Direct observation of the effects of chemical fixation in MNT-1 cells: A SE-ADM and Raman study

Abstract

Aldehydes fixation was accidentally discovered in the early 20th century and soon became a widely adopted practice in the histological field, due to an excellent staining enhancement in tissues imaging. However, the fixation process itself entails cell proteins denaturation and crosslinking. The possible presence of artifacts, that depends on the specific system under observation, must therefore be considered to avoid data misinterpretation. This contribution takes advantage of scanning electron assisted-dielectric microscopy (SE-ADM) and Raman 2D imaging to reveal the possible presence and the nature of artifacts in unstained, and paraformldehyde, PFA, fixed MNT-1 cells. The high resolution of the innovative SE-ADM technique allowed the identification of globular protein clusters in the cell cytoplasm, formed after protein denaturation and crosslinking. Concurrently, SE-ADM images showed a preferential melanosome adsorption on the cluster’s outer surface. The micron-sized aggregates were discernible in Raman 2D images, as the melanosomes signal, extracted through 2D principal component analysis, unequivocally mapped their location and distribution within the cells, appearing randomly distributed in the cytoplasm. Protein clusters were not observed in living MNT-1 cells. In this case, mature melanosomes accumulate preferentially at the cell periphery and are more closely packed than in fixed cells. Our results show that, although PFA does not affect the melanin structure, it disrupts melanosome distribution within the cells. Proteins secondary structure, conversely, is partially lost, as shown by the Raman signals related to α-helix, β-sheets, and specific amino acids that significantly decrease after the PFA treatment.

Quantitative Evaluation of Iron-Containing Proteins Bound to Mesoporous Silica Microspheres by Inductively Coupled Plasma Mass Spectrometry and Confocal Laser Raman Microscopy

“…The amount of LF bound to SBA24 was measured using CLRM. CLRM allows compositional analysis of samples by analyzing the Raman spectrum obtained when a sample is irradiated with laser light [ 17 , 18 , 19 , 20 ]. In particular, during the analysis of biological samples, the portions of lipids and proteins in the biological sample can be identified from peaks in the Raman spectrum.…”

Quantitative Evaluation of Iron-Containing Proteins Bound to Mesoporous Silica Microspheres by Inductively Coupled Plasma Mass Spectrometry and Confocal Laser Raman Microscopy

“…The amount of LF bound to SBA24 was measured using CLRM. CLRM allows compositional analysis of samples by analyzing the Raman spectrum obtained when a sample is irradiated with laser light [ 17 , 18 , 19 , 20 ]. In particular, during the analysis of biological samples, the portions of lipids and proteins in the biological sample can be identified from peaks in the Raman spectrum.…”

Monitoring Molecular Interactions with Cell Membranes Using Time-Dependent Second Harmonic Generation Microscopy

“…In paraformaldehyde fixed cells, after protein denaturation and cross-linking, active transport is no longer observed, as noted by other researchers. 61 In living cells, the active transport of dyes through protein channels and/or lipid rafts is facilitated, whereas passive transport is most likely the dominant mode of transport across membranes in fixed cells. Here, time-dependent SHG microscopy is shown to be a powerful method for characterizing these complicated interactions between cationic molecules and cellular membranes.…”

Photoinduced Actin Aggregation Involves Cell Death: A Mechanism of Cancer Cell Cytotoxicity after Near-Infrared Photoimmunotherapy

“…We have confirmed that the black particles in SE-ADM images are protein aggregates by previous observations of biological samples and cultured cells. 29−32 For example, cell adhesion cores, 29 casein micelles in milk, 30 melanin pigment vesicles, 31 and mineralizing nanovesicles 32 are densely packed with proteins, and all of these are observed as clear black contrasts. Therefore, the black particles observed in this experiment are also thought to be protein aggregates.…”

Quantitative Evaluation of Iron-Containing Proteins Bound to Mesoporous Silica Microspheres by Inductively Coupled Plasma Mass Spectrometry and Confocal Laser Raman Microscopy

“…The amount of LF bound to SBA24 was measured using CLRM. CLRM allows compositional analysis of samples by analyzing the Raman spectrum obtained when a sample is irradiated with laser light [ 17 , 18 , 19 , 20 ]. In particular, during the analysis of biological samples, the portions of lipids and proteins in the biological sample can be identified from peaks in the Raman spectrum.…”

Monitoring Molecular Interactions with Cell Membranes Using Time-Dependent Second Harmonic Generation Microscopy

“…In paraformaldehyde fixed cells, after protein denaturation and cross-linking, active transport is no longer observed, as noted by other researchers. 61 In living cells, the active transport of dyes through protein channels and/or lipid rafts is facilitated, whereas passive transport is most likely the dominant mode of transport across membranes in fixed cells. Here, time-dependent SHG microscopy is shown to be a powerful method for characterizing these complicated interactions between cationic molecules and cellular membranes.…”

Photoinduced Actin Aggregation Involves Cell Death: A Mechanism of Cancer Cell Cytotoxicity after Near-Infrared Photoimmunotherapy

“…We have confirmed that the black particles in SE-ADM images are protein aggregates by previous observations of biological samples and cultured cells. 29−32 For example, cell adhesion cores, 29 casein micelles in milk, 30 melanin pigment vesicles, 31 and mineralizing nanovesicles 32 are densely packed with proteins, and all of these are observed as clear black contrasts. Therefore, the black particles observed in this experiment are also thought to be protein aggregates.…”

Quantitative Evaluation of Iron-Containing Proteins Bound to Mesoporous Silica Microspheres by Inductively Coupled Plasma Mass Spectrometry and Confocal Laser Raman Microscopy

“…The amount of LF bound to SBA24 was measured using CLRM. CLRM allows compositional analysis of samples by analyzing the Raman spectrum obtained when a sample is irradiated with laser light [ 17 , 18 , 19 , 20 ]. In particular, during the analysis of biological samples, the portions of lipids and proteins in the biological sample can be identified from peaks in the Raman spectrum.…”

Monitoring Molecular Interactions with Cell Membranes Using Time-Dependent Second Harmonic Generation Microscopy

“…In paraformaldehyde fixed cells, after protein denaturation and cross-linking, active transport is no longer observed, as noted by other researchers. 61 In living cells, the active transport of dyes through protein channels and/or lipid rafts is facilitated, whereas passive transport is most likely the dominant mode of transport across membranes in fixed cells. Here, time-dependent SHG microscopy is shown to be a powerful method for characterizing these complicated interactions between cationic molecules and cellular membranes.…”

Photoinduced Actin Aggregation Involves Cell Death: A Mechanism of Cancer Cell Cytotoxicity after Near-Infrared Photoimmunotherapy

“…We have confirmed that the black particles in SE-ADM images are protein aggregates by previous observations of biological samples and cultured cells. 29−32 For example, cell adhesion cores, 29 casein micelles in milk, 30 melanin pigment vesicles, 31 and mineralizing nanovesicles 32 are densely packed with proteins, and all of these are observed as clear black contrasts. Therefore, the black particles observed in this experiment are also thought to be protein aggregates.…”