison with the nonconductive adhesive film. We divided the frozen sections into two groups, a freeze-dried group and a thawed group, to examine the freeze-thaw effect on the signals of representative compounds of amino acids, cholesterol, and phosphatidylcholines. The freeze-dried samples were found to be useful for the analysis. These results indicate that the sections made with the conductive adhesive film under a freeze-dried condition can expand the utility of the MALDI-MSI analysis.

Preparation of thin frozen sections from nonfixed and undecalcified hard tissues using Kawamot's film method (2012).

A method for preparing hard tissue sections by using an adhesive film is described. The method produces very thin (to one-micrometer thick) frozen sections from adult mouse and rat bone. The bone tissue is freeze-embedded with water-soluble medium and then cut with a disposable tungsten carbide blade after mounting the adhesive film onto the cut surface. The sections are stained on the adhesive film and preserved between the adhesive film and glass slide. All the steps including the embedding, cutting, staining, and mounting are completed within only 20 min. The soft and hard tissues are preserved satisfactorily and the bone marrow is also preserved perfectly. Cells such as osteoblasts, fibroblasts, and osteoclasts are clearly identified, and the osteoid layer of bone is clearly observed. The sections are applicable to many types of staining such as histology, histochemistry, enzyme histochemistry, immunohistochemistry, and in situ hybridization. The immunohistochemistry can be carried out with nonfixed and undecalcified sections. In addition to these applications, the sections are used for observing the PGF fluorescence. The sections are also usable for studying the distribution of water-soluble materials in the tissues. Furthermore, the sections are very useful for gene analysis using LMD technique and for imaging mass spectrometry.