Monitoring cell cycle distributions in MCF-7 cells using near-field photothermal microspectroscopy.

Microspectroscopic techniques such as Fourier transform infrared (FTIR) have played an important role in "fingerprinting" the biochemical composition of cellular components. Based on structure and function, complex biomolecules absorb energy in the mid-infrared ( = 2–20 µm) yielding characteristic vibrational infrared (IR) spectra. However, optical detection FTIR microspectroscopy may not be suitable for IR-absorbing sample materials. Photothermal microspectroscopy (PTMS) permits the direct measurement of heat generated as a result of sample material absorbing radiation. This approach generates true absorption spectra and is implemented by interfacing a scanning probe microscope and an FTIR spectrometer. Detection is performed using a near-field ultra-miniaturized temperature sensor. Employing PTMS, IR spectra of MCF-7 cells were examined in spectral regions (900–2000 cm–1) corresponding to proteins, DNA, RNA, glycoproteins, carbohydrates, lipids, and levels of protein phosphorylation. As a cell passes through the cell cycle, its nuclear material decondenses and condenses and this has led to ambiguity as to whether the intensity of such spectral regions may be associated with the G1-, S- or G2-phases of the cell cycle. Cultured cells were tracked over a time course known to correspond to marked alterations in cell-cycle distributions, as determined using flow cytometry. Experiments were carried out in the absence or presence of lindane, a pesticide known to induce G1-arrest in MCF-7 cells. Significant (P < 0.05) elevations in spectral intensities were associated with exponentially growing cell populations, predominantly in S-phase or G2-phase, compared to more quiescent populations predominantly in G1-phase. Increases in the absorption band at 970 cm–1, associated with elevated protein phosphorylation, were observed in vibrational spectra of exponentially growing cell populations compared to those exhibiting a slowing in their growth kinetics. These results seem to suggest that intracellular bulk changes, associated with transit through the cell cycle, can be tracked using PTMS

Discrimination of human stem cells by photothermal microspectroscopy

Stem cells have great potential in clinical medicine. Sensitive methods for stem cell identification are a requirement for the development of medical interventions involving these cells. To date, a definitive stem cell marker has not been discovered. We are exploring the use of photothermal microspectroscopy (PTMS) for the purpose of stem cell characterisation and identification in human corneal epithelium. PTMS measures heat fluctuations associated with infrared radiation absorption. The technique is advantageous over existing Fourier transform infrared (FTIR) spectroscopy methods in having a spatial resolution which is not diffraction limited, thus allowing examination at a sub-cellular scale. PTMS measurements are unaffected by IR opacity of the sample, giving the method a further edge in comparison to FTIR spectroscopy. We show that PTMS spectra can be used for the characterisation of stem cells and differentiated cells in the human corneal stem cell model. We demonstrate for the first time that PTMS spectra derived from these cell types segregate into separate data clusters after principal component analysis. The predominant wavenumbers responsible for this separation appear to be associated with nucleic acid structure and function. PTMS offers great promise as a technique for stem cell identification in tissue samples where spatial resolution at the cellular scale or better is required. (C) 2008 Elsevier B.V. All rights reserved

Imaging Joule heating in a conjugated-polymer light-emitting diode using a scanning thermal microscope.

We have used a scanning thermal microscope to image Joule heating in a conjugated-polymer light-emitting diode (LED). Our LEDs had an active diameter of 100 µm, which was defined using an insulating layer of silicon nitride patterned onto the LED anode. At an average power input of 0.2 mW into the LED, we find that the center of the cathode is some 0.2 K warmer than its periphery. The observed temperature distribution across the pixel is slightly asymmetric, an effect which may be correlated with spatial inhomogeneity in the local current density across the device. We present a finite element analysis thermal model which is able to accurately describe the observed temperature distribution across the LED cathode

FTIR Microspectroscopy Coupled with Two-Class Discrimination Segregates Markers Responsible for Inter- and Intra-Category Variance in Exfoliative Cervical Cytology.

Infrared (IR) absorbance of cellular biomolecules generates a vibrational spectrum, which can be exploited as a “biochemical fingerprint” of a particular cell type. Biomolecules absorb in the mid-IR (2–20 μm) and Fourier-transform infrared (FTIR) microspectroscopy applied to discriminate different cell types (exfoliative cervical cytology collected into buffered fixative solution) was evaluated. This consisted of cervical cytology free of atypia (i.e. normal; n = 60), specimens categorised as containing low-grade changes (i.e. CIN1 or LSIL; n = 60) and a further cohort designated as high-grade (CIN2/3 or HSIL; n = 60). IR spectral analysis was coupled with principal component analysis (PCA), with or without subsequent linear discriminant analysis (LDA), to determine if normal versus low-grade versus high-grade exfoliative cytology could be segregated. With increasing severity of atypia, decreases in absorbance intensity were observable throughout the 1,500 cm−1 to 1,100 cm−1 spectral region; this included proteins (1,460 cm−1), glycoproteins (1,380 cm−1), amide III (1,260 cm−1), asymmetric (νas) PO2 − (1,225 cm−1) and carbohydrates (1,155 cm−1). In contrast, symmetric (νs) PO2 − (1,080 cm−1) appeared to have an elevated intensity in high-grade cytology. Inter-category variance was associated with protein and DNA conformational changes whereas glycogen status strongly influenced intra-category. Multivariate data reduction of IR spectra using PCA with LDA maximises inter-category variance whilst reducing the influence of intra-class variation towards an objective approach to class cervical cytology based on a biochemical profile

Non-contact micro-cantilevers detect photothermally induced vibrations that can segregate different categories of exfoliative cervical cytology.

We implemented a non-contact photo-thermo-mechanical recording method whereby a silicon nitride atomic force microscopy cantilever is placed several micrometer above the surface of samples. Samples were illuminated with infrared (IR) radiation after which, cantilever mechanical vibrations were optically sensed. Following spectrometric acquisition and Fourier transformation, true IR absorption spectra were obtained. With multivariate analysis, segregation between different categories of exfoliative cervical cytology was obtained. This approach points towards the implementation of a novel near-field system that allows IR spectral analysis without probe contamination

Characterisation of human corneal stem cells by synchrotron infrared micro-spectroscopy.

Purpose: The purpose of this study was to use high resolution synchrotron radiation-based Fourier Transform Infrared (FTIR) micro-spectroscopy coupled with multivariate analysis to investigate the characteristics of adult stem cell (SC) and transit amplifying (TA) cell populations of the human corneal epithelium. Methods: Spectra of individual SC and TA cells in situ from cryosections of human cornea were collected using a synchrotron micro-spectroscopy facility at Daresbury laboratory, UK. Multivariate analysis and Mann Whitney U tests were used to analyse the spectral data from the SC and TA cell populations. Results: There were marked differences between the median spectra of the two cell populations. This correlated with their level of differentiation and functional specialization. Multivariate (principal component) analysis revealed that the cell populations could be segregated into distinct clusters, with only slight overlap between the two cell types. Significant (p<0.05) spectral differences were found in the spectral regions associated with nucleic acid, protein and lipids. Conclusions: Synchrotron FTIR micro-spectroscopy together with principal component analysis is able to discriminate between SC and TA cell populations. Our results also suggest a small sub-population of corneal epithelial cells in the SC niche have TA cell-like characteristics. Many of the spectral differences between the SC and TA cell populations relate to differences in nucleic acid conformation

Characterization of human corneal stem cells by synchrotron infrared micro-spectroscopy.

Purpose: The purpose of this study was to use high resolution synchrotron radiation-based Fourier Transform Infrared (FTIR) micro-spectroscopy coupled with multivariate analysis to investigate the characteristics of adult stem cell (SC) and transit amplifying (TA) cell populations of the human corneal epithelium. Methods: Spectra of individual SC and TA cells in situ from cryosections of human cornea were collected using a synchrotron micro-spectroscopy facility at Daresbury laboratory, UK. Multivariate analysis and Mann Whitney U tests were used to analyse the spectral data from the SC and TA cell populations. Results: There were marked differences between the median spectra of the two cell populations. This correlated with their level of differentiation and functional specialization. Multivariate (principal component) analysis revealed that the cell populations could be segregated into distinct clusters, with only slight overlap between the two cell types. Significant (p<0.05) spectral differences were found in the spectral regions associated with nucleic acid, protein and lipids. Conclusions: Synchrotron FTIR micro-spectroscopy together with principal component analysis is able to discriminate between SC and TA cell populations. Our results also suggest a small sub-population of corneal epithelial cells in the SC niche have TA cell-like characteristics. Many of the spectral differences between the SC and TA cell populations relate to differences in nucleic acid conformation

Infrared Spectroscopy with Multivariate Analysis Potentially Facilitates the Segregation of Different Types of Prostate Cell.

The prostate gland is conventionally divided into zones or regions. This morphology is of clinical significance as prostate cancer (CaP) occurs mainly in the peripheral zone (PZ). We obtained tissue sets consisting of paraffin-embedded blocks of cancer-free transition zone (TZ) and PZ and adjacent CaP from patients (n = 6) who had undergone radical retropubic prostatectomy; a seventh tissue set of snap-frozen PZ and TZ was obtained from a CaP-free gland removed after radical cystoprostatectomy. Paraffin-embedded tissue slices were sectioned (10-µm thick) and mounted on suitable windows to facilitate infrared (IR) spectra acquisition before being dewaxed and air dried; cryosections were dessicated on BaF2 windows. Spectra were collected employing synchrotron Fourier-transform infrared (FTIR) microspectroscopy in transmission mode or attenuated total reflection-FTIR (ATR) spectroscopy. Epithelial cell and stromal IR spectra were subjected to principal component analysis to determine whether wavenumber-absorbance relationships expressed as single points in "hyperspace" might on the basis of multivariate distance reveal biophysical differences between cells in situ in different tissue regions. After spectroscopic analysis, plotted clusters and their loadings curves highlighted marked variation in the spectral region containing DNA/RNA bands (1490–1000 cm–1). By interrogating the intrinsic dimensionality of IR spectra in this small cohort sample, we found that TZ epithelial cells appeared to align more closely with those of CaP while exhibiting marked structural differences compared to PZ epithelium. IR spectra of PZ stroma also suggested that these cells are structurally more different to CaP than those located in the TZ. Because the PZ exhibits a higher occurrence of CaP, other factors (e.g., hormone exposure) may modulate the growth kinetics of initiated epithelial cells in this region. The results of this pilot study surprisingly indicate that TZ epithelial cells are more likely to exhibit what may be a susceptibility-to-adenocarcinoma spectral signature. Thus, IR spectroscopy on its own may not be sufficient to identify premalignant prostate epithelial cells most likely to progress to CaP