A novel cryo-embedding method for in-depth analysis of craniofacial mini pig bone specimens

Ticha, Pavla; Pilawski, Igor; Yuan, Xue; Pan, Jie; Tulu, U.S.; Coyac, Benjamin R.; Hoffmann, Waldemar; Helms, Jill A.

doi:10.1038/s41598-020-76336-3

Cited by 5 publications

(3 citation statements)

References 45 publications

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“…Cryofilm 3C 16UF was predicted to be nonconductive since it is comprised of a polyvinylidene chloride film and acrylic resin cryoglue. 34 For MALDI IMS, a charged surface enhances ion acceleration and dissipates charge accumulation on the tissue surface, which improves ionization and signal. Therefore, alternative conductive adhesives have been explored for imaging mass spectrometry.…”

Section: Resultsmentioning

confidence: 99%

High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone

Neumann

Butrico

et al. 2021

Preprint

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Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to pathological alterations giving rise to debilitating diseases like rheumatoid arthritis, osteoporosis, osteomyelitis, and cancer. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) enable the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of native osseous tissue for MALDI IMS has been difficult due to the mineralized composition and heterogenous morphology of the tissue, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs allowing the distribution of endogenous lipids to be linked to specific tissue structures and cell types. Adhesive-bound bone sections were mounted onto ITO coated glass slides with a microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. Subliming matrix does not induce further bone marrow cracks, and recrystallizing the deposited matrix improves lipid signal. High spatial resolution (10 μm) MALDI IMS was leveraged to characterize lipid distributions in fresh-frozen bone, and complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to bone marrow, adipose tissue, marrow adipose tissue, and muscle. Furthermore, we discovered that [sphingomyelin(42:1) + H]+ was abundant in megakaryocytes, whereas [sphingomyelin(42:2) + H]+ was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Therefore, this application of multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage in a preserved native bone microenvironment.

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Section: Resultsmentioning

confidence: 99%

High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone

Neumann

Butrico

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…Cryofilm 3C 16UF was predicted to be nonconductive since it comprises a polyvinylidene chloride film and acrylic resin cryoglue. 35 For MALDI IMS, a charged surface enhances ion acceleration and dissipates charge accumulation on the tissue surface, which improves the ionization and signal. Therefore, alternative conductive adhesives have been explored for imaging mass spectrometry.…”

Section: ■ Methodsmentioning

confidence: 99%

High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone

et al. 2022

View full text Add to dashboard Cite

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis and osteomyelitis. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) facilitate the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of osseous tissue for MALDI IMS has been difficult due to its mineralized composition and heterogeneous morphology, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs, allowing the distribution of endogenous lipids to be linked to tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. High spatial resolution (10 μm) MALDI IMS was employed to characterize lipid distributions, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to the bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment.

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“…However, due to the seed's resistance to crack, the superglue was insufficient to hold the globose seed when the steel blade forced its surface, and the sample dropped from the holder. Next, a strategy to use embedding media to facilitate sectioning with a microtome was performed, as previous studies with tooth 34 , craniofacial pig bone 35 , and botanical samples [36][37][38] successfully applied this method. Paraffin was first evaluated, as it is reported to facilitate sectioning using a microtome at room temperature.…”

Section: Sample Preparation Methodsmentioning

confidence: 99%

Acquiring thin histological sections of açaí (Euterpe oleracea Mart.) seeds for molecular mapping by mass spectrometry imaging

Brum

Martins

Mohana‐Borges

et al. 2022

Preprint

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RATIONALE: Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) of tissues became popular in the last decade. Consequently, adapting sample preparation methods for different materials turned to be a pivotal step for a successful analysis, due to the requirement of samples slices of 12-20 µm thickness. However, preparing thin sections compatible with MALDI-IMS for unusual samples is challenging, as existing histological protocols may not be suitable, thus requiring new methods. Açaí (Euterpe oleracea Mart.) seed is an example of a challenging material due to its toughness and resistance to crack. Therefore, our goal was to develop a methodology to obtain thin (<20 µm) and entire longitudinal sections of açaí seeds for MALDI-IMS analysis. METHODS: Different strategies were evaluated for obtaining thin cuts of seeds, being the combination of the following steps the most suitable option: (i) softening of seeds by water immersion for 24 h; (ii) longitudinal cut of seeds to obtain half-seeds using a razor blade and a hammer; (iii) half-seeds imbibition in gelatin; (iv) sectioning using a cryostat at -20 °C to obtain samples with <20 µm thickness; and (v) collection of samples in an indium tin oxide coated glass slide covered by a double-face copper tape to avoid sample wrapping and ensure adhesion after unfreezing. (iv) storage of samples in a -80 °C freezer, if necessary. RESULTS: As a result, this adapted sample preparation method enabled the analysis of açaí seeds by MALDI-IMS providing spatial distribution of carbohydrates in the endosperm. CONCLUSIONS: The adaptations developed for sample preparation will help investigate the metabolic and physiological properties of açaí seeds in future studies.

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A novel cryo-embedding method for in-depth analysis of craniofacial mini pig bone specimens

Cited by 5 publications

References 45 publications

High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone

High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone

High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone

Acquiring thin histological sections of açaí (Euterpe oleracea Mart.) seeds for molecular mapping by mass spectrometry imaging

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