Fixation protocols for subcellular imaging by synchrotron‐based Fourier transform infrared microspectroscopy

Gazi, Ehsan; Dwyer, John; Lockyer, Nicholas P.; Miyan, Jaleel; Gardner, Peter; Hart, Claire A.; Brown, Michael D.; Clarke, Noel W.

doi:10.1002/bip.20167

Cited by 131 publications

(130 citation statements)

References 11 publications

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“…[22][23][24] The use of ethanol in both H&E and Pap staining procedures would explain the decrease in the intensity of these two peaks. 15,16,25 This is further confirmed by the fact that physical removal of membrane and cytoplasmic lipids caused a reduction in the intensity of these two peaks. 6 On the other hand, we did not see any differences in the bands around 2870 and 2960 cm À1 , which originate from the CH 3 groups not only of lipids and proteins, but also of nucleic acids.…”

Section: Discussionmentioning

confidence: 67%

“…So far, FTIR spectroscopy using synchrotron or bench-top spectrometers has concentrated on the study of dried or fixed cells, 15,16 and tissue either frozen, 17,18 in paraffin, 18,19 deparaffinised, 3,20 or prepared by touch-imprint cytology. 21 However, in all these cases, samples had been studied without prior staining.…”

Section: Discussionmentioning

confidence: 99%

“…It has been described previously that ethanol may remove lipids that have not been appropriately fixed by formalin. 16 While the fixation protocol of cells growing on MirrIR low-e microscope slides was the same as the fixation protocol for cytospins and smears, it could be argued that fixation of lung cancer cells might have been more successful in the case of cytospin and smear rather than in the case of cancer cells growing on MirrIR low-e microscope slides.…”

Section: Discussionmentioning

confidence: 99%

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Synchrotron-based FTIR spectra of stained single cells. Towards a clinical application in pathology

Pijanka

Sockalingum

Köhler

et al. 2010

Laboratory Investigation

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Over the last few years, FTIR spectroscopy has become a potential analytical method in tissue and cell studies for cancer diagnosis. This has opened a way towards clinical applications such as a tool that would scan samples to assess the presence or absence of malignant cells in biopsies, or as an aid to help pathologists to better characterise those cells that are suspicious but not diagnostic for cancer. The latter application has the problem that in order to assess these cells pathologists would have already dealt with stained samples. Therefore, it is important to understand how staining would affect the spectra of cells. To this purpose, we have conducted this study in order to clarify, first, how haematoxylin and eosin (H&E) and Papanicolau (Pap) stainings affect the spectra of single cells and, second, whether FTIR spectroscopy could differentiate between stained lung cancer cells and their normal counterparts. Furthermore, different cell preparations (cytospin, and smear) used in cytological diagnosis were assessed. Experiments performed using a bright infrared (IR) source (synchrotron) showed that both H&E and Pap staining induced marked changes in the lipid and amide-II band regions. Despite this, FTIR spectroscopy of already stained cells is capable of differentiating between lung cancer cells and their normal counterparts. The clinical applications of this methodology are discussed.

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Section: Discussionmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Synchrotron-based FTIR spectra of stained single cells. Towards a clinical application in pathology

Pijanka

Sockalingum

Köhler

et al. 2010

Laboratory Investigation

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“…Formalin is a widespread chemical xative that causes crosslinking between the aldehyde and the primary and secondary amine groups of cellular proteins (via methylene bridges), creating a gel and thus retaining the cellular constituents in a close to in vivo relationship. While no considerable effects of this xation procedure have been observed in the IR spectra of single cells, 55 it is expected to induce some reduction in vibrational band intensity, resulting from the disruption of lipid assembly and from changes in protein conformation (mainly reected in the amide I and II bands). Nevertheless, formalin xation has been found to be the best method for preserving cellular integrity with a weak impact on the overall molecular content and yielding the spectral prole closest to that observed in the live state, while hardly affecting discrimination between cells under distinct conditions (namely control and drugexposed).…”

Section: Resultsmentioning

confidence: 99%

Chemotherapeutic response to cisplatin-like drugs in human breast cancer cells probed by vibrational microspectroscopy

et al. 2016

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Studies of drug-cell interactions in cancer model systems are essential in the preclinical stage of rational drug design, which relies on a thorough understanding of the mechanisms underlying cytotoxic activity and biological effects, at a molecular level.This study aimed at applying complementary vibrational spectroscopy methods to evaluate the cellular impact of two Pt(II) and Pd(II) dinuclear chelates with spermine (Pt 2 Spm and Pd 2 Spm), using cisplatin (cis-Pt(NH 3 ) 2 Cl 2 ) as a reference compound. Their effects on cellular metabolism were monitored in a human triple-negative metastatic breast cancer cell line (MDA-MB-231) by Raman and synchrotron-radiation infrared microspectroscopies, for different drug concentrations (2-8 mM) at 48 h exposure.Multivariate data analysis was applied (unsupervised PCA), unveiling drug-and concentration-dependent effects: apart from discrimination between control and drugtreated cells, a clear separation was obtained for the different agents studiedmononuclear vs. polynuclear, and Pt(II) vs. Pd(II). Spectral biomarkers of drug action were identified, as well as the cellular response to the chemotherapeutic insult. The main effect of the tested compounds was found to be on DNA, lipids and proteins, the Pd(II) agent having a more significant impact on proteins while its Pt(II) homologue affected the cellular lipid content at lower concentrations, which suggests the occurrence of distinct and unconventional pathways of cytotoxicity for these dinuclear polyamine complexes. Raman and FTIR microspectroscopies were confirmed as powerful non-invasive techniques to obtain unique spectral signatures of the biochemical impact and physiological reaction of cells to anticancer agents.

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“…Fourier transform infrared (FTIR) spectroscopy has been demonstrated as a potential tool for disease diagnostics in tissue [1][2][3] and in its microscopical form, for cellular analysis [4][5][6][7][8][9][10][11][12]. The use of synchrotron sources and attenuated total reflection imaging have achieved subcellular resolution thus promising a 20 powerful technique for the analysis of the biochemical origin of disease [7,13,14].…”

mentioning

confidence: 99%

Reflection contributions to the dispersion artefact in FTIR spectra of single biological cells

Bassan

Byrne²,

Lee

et al. 2009

Analyst

118

112

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Fourier transform infrared spectra of a single cell in transflection geometry are seen to vary significantly with position on the cell, showing a distorted derivative-like lineshape in the region of the optically dense nucleus. A similar behaviour is observable in a model system of the protein albumin doped in a potassium bromide disk. It is demonstrated that the spectrum at 10 any point is a weighted sum of the sample reflection and transmission and that the dominance of the reflection spectrum in optically dense regions can account for some of the spectral distortions previously attributed to dispersion artefacts. Rather than being an artefact, the reflection contribution is ever present in transflection spectra and it is further demonstrated that the reflection characteristics can be used for cellular mapping.

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Fixation protocols for subcellular imaging by synchrotron‐based Fourier transform infrared microspectroscopy

Abstract: Synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy is a powerful bioanalytical

Cited by 131 publications

References 11 publications

Synchrotron-based FTIR spectra of stained single cells. Towards a clinical application in pathology

Synchrotron-based FTIR spectra of stained single cells. Towards a clinical application in pathology

Chemotherapeutic response to cisplatin-like drugs in human breast cancer cells probed by vibrational microspectroscopy

Reflection contributions to the dispersion artefact in FTIR spectra of single biological cells

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