Surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) in label-free characterization of erythrocyte membranes and extracellular vesicles at the nano-scale and molecular level.

The manuscript presents the potential of surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS) for label-free characterization of extracellular microvesicles (EVs) and their isolated membranes derived from red blood cells (RBCs) at the nanoscale and at the single-molecule level, providing detection of a few individual amino acids, protein and lipid membrane compartments. The study shows future directions for research, such as investigating the use of the mentioned techniques for the detection and diagnosis of diseases. We demonstrate that SERS and TERS are powerful techniques for identifying the biochemical composition of EVs and their membranes, allowing the detection of small molecules, lipids, and proteins. Furthermore, extracellular vesicles released from red blood cells (REVs) can be broadly classified into exosomes, microvesicles, and apoptotic bodies, based on their size and biogenesis pathways. Our study specifically focuses on microvesicles that range from 100 to 1000 nanometres in diameter, as presented in AFM images. Using SERS and TERS spectra obtained for REVs and their membranes, we were able to characterize the chemical and structural properties of microvesicle membranes with high sensitivity and specificity. This information may help better distinguish and categorize different types of EVs, leading to a better understanding of their functions and potential biomedical applications.

Secondary structure alterations of RBC assessed by FTIR-ATR in correlation to 2,3-DPG levels in ApoE/LDLR-/- Mice.

In the present study, we characterized the secondary structure alterations of intact red blood cells (RBCs) cytosol with special attention to the sex-related alterations in 8- and 24-week-old female and male ApoE/LDLR-/- mice, compared to age-matched female and male C57BL/6J control animals. Results were obtained with previously established methodology based on Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Additionally, we evaluated 2,3-DPG levels in the RBCs and showed its potential link to the hemoglobin (Hb) secondary structure alterations. Considering Hb structure alterations probed by FTIR-ATR, the ratio of turns to α-helices in 8-week-old ApoE/LDLR-/- mice suggested more pronounced secondary structure alterations within the RBCs than in the age-matched control. Sex-related differences were observed solely in 24-week-old male ApoE/LDLR-/- mice, which showed statistically significant increase in the secondary structure alterations compared to 24-week-old female ApoE/LDLR-/- mice. Similar to the secondary structure alterations, no sex-related differences were observed in the levels of 2,3-DPG in RBCs, except for 24-week-old male ApoE/LDLR-/- mice, which showed significantly higher levels compared to the age-matched female ApoE/LDLR-/- mice. Considering the age-related alterations, we observed significant increases in the intracellular 2,3-DPG of RBCs with animals' age in all studied groups, except for female ApoE/LDLR-/- mice, where a significant difference was not reported. This suggests the clear correlation between secondary structure of Hb alterations and 2,3-DPG levels for male and female murine RBC and proves a higher resistance of older female RBCs to the secondary structure changes with progression of atherosclerosis. Moreover, it may be concluded that higher 2,3-DPG levels in RBCs occurred in response to the secondary structure alterations of Hb in ApoE/LDLR-/- mice.

Label-free testing strategy to evaluate packed red blood cell quality before transfusion to leukemia patients.

Patients worldwide require therapeutic transfusions of packed red blood cells (pRBCs), which is applied to the high-risk patients who need periodic transfusions due to leukemia, lymphoma, myeloma and other blood diseases or disorders. Contrary to the general hospital population where the transfusions are carried out mainly for healthy trauma patients, in case of high-risk patients the proper quality of pRBCs is crucial. This leads to an increased demand for efficient technology providing information on the pRBCs alterations deteriorating their quality. Here we present the design of an innovative, label-free, noninvasive, rapid Raman spectroscopy-based method for pRBCs quality evaluation, starting with the description of sample measurement and data analysis, through correlation of spectroscopic results with reference techniques' outcomes, and finishing with methodology verification and its application in clinical conditions. We have shown that Raman spectra collected from the pRBCs supernatant mixture with a proper chemometric analysis conducted for a minimum one ratio of integral intensities of the chosen Raman marker bands within the spectrum allow evaluation of the pRBC quality in a rapid, noninvasive, and free-label manner, without unsealing the pRBCs bag. Subsequently, spectroscopic data were compared with predefined reference values, either from pRBCs expiration or those defining the pRBCs quality, allowing to assess their utility for transfusion to patients with acute myeloid leukemia (AML) and lymphoblastic leukemia (ALL).

Sex-Specific Differences of Adenosine Triphosphate Levels in Red Blood Cells Isolated From ApoE/LDLR Double-Deficient Mice.

In this study for the first time, we investigated the correlation between sex-specific differences in adenosine triphosphate (ATP) levels in red blood cells (RBCs) and their mechanical, biochemical, and morphological alterations during the progression of atherosclerosis in ApoE/LDLR double-deficient (ApoE/LDLR-/-) mice. Our results indicate that both sex and age affect alterations in RBCs of both ApoE/LDLR-/- and C57BL/6J mice. When compared with male RBCs, female RBCs were characterized by lower basal ATP and mean corpuscular hemoglobin concentration (MCHC), higher hemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), deformability, and phosphatidylserine (PS) exposure levels, regardless of age in both, ApoE/LDLR-/- and C57BL/6J mice. ApoE/LDLR-/- mice compared with age-matched controls showed lower basal ATP levels regardless of age and sex. Intracellular ATP level of RBCs was decreased solely in senescent female C57BL/6J mice, while it was elevated in males. Basal extracellular ATP levels were 400 times lower than corresponding intracellular level. In conclusion, basal ATP levels, RBC morphology, deformability, PS exposure levels alterations are sex-dependent in mice. Changes in basal ATP levels were correlated with PS exposure and trends of changes in MCV. Trends of changes of the most RBC parameters were similar in both sexes of ApoE/LDLR-/- mice compared with age-matched controls; however, their kinetics and levels vary greatly between different stages of disease progression.

Temporal relationship between systemic endothelial dysfunction and alterations in erythrocyte function in a murine model of chronic heart failure.

AIMS: Endothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterize the relationship between the development of peripheral ED and the occurrence of structural nanomechanical and biochemical changes in red blood cells (RBCs). METHODS AND RESULTS: Systemic ED was detected in vivo in 8-month-old Tgαq*44 mice, as evidenced by impaired acetylcholine-induced vasodilation in the aorta and increased endothelial permeability in the brachiocephalic artery. ED in the aorta was associated with impaired nitric oxide (NO) production in the aorta and diminished systemic NO bioavailability. ED in the aorta was also characterized by increased superoxide and eicosanoid production. In 4- to 6-month-old Tgαq*44 mice, RBC size and membrane composition displayed alterations that did not result in significant changes in their nanomechanical and functional properties. However, 8-month-old Tgαq*44 mice presented greatly accentuated structural and size changes and increased RBC stiffness. In 12-month-old Tgαq*44 mice, the erythropathy was featured by severely altered RBC shape and elasticity, increased RDW, impaired RBC deformability, and increased oxidative stress (gluthatione (GSH)/glutathione disulfide (GSSG) ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, coincubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor [2(S)-amino-6-boronohexanoic acid]. CONCLUSION: In the Tgαq*44 murine model of HF, systemic ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic ED in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic ED in HF.

High-Resolution Fourier Transform Infrared (FT-IR) Spectroscopic Imaging for Detection of Lung Structures and Cancer-Related Abnormalities in a Murine Model.

Label-free molecular imaging is a promising utility to study tissues in terms of the identification of their compartments as well as chemical features and alterations induced by disease. The aim of this work was to assess if higher magnification of optics in the Fourier transform infrared (FT-IR) microscope coupled with the focal plane detector resulted in better resolution of lung structures and if the histopathological features correlated with clustering of spectral images. FT-IR spectroscopic imaging was performed on paraffinized lung tissue sections from mice with optics providing a total magnification of 61× and 36×. Then, IR images were subjected to unsupervised cluster analysis and, subsequently, cluster maps were compared with hematoxylin and eosin staining of the same tissue section. Based on these results, we observed minute features such as cellular compartments in single alveoli and bronchiole, blood cells and megakaryocytes in a vessel as well as atelectasis of the lung. In the case of the latter, differences in composition were also noted between the tissue from the non-cancerous and cancerous specimen. This study demonstrated the ability of high-definition FT-IR imaging to evaluate the chemical features of well-resolved lung structures that could complement the histological examination widely used in animal models of disease.

Tauroursodeoxycholic Acid (TUDCA)-Lipid Interactions and Antioxidant Properties of TUDCA Studied in Model of Photoreceptor Membranes.

Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid containing taurine conjugated with the ursodeoxycholic acid (UDCA), has been known and used from ancient times as a therapeutic compound in traditional Chinese medicine. TUDCA has recently been gaining significant interest as a neuroprotective agent, also exploited in the visual disorders. Among several mechanisms of TUDCA's protective action, its antioxidant activity and stabilizing effect on mitochondrial and plasma membranes are considered. In this work we investigated antioxidant activity of TUDCA and its impact on structural properties of model membranes of different composition using electron paramagnetic resonance spectroscopy and the spin labeling technique. Localization of TUDCA molecules in a pure POPC bilayer has been studied using a molecular dynamics simulation (MD). The obtained results indicate that TUDCA is not an efficient singlet oxygen (1O2 (1Δg)) quencher, and the determined rate constant of its interaction with 1O2 (1Δg) is only 1.9 × 105 M-1s-1. However, in lipid oxidation process induced by a Fenton reaction, TUDCA reveals substantial antioxidant activity significantly decreasing the rate of oxygen consumption in the system studied. In addition, TUDCA induces slight, but noticeable changes in the polarity and fluidity of the investigated model membranes. The results of performed MD simulation correspond very well with the experimental results.

Spectroscopic Signature of Red Blood Cells in a D-Galactose-Induced Accelerated Aging Model.

This work presents a semi-quantitative spectroscopic approach, including FTIR-ATR and Raman spectroscopies, for the biochemical analysis of red blood cells (RBCs) supported by the biochemical, morphological and rheological reference techniques. This multi-modal approach provided the description of the RBC alterations at the molecular level in a model of accelerated aging induced by administration of D-galactose (D-gal), in comparison to natural aging. Such an approach allowed to conclude that most age-related biochemical RBC membrane changes (a decrease in lipid unsaturation and the level of phospholipids, or an increase in acyl chain shortening) as well as alterations in the morphological parameters and RBC deformability are well reflected in the D-gal model of accelerated aging. Similarly, as in natural aging, a decrease in LDL level in blood plasma and no changes in the fraction of glucose, creatinine, total cholesterol, HDL, iron, or triglycerides were observed during the course of accelerated aging. Contrary to natural aging, the D-gal model led to an increase in cholesterol esters and the fraction of total esterified lipids in RBC membranes, and evoked significant changes in the secondary structure of the membrane proteins. Moreover, a significant decrease in the phosphorous level of blood plasma was specific for the D-gal model. On the other hand, natural aging induced stronger changes in the secondary structures of the proteins of the RBCs' interior. This work proves that research on the aging mechanism, especially in circulation-related diseases, should employ the D-gal model with caution. Nonetheless, the D-gal model enables to imitate age-related rheological alterations in RBCs, although they are partially derived from different changes observed in the RBC membrane at the molecular level.

Probing Heme Active Sites of Hemoglobin in Functional Red Blood Cells Using Resonance Raman Spectroscopy.

The UV-vis absorption, Raman imaging, and resonance Raman (rR) spectroscopy methods were employed to study cyanohemoglobin (HbCN) adducts inside living functional red blood cells (RBCs). The cyanide ligands are especially optically sensitive probes of the active site environment of heme proteins. The rR studies of HbCN and its isotopic analogues (13CN-, C15N-, and 13C15N-), as well as a careful deconvolution of spectral data, revealed that the ν(Fe-CN) stretching, δ(Fe-CN) bending, and ν(C≡N) stretching modes occur at 454, 382, and 2123 cm-1, respectively. Interestingly, while the ν(Fe-CN) modes exhibit the same frequencies in both the isolated and RBC-enclosed hemoglobin molecules, small frequency differences are observed in the δ(Fe-CN) bending modes and the values of their isotopic shifts. These studies show that even though the overall tilted conformation of the Fe-C≡N fragment in the isolated HbCN is preserved in the HbCN enclosed within living cells, there is a small difference in the degree of distortion of the Fe-C≡N fragment. The slight changes in the ligand geometry can be reasonably attributed to the high ordering and tight packing of Hb molecules inside RBCs.

Age-related and atherosclerosis-related erythropathy in ApoE/LDLR-/- mice.

In this work we applied a multimodal approach to define the age- and atherosclerosis-related biochemical and functional alterations in red blood cells (RBCs) in ApoE/LDLR-/- mice. Our results revealed that age-related changes in RBCs, such as decreases in RBC deformability and mean height, were more pronounced in ApoE/LDLR-/- mice than in age-matched control mice (C57BL/6J). The decreases in phospholipid content and level of lipid unsaturation were accompanied by an increase in cholesterol esters and esterified lipids in RBC membranes in aged C57BL/6J mice. The age-related decrease in the phospholipid content was more pronounced in ApoE/LDLR-/- mice. In contrast, the increase in the total lipid content in RBC membranes occurred only in ApoE/LDLR-/- mice with advanced atherosclerosis. The age-related alterations also included a decrease in the ratio of turns to α-helices in the secondary structure of hemoglobin (Hb) inside intact RBCs. On the other hand, an increase in the ratio of unordered conformations to α-helices of Hb was observed only in ApoE/LDLR-/- mice and occurred already at the age of 5-weeks. This was related to hypercholesterolemia and resulted in an increased oxygen-carrying capacity. In conclusion, progressive mechanical and functional alterations of RBCs in aged ApoE/LDLR-/- mice were more pronounced than in age-matched C57BL/6J mice. Although, several biochemical changes in RBCs in aged ApoE/LDLR-/- mice recapitulated age-dependent changes observed in control mice, some biochemical features of RBC membranes attributed to hypercholesterolemia were distinct and could contribute to the accelerated deterioration of RBC function in ApoE/LDLR-/- mice.

Irreversible alterations in the hemoglobin structure affect oxygen binding in human packed red blood cells.

The ability of hemoglobin (Hb) to transport respiratory gases is directly linked to its quaternary structure properties and reversible changes between T (tense) and R (relax) state. In this study we demonstrated that packed red blood cells (pRBCs) storage resulted in a gradual increase in the irreversible changes in the secondary and quaternary structures of Hb, with subsequent impairment of the T↔R transition. Such alteration was associated with the presence of irreversibly settled in the relaxed form, quaternary structure of Hb, which we termed R'. On the secondary structure level, disordered protein organization involved formation of ß-sheets and a decrease in α-helices related to the aggregation process stabilized by strong intermolecular hydrogen bonding. Compensatory changes in RBCs metabolism launched to preserve reductive microenvironment were disclosed as an activation of nicotinamide adenine dinucleotide phosphate (NADPH) production and increased reduced to oxidized glutathione (GSH/GSSG) ratio. For the first time we showed the relationship between secondary structure changes and the occurrence of newly discovered R', which through an artificial increase in oxyhemoglobin level altered Hb ability to bind and release oxygen.

Comparison of standard and HD FT-IR with multimodal CARS/TPEF/SHG/FLIMS imaging in the detection of the early stage of pulmonary metastasis of murine breast cancer.

Lungs, due to their high oxygen availability and vascularization, are an ideal environment for cancer cell migration, metastasis and tumour formation. These processes are directly connected with extracellular matrix (ECM) remodelling, resulting from cancer cell infiltration and preparation of the environment suitable for tumour growth. Herein, we compare the potential of fast, label-free and non-destructive methods of Fourier-transform infrared spectroscopy (FT-IR) in standard and high definition (HD) modes with nonlinear coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF) and a fluorescence lifetime imaging (FLIM) technique for lung metastasis detection. We show their potential in the detection of lung macrometastasis, in which we already observed the ECM remodelling. The CARS image revealed a dense cell fraction typical of ECM remodeling and reduction of the TPEF signal together with an increase of fluorescence lifetime predominantly due to NAD(P)H suggesting metabolic changes in the metastatic foci. FT-IR spectroscopy allowed not only for macrometastasis detection but also their stage definition based mainly on the analysis of proteins, RNA and glycogen fractions. The multimodal approach additionally suggested partial enzymatic degradation of elastin in ECM and collagen remodelling.

Probing the structure-function relationship of hemoglobin in living human red blood cells.

Hemoglobin (Hb) is a key component of respiratory system and as such plays important role in human physiology. The studies of Hb's structure and functions are usually performed on cell-free protein; however, it has been shown that there are functionally relevant differences between isolated Hb and Hb present inside red blood cells (RBCs). It is clear that new experimental approaches are needed to understand the origin of these differences and to gain insight into the structure-function relationship of Hb within intact living cells. In this work we present a novel application of Resonance Raman spectroscopy to study heme active site of different forms of human Hb within living RBCs using laser excitation lines in resonance with their Soret absorption bands. These studies revealed that there are no significant changes in the disposition of the Fe-O-O fragment or the Fe-NHis linkage for Hb molecules enclosed in RBCs and these in free isolated states. However, some changes in the orientation of the heme vinyl groups were observed which might account for the differences in the protein activity and ligand affinity. This work highlights importance of protein-based studies and presents a new opportunity to translate these results to physiological cell systems.

Temporal sequence of the human RBCs' vesiculation observed in nano-scale with application of AFM and complementary techniques.

Based on the multimodal characterization of human red blood cells (RBCs), the link between the storage-related sequence of the nanoscale changes in RBC membranes in the relation to their biochemical profile as well as mechanical and functional properties was presented. On the background of the accumulation of RBCs waste products, programmed cell death and impaired rheological properties, progressive alterations in the RBC membranes including changes in their height and diameter as well as the in situ characterization of RBC-derived microparticles (RMPs) on the RBCs surface were presented. The advantage of atomic force microscopy (AFM) in RMPs visualization, even at the very early stage of vesiculation, was shown based on the results revealed by other reference techniques. The nanoscale characterization of RMPs was correlated with a decrease in cholesterol and triglycerides levels in the RBC membranes, proving the link between the lipids leakage from RBCs and the process of vesiculation.

Tracking Extracellular Matrix Remodeling in Lungs Induced by Breast Cancer Metastasis. Fourier Transform Infrared Spectroscopic Studies.

This work focused on a detailed assessment of lung tissue affected by metastasis of breast cancer. We used large-area chemical scanning implemented in Fourier transform infrared (FTIR) spectroscopic imaging supported with classical histological and morphological characterization. For the first time, we differentiated and defined biochemical changes due to metastasis observed in the lung parenchyma, atelectasis, fibrous, and muscle cells, as well as bronchi ciliate cells, in a qualitative and semi-quantitative manner based on spectral features. The results suggested that systematic extracellular matrix remodeling with the progress of the metastasis process evoked a decrease in the fraction of the total protein in atelectasis, fibrous, and muscle cells, as well as an increase of fibrillar proteins in the parenchyma. We also detected alterations in the secondary conformations of proteins in parenchyma and atelectasis and changes in the level of hydroxyproline residues and carbohydrate moieties in the parenchyma. The results indicate the usability of FTIR spectroscopy as a tool for the detection of extracellular matrix remodeling, thereby enabling the prediction of pre-metastatic niche formation.

Multimodal detection and analysis of a new type of advanced Heinz body-like aggregate (AHBA) and cytoskeleton deformation in human RBCs.

A new type of aggregate, formed in human red blood cells (RBCs) in response to glutaraldehyde treatment, was discovered and analyzed with the classical and advanced biomolecular imaging techniques. Advanced Heinz body-like aggregates (AHBA) formed in a single human RBC are characterized by a higher level of hemoglobin (Hb) degradation compared to typical Heinz bodies, which consist of hemichromes. The complete destruction of the porphyrin structure of Hb and the aggregation of the degraded proteins in the presence of Fe3+ ions are observed. The presence of such aggregated, highly degraded proteins inside RBCs, without cell membrane destruction, has been never reported before. For the first time the spatial differentiation of two kinds of protein mixtures inside a single RBC, with different phenylalanine (Phe) conformations, is visualized. The non-resonant Raman spectra of altered RBCs with AHBA are characterized by the presence of a strong band located at 1037 cm-1, which confirms that glutaraldehyde interacts strongly with Phe. The shape-shifting of RBCs from a biconcave disk to a spherical structure and sinking of AHBA to the bottom of the cell are observed. Results reveal that the presence of AHBA should be considered when fixing RBCs and indicate the analytical potential of Raman spectroscopy, atomic force microscopy and scanning near-field optical microscopy in AHBA detection and analysis.

FTIR, Raman and AFM characterization of the clinically valid biochemical parameters of the thrombi in acute ischemic stroke.

The significance and utility of innovative imaging techniques in arterial clot analysis, which enable far more detailed and automated analysis compared to standard methods, are presented. The examination of two types of human thrombi is shown, representing the main ischemic stroke etiologies: fibrin-predominant clot of large vessel origin and red blood cells-rich clot of cardioembolic origin. The synergy effect of Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy (RS) and atomic force microscopy (AFM) techniques supported by chemometrics in comparison with reference histological staining was presented. The main advantage of such approach refers to free-label and non-destructive quantitative imaging of clinically valid, biochemical parameters in whole sample (FTIR-low resolution) and selected regions (RS-ultra-high resolution). We may include here analysis of lipid content, its distribution and total degree of unsaturation as well as analysis of protein content (mainly fibrin and hemoproteins). The AFM studies enhanced the vibrational data, showed clearly shape and thickness of clot features as well as visualized the fibrin framework. The extraordinary sensitivity of FTIR and RS imaging toward detection and discrimination of clinically valid parameters in clot confirms its applicability in assessment of thrombi origin.

An Analysis of Isolated and Intact RBC Membranes-A Comparison of a Semiquantitative Approach by Means of FTIR, Nano-FTIR, and Raman Spectroscopies.

This work presents the potential of vibrational spectroscopy, Vis and NIR Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR) in reflection and transmission modes, and nano-FTIR microscopy to study the biochemical alterations in membranes of isolated and intact red blood cells (RBCs). The main goal was to propose the best spectroscopic method which enabled following biochemical alterations in the RBC membranes and then to translate this spectroscopic signature of degradation to in situ analysis of RBCs. Two models corresponding to two distinct cases of RBC membrane conditions were employed, and they were derived from healthy and young mice and mature mice with advanced atherosclerosis. It was shown that each technique provided essential information about biochemical alterations of the isolated membranes as well as membranes in the intact RBCs, which can be used in the development of a rapid and in situ analytical technology. Finally, we proposed that the combination of macro- and nanoprobing implemented in IR spectroscopy provided a wide chemical characterization of the RBC membranes, including alterations in lipid and protein fractions. This study also examined the effect of the sample preparation to determine destructive factors influencing a spectroscopic analysis of isolated membranes and intact RBCs derived from healthy and disease-affected mice.

High and ultra-high definition of infrared spectral histopathology gives an insight into chemical environment of lung metastases in breast cancer.

Using high definition (HD) and ultra-high definition (UHD) of Fourier-transform infrared (FTIR) spectroscopic imaging, we characterized spectrally pulmonary metastases in a murine model of breast cancer comparing them with histopathological results (Hematoxylin and eosin [H&E] staining). This comparison showed excellent agreement between the methods in case of localization of metastases with size below 1 mm and revealed that label-free HD and UHD IR spectral histopathology distinguish the type of neoplastic cells. We primary focused on differentiation between metastatic foci in the pleural cavity from cancer cells present in lung parenchyma and inflamed cells present in extracellular matrix of lungs due to growing of advanced metastases. In addition, a combination of unsupervised clustering and IR imaging indicated the high sensitivity of FTIR spectroscopy to identify chemical features of small macrometastases located under the pleural cavity and during epithelial-mesenchymal transition. FTIR-based spectral histopathology was proved to detect not only phases of breast cancer metastasis to lungs but also to differentiate various origins of metastases seeded from breast cancer.