Blue-violet excited autofluorescence spectroscopy and imaging of normal and cancerous human bronchial tissue after formalin fixation

Autofluorescence (AF) imaging is a powerful tool for the detection of (pre-)neoplastic lesions in the bronchi. Several endoscopic imaging systems exploit the spectral and intensity contrast of AF between healthy and (pre-)neoplastic bronchial tissues, yet, the mechanisms underlying these contrasts are poorly understood. In this report, the effect of formalin fixation on the human bronchi AF, hence on the contrast, was studied by spectrofluorometric point measurements and DAFE (Diagnostic AutoFluorescence Endoscopy) broad field imaging. Generally, formalin-fixed samples have higher AF intensity than in vivo, whereas the emission spectral shape is similar. Additionally, the spectrofluorometric data showed a moderate decrease of the AF intensity on (pre-)neoplastic lesions relative to the healthy bronchial samples. However, this decrease was lower than that reported from in vivo measurements. Neither spectral measurements nor imaging revealed spectral contrast between healthy bronchial tissue and (pre-)neoplastic lesions in formalin. These results indicate that epithelial thickening and blood supply in the adjacent lamina propria are likely to play a key role in the generation of the AF contrast in bronchial tissues. Our results show that the AF contrast in bronchial tissues was significantly affected by standard, 10% buffered, formalin fixation. Therefore, these samples are not suited to AF contrast studies.     

Preparation of tissue samples for X-ray fluorescence microscopy

As is well-known, trace elements, especially metals, play an important role in the pathogenesis of many disorders. The topographic and quantitative elemental analysis of pathologically changed tissues may shed some new light on processes leading to the degeneration of cells in the case of selected diseases. An ideal and powerful tool for such purpose is the Synchrotron Microbeam X-ray Fluorescence technique. It enables the carrying out of investigations of the elemental composition of tissues even at the single cell level.

The tissue samples for histopathological investigations are routinely fixed and embedded in paraffin. The authors try to verify the usefulness of such prepared tissue sections for elemental analysis with the use of X-ray fluorescence microscopy. Studies were performed on rat brain samples. Changes in elemental composition caused by fixation in formalin or paraformaldehyde and embedding in paraffin were examined.

Measurements were carried out at the bending magnet beamline L of the Hamburger Synchrotronstrahlungslabor HASYLAB in Hamburg.

The decrease in mass per unit area of K, Br and the increase in P, S, Fe, Cu and Zn in the tissue were observed as a result of the fixation. For the samples embedded in paraffin, a lower level of most elements was observed. Additionally, for these samples, changes in the composition of some elements were not uniform for different analyzed areas of rat brain.     

N2 laser excited autofluorescence spectroscopy of formalin-fixed human breast tissue

The paper reports results of an in vitro study on autofluorescence spectroscopy of fresh and formalin-fixed human breast tissue samples to investigate the effect of formalin fixation on the measured autofluorescence spectra. It also explores the applicability of the approach in discriminating cancerous from the uninvolved sites of the formalin-fixed breast tissues based on their autofluorescence spectra. A probability-based diagnostic algorithm, making use of the theory of relevance vector machine (RVM), a powerful recent approach for statistical pattern recognition, was developed for that purpose. The algorithm provided sensitivity values of up to 97% and specificity values of up to 100% towards cancer for both the independent validation data set as well as for the training data set based on leave-one-out cross-validation. These results suggest that autofluorescence spectroscopy may prove to be a valuable additional in vitro diagnostic modality in clinical pathology setting for discriminating cancerous tissue sites from normal sites.     

MALDI tissue imaging: from biomarker discovery to clinical applications

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for the generation of multidimensional spatial expression maps of biomolecules directly from a tissue section. From a clinical proteomics perspective, this method correlates molecular detail to histopathological changes found in patient-derived tissues, enhancing the ability to identify candidates for disease biomarkers. The unbiased analysis and spatial mapping of a variety of molecules directly from clinical tissue sections can be achieved through this method. Conversely, targeted IMS, by the incorporation of laser-reactive molecular tags onto antibodies, aptamers, and other affinity molecules, enables analysis of specific molecules or a class of molecules. In addition to exploring tissue during biomarker discovery, the integration of MALDI-IMS methods into existing clinical pathology laboratory practices could prove beneficial to diagnostics. Querying tissue for the expression of specific biomarkers in a biopsy is a critical component in clinical decision-making and such markers are a major goal of translational research. An important challenge in cancer diagnostics will be to assay multiple parameters in a single slide when tissue quantities are limited. The development of multiplexed assays that maximize the yield of information from a small biopsy will help meet a critical challenge to current biomarker research. This review focuses on the use of MALDI-IMS in biomarker discovery and its potential as a clinical diagnostic tool with specific reference to our application of this technology to prostate cancer.     

Imaging of Phospholipids in Formalin Fixed Rat Brain Sections by Matrix Assisted Laser Desorption/Ionization Mass Spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a valuable tool for the analysis of molecules directly from tissue. Imaging of phospholipids is gaining widespread interest, particularly as these lipids have been implicated in a variety of pathologic processes. Formalin fixation (FF) is the standard protocol used in histology laboratories worldwide to preserve tissue for analysis, in order to aid in the diagnosis and prognosis of diseases. This study assesses MALDI imaging of phospholipids directly in formalin fixed tissue, with a view to future analysis of archival tissue. This investigation proves the viability of MALDI-MSI for studying the distribution of lipids directly in formalin fixed tissue, without any pretreatment protocols. High quality molecular images for several phosphatidylcholine (PC) and sphingomyelin (SM) species are presented. Images correspond well with previously published data for the analysis of lipids directly from freshly prepared tissue. Different ionization pathways are observed when analyzing fixed tissue compared with fresh, and this change was found to be associated with formalin buffers employed in fixation protocols. The ability to analyze lipids directly from formalin fixed tissue opens up new doors in the investigation of disease profiles. Pathologic specimens taken for histologic investigation can be analyzed by MALDI-MS to provide greater information on the involvement of lipids in diseased tissue.     

Applications of condensed matter understanding to medical tissues and disease progression: Elemental analysis and structural integrity of tissue scaffolds

The investigations reported herein link tissue structure and elemental presence with issues of environmental health and disease, exemplified by uptake and storage of potentially toxic elements in the body, the osteoarthritic condition and malignancy in the breast and other soft tissues. Focus is placed on application of state-of-the-art ionizing radiation techniques, including, micro-synchrotron X-ray fluorescence (μ-SXRF) and particle-induced X-ray emission/Rutherford backscattering mapping (μ-PIXE/RBS), coherent small-angle X-ray scattering (cSAXS) and X-ray phase-contrast imaging, providing information on elemental make-up, the large-scale organisation of collagen and anatomical features of moderate and low atomic number media. For the particular situations under investigation, use of such facilities is allowing information to be obtained at an unprecedented level of detail, yielding new understanding of the affected tissues and the progression of disease.     

Removing Formaldehyde-Induced Peptidyl Crosslinks Enables Mass Spectrometry Imaging of Peptide Hormone Distributions from Formalin-Fixed Paraffin-Embedded Tissues

Linking molecular and chemical changes to human disease states depends on the availability of appropriate clinical samples, mostly preserved as formalin-fixed paraffin-embedded (FFPE) specimens stored in tissue banks. Mass spectrometry imaging (MSI) enables the visualization of the spatiotemporal distribution of molecules in biological samples. However, MSI is not effective for imaging FFPE tissues because of the chemical modifications of analytes, including complex crosslinking between nucleophilic moieties. Here we used an MS-compatible inorganic nucleophile, hydroxylamine hydrochloride, to chemically reverse inter- and intra-crosslinks from endogenous molecules. The analyte restoration appears specific for formaldehyde-reactive amino acids. This approach enabled the MSI-assisted localization of pancreatic peptides expressed in the alpha, beta, and gamma cells. Pancreatic islet-like distributions of islet hormones were observed in human FFPE tissues preserved for more than five years, demonstrating that samples from biobanks can effectively be investigated with MSI.     

Sucrose cryo‐protection facilitates imaging of whole eye sections by MALDI mass spectrometry

Sucrose is used as a cryo‐preservation agent on large mammalian eyes post formalin fixation and is shown to reduce freezing artefacts allowing the collection of 12‐µm thick sections from these large aqueous samples. The suitability of this technique for use in MALDI imaging experiments is demonstrated by the acquisition of the first images of lipid distributions within whole sagittal porcine eye sections. Copyright © 2012 John Wiley & Sons, Ltd.     

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

The in vitro study of cellular species using Raman spectroscopy has proven a powerful non-invasive modality for the analysis of cell constituents and processes. This work uses micro-Raman spectroscopy to study the chemical fixation mechanism in three human cell lines (normal skin, normal bronchial epithelium, and lung adenocarcinoma) employing fixatives that preferentially preserve proteins (formalin), and nucleic acids (Carnoy’s fixative and methanol–acetic acid). Spectral differences between the mean live cell spectra and fixed cell spectra together with principal components analysis (PCA), and clustering techniques were used to analyse and interpret the spectral changes. The results indicate that fixation in formalin produces spectral content that is closest to that in the live cell and by extension, best preserves the cellular integrity. Nucleic acid degradation, protein denaturation, and lipid leaching were observed with all fixatives and for all cell lines, but to varying degrees. The results presented here suggest that the mechanism of fixation for short fixation times is complex and dependent on both the cell line and fixative employed. Moreover, important spectral changes occur with all fixatives that have consequences for the interpretation of biochemical processes within fixed cells. The study further demonstrates the potential of vibrational spectroscopy in the characterization of complex biochemical processes in cells at a molecular level.     

Fourier transform infrared imaging analysis in discrimination studies of squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) of the oral cavity and oropharynx represents more than 95% of all malignant neoplasms in the oral cavity. Histomorphological evaluation of this cancer type is invasive and remains a time consuming and subjective technique. Therefore, novel approaches for histological recognition are necessary to identify malignancy at an early stage. Fourier transform infrared (FTIR) imaging has become an essential tool for the detection and characterization of the molecular components of biological processes, such as those responsible for the dynamic properties of tumor progression. FTIR imaging is a modern analytical technique enabling molecular imaging of a complex biological sample and is based on the absorption of IR radiation by vibrational transitions in covalent bonds. One major advantage of this technique is the acquisition of local molecular expression profiles, while maintaining the topographic integrity of the tissue and avoiding time-consuming extraction, purification, and separation steps. With this imaging technique, it is possible to obtain unique images of the spatial distribution of proteins, lipids, carbohydrates, cholesterols, nucleic acids, phospholipids, and small molecules with high spatial resolution. Analysis and visualization of FTIR imaging datasets are challenging and the use of chemometric tools is crucial in order to take advantage of the full measurement. Therefore, methodologies for this task based on the novel developed algorithm for multivariate image analysis (MIA) are often necessary. In the present study, FTIR imaging and data analysis methods were combined to optimize the tissue measurement mode after deparaffinization and subsequent data evaluation (univariate analysis and MIAs). We demonstrate that it is possible to collect excellent IR spectra from formalin-fixed paraffin-embedded (FFPE) tissue microarrays (TMAs) of OSCC tissue sections employing an optimised analytical protocol. The correlation of FTIR imaging to the morphological tissue features obtained by histological staining of the sections demonstrated that many histomorphological tissue patterns can be visualized in the colour images. The different algorithms used for MIAs of FTIR imaging data dramatically increased the information content of the IR images from squamous cell tissue sections. These findings indicate that intra-operative and surgical specimens of squamous cell carcinoma tissue can be characterized by FTIR imaging.     

High-resolution laser ablation-inductively coupled plasma-mass spectrometry imaging of cisplatin-induced nephrotoxic side effects

Two-dimensional elemental mapping (bioimaging) via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was performed on 5 μm thick formalin-fixed, paraffin-embedded kidney tissue sections from Cynomolgus monkeys administered with increasing pharmacological doses of cisplatin. Laterally resolved pixels of 1 μm were achieved, enabling elemental analysis on a (sub-)cellular level. Zones of high Pt response were observed in the renal cortex, where proximal tubules are present, the epithelium of which is responsible for partial reabsorption of cisplatin. Histopathological evaluation, of hematoxylin and eosin-stained serial sections, adjacent to the sections probed via LA-ICP-MS, revealed minimal to mild cisplatin-related lesions (<100 μm) in the renal cortex. Necrotic proximal tubules with sloughed epithelial cells in their lumen could be linked directly to the areas with the highest accumulation of cisplatin, indicating a direct link between cellular concentration and toxicity, thereby providing more insight into the mechanisms through which renal damage occurs.     

FTIR and Raman microspectroscopy of normal,benign, and malignant formalin-fixed ovarian tissues

Ovarian cancer is the sixth most common cancer among women worldwide, and mortality rates from this cancer are higher than for other gynecological cancers. This is attributed to a lack of reliable screening methods and the inadequacy of treatment modalities for the advanced stages of the disease. FTIR and Raman spectroscopic studies of formalin-fixed normal, benign, and malignant ovarian tissues have been undertaken in order to investigate and attempt to understand the underlying biochemical changes associated with the disease, and to explore the feasibility of discriminating between these different tissue types. Raman spectra of normal tissues indicate the dominance of proteins and lower contents of DNA and lipids compared to malignant tissues. Among the pathological tissues studied, spectra from benign tissues seem to contain more proteins and less DNA and lipids compared to malignant tissue spectra. FTIR studies corroborate these findings. FTIR and Raman spectra of both normal and benign tissues showed more similarities than those of malignant tissues. Cluster analysis of first-derivative Raman spectra in the 700–1700 cm⁻¹ range gave two clear groups, one corresponding to malignant and the other to normal+benign tissues. At a lower heterogeneity level, the normal+benign cluster gave three nonoverlapping subclusters, one corresponding to normal and two for benign tissues. Cluster analysis of second-derivative FTIR spectra in the combined spectral regions of 1540–1680 and 1720–1780 cm⁻¹ resulted into two clear clusters corresponding to malignant and normal+benign tissues. The cluster corresponding to normal+benign tissues produced nonoverlapping subclusters for normal and benign tissues at a lower heterogeneity level. The findings of this study demonstrate the feasibility of Raman and FTIR microspectroscopic discrimination of formalin-fixed normal, benign, and malignant ovarian tissues.     

Probing cell proliferation in the human colon using vibrational spectroscopy: a novel use of FTIR-microspectroscopy

Recently, Fourier transform infrared (FTIR)-spectroscopy has been used to monitor cell growth by several works. Conventionally, the study of cell and tissue dynamics at molecular levels is carried out through various approaches like histochemical methods, application of molecular biology and immunology. Colonic crypts display a pattern in cell growth along their height. Histologically normal sections obtained from formalin fixed biopsies of colon cancer patients were studied in the present work through vibrational spectroscopy. The evolution and development of the normal human colonic crypts manifested in Fourier transform infrared-microspectroscopy (FTIR-MSP) as spectral changes in the levels of nucleic acids, proteins, carbohydrates and lipids. The results indicate that the level of carbohydrates, nucleic acids and lipids increases only till the middle of the crypt up to which the maturation zone is restricted and thereafter decreases till the top where the cells are exfoliated. These observations are in coherence with earlier reports on crypt proliferation. We identify the normal pattern of various biochemicals along the colonic crypt based on data analyzed from FTIR-MSP. This study affords an important example of the application of microscopic vibrational spectroscopy for understanding basic cell processes from formalin fixed tissues where in vivo studies and immunological methods are not feasible.     

MALDI imaging in human skin tissue sections: focus on various matrices and enzymes

Matrix-assisted laser/desorption ionization (MALDI) mass-spectrometric imaging (MSI), also known as MALDI imaging, is a powerful technique for mapping biological molecules such as endogenous proteins and peptides in human skin tissue sections. A few groups have endeavored to apply MALDI-MSI to the field of skin research; however, a comprehensive article dealing with skin tissue sections and the application of various matrices and enzymes is not available. Our aim is to present a multiplex method, based on MALDI-MSI, to obtain the maximum information from skin tissue sections. Various matrices were applied to skin tissue sections: (1) 9-aminoacridine for imaging metabolites in negative ion mode; (2) sinapinic acid to obtain protein distributions; (3) α-cyano-4-hydroxycinnamic acid subsequent to on-tissue enzymatic digestion by trypsin, elastase, and pepsin, respectively, to localize the resulting peptides. Notably, substantial amounts of data were generated from the distributions retrieved for all matrices applied. Several primary metabolites, e.g. ATP, were localized and subsequently identified by on-tissue postsource decay measurements. Furthermore, maps of proteins and peptides derived from on-tissue digests were generated. Identification of peptides was achieved by elution with different solvents, mixing with α-cyano-4-hydroxycinnamic acid, and subsequent tandem mass spectrometry (MS/MS) measurements, thereby avoiding on-tissue MS/MS measurements. Highly abundant peptides were identified, allowing their use as internal calibrants in future MALDI-MSI analyses of human skin tissue sections. Elastin as an endogenous skin protein was identified only by use of elastase, showing the high potential of alternative enzymes. The results show the versatility of MALDI-MSI in the field of skin research. This article containing a methodological perspective depicts the basics for a comprehensive comparison of various skin states.

Raman mapping and FTIR imaging of lung tissue: congenital cystic adenomatoid malformation

Congenital cystic adenomatoid malformations (CCAMs) are benign masses of non-functional lung tissue developing from an overgrowth of the terminal bronchioles with subsequent suppressing of alveolar growth. For the first time CCAMs are studied by Raman mapping and Fourier transform infrared (FTIR) imaging. Both vibrational spectroscopic methods are able to analyze the biochemical composition of tissues and their pathological changes at the molecular level. Cryosections were prepared on calcium fluoride substrates from CCAMs and from normal lung tissue of two infant patients who underwent surgery. Raman maps were collected at a step size of 100 microm in order to assess the whole tissue section and at a smaller step size of 10 microm in order to resolve details in selected areas. FTIR images were collected in the macroscopic and microscopic modes. Data sets were segmented by cluster analysis and the mean spectra of each cluster were compared. At low lateral resolution a lower red blood cell content and higher lipid content were found in CCAMs than in normal lung tissue. At higher lateral resolution, accumulations of lipids and glycogen were identified in CCAMs. The lipid aggregates contain a high concentration of phosphatidylcholine. It is discussed how the combined application of Raman mapping and FTIR imaging might improve the differential diagnosis of lung malformations and how both modalities might be applied to other bioanalytical and biomedical problems in the future.     

Combination of FTIR spectral imaging and chemometrics for tumour detection from paraffin-embedded biopsies

FTIR spectral imaging was applied on formalin-fixed paraffin-embedded biopsies from colon and skin cancerous lesions. These samples were deposited onto different substrates (zinc selenide and calcium fluoride respectively) and embedded using two types of paraffin. Formalin fixation followed by paraffin embedding is the gold standard in tissue storage. It can preserve molecular structures and it is compatible with immunohistochemistry. However, paraffin absorption bands are significant in the mid-infrared region and can mask some molecular vibrations of the tissue. Direct data processing was applied on spectral images without any chemical dewaxing of the tissues. Extended Multiplicative Signal Correction was used to correct the spectral contribution from paraffin. For this purpose, the signal of paraffin was modelled using Principal Component Analysis and paraffin spectra were removed from the raw images based on an outlier detection. Then, pseudo-colour images were computed by K-means clustering in order to highlight histological structures of interest. This robust chemometrics methodology was applied on the two samples. Tumour areas were successfully demarcated from the rest of the tissue in both colon and skin independently of the embedding material and of the substrate.     

The Effects of ex vivo Handling Procedures on the Near-Infrared Raman Spectra of Normal Mammalian Tissues

Recent studies in the literature have investigated the feasibility of tissue diagnostics based on Raman spectroscopy. The majority of these compare the ex vivo spectra of normal and diseased tissue. Due to the time lapse between tissue excision and spectroscopic examination, samples must be frozen or otherwise preserved to maintain their native biochemical states. In order to establish optimum procedures for ex vivo Raman spectroscopy of tissue, the effects of tissue drying, formalin fixing, snap freezing, tissue freezing in optimal cutting temperature (OCT) medium and extended post-thaw durations were studied to determine if any of these handling procedures introduced spectral artifacts. Experiments on representative tissues indicated that tissue heating due to the excitation light did not change the spectra significantly. With minor exceptions, OCT and formalin did not contaminate tissue spectra, so that samples stored for histological examination could also be studied with Raman spectroscopy. Tissue dehydration caused disruption of the protein vibrational modes, which caused spectral artifacts. It is concluded that ex vivo tissue samples should be frozen in OCT. Prior to spectral analysis, the tissue should then be acclimatized at room temperature in phosphate-buffered saline (PBS) and immersed in PBS during spectroscopic examination.     

Gold internal standard correction for elemental imaging of soft tissue sections by LA-ICP-MS: element distribution in eye microstructures

Laser ablation coupled to inductively coupled plasma mass spectrometry has been developed for the elemental imaging of Mg, Fe and Cu distribution in histological tissue sections of fixed eyes, embedded in paraffin, from human donors (cadavers). This work presents the development of a novel internal standard correction methodology based on the deposition of a homogeneous thin gold film on the tissue surface and the use of the ¹⁹⁷Au⁺ signal as internal standard. Sample preparation (tissue section thickness) and laser conditions were carefully optimized, and internal normalisation using ¹⁹⁷Au⁺ was compared with ¹³C⁺ correction for imaging applications. ²⁴Mg⁺, ⁵⁶Fe⁺ and ⁶³Cu⁺ distributions were investigated in histological sections of the anterior segment of the eye (including the iris, ciliary body, cornea and trabecular meshwork) and were shown to be heterogeneously distributed along those tissue structures. Reproducibility was assessed by imaging different human eye sections from the same donor and from ten different eyes from adult normal donors, which showed that similar spatial maps were obtained and therefore demonstrate the analytical potential of using ¹⁹⁷Au⁺ as internal standard. The proposed analytical approach could offer a robust tool with great practical interest for clinical studies, e.g. to investigate trace element distribution of metals and their alterations in ocular diseases.

A study of the effect of formalin preservation on normal and cancerous breast tissues using small angle X-ray scattering (SAXS)

Small angle X-ray scattering (SAXS) has the ability to provide information on a molecular and supra-molecular scale from biological tissue specimens. It has been postulated that this information will be useful in providing histopathological diagnoses for certain diseases of the breast. In this category, we include cancer, a major health problem for a number of populations around the world. So far studies in our group have been made using flash-frozen tissue samples. This limits the range and ease of use of the technique. If we were able to obtain the same information from preserved tissues then a more extensive use of SAXS diagnosis would be possible. Here we report on the first investigations into this possibility. In the research reported in this paper, 84 human breast biopsies including cancer and normal tissues were obtained from human patients. Small angle scatter data were collected at station 2.1 of the SRS at the Daresbury Laboratory, UK using a beam size of 0.25 mm² at the sample and a wavelength of 1.54 Å. The sample to detector distance was 2000 mm.The results verify that there is a quantifiable difference between the scatter curves from flash-frozen cancer and normal breast tissue in the range of scatter vector Q between 0.4 and 0.7 nm⁻¹. After preserving the tissues in formalin, the difference between the normal and cancerous tissues is less marked. The preservation of the tissue in formalin can essentially mask the effects that disease would have on the tissue supra-molecular structure rendering the preserved specimens of less useful for this histopathology technique.     

Application of Deuteporfin in the Metastatic Lymph Node Mapping of Pancreatic Cancer: An in vivo Study

For most cancer patients, the presence of metastatic lymph nodes usually indicates regional recurrence and poor prognosis. Therefore, lymph node mapping is a requisite for disease staging, prognosis prediction and decision making in the treatment of cancer. Deuteporfin, a second‐generation photosensitizer, has a maximum excitation wavelength that can reach the near infrared (NIR) region (650–700 nm). We aimed to take advantage of these aspects of deuteporfin and use it as a fluorescent probe for metastatic lymph node mapping in vivo using NIR fluorescent imaging. In our study, we further investigated whether a photosensitizer could be used as a tracer for metastatic lymph node mapping of pancreatic cancer based on previous reports. Compared to normal tissues, tumor tissues including primary tumors and metastatic lymph nodes had a higher uptake ability of deuteporfin (P < 0.05). Our research confirmed this targeting property of deuteporfin using in vivo fluorescent imaging. Consistent with observations from in vivo imaging experiments, frozen sections of metastatic lymph nodes intuitively displayed significantly higher and wider distributions of deuteporfin than normal sections.