Infrared spectroscopy and microscopy in cancer research and diagnosis

Abstract

Since the middle of 20(th) century infrared (IR) spectroscopy coupled to microscopy (IR microspectroscopy) has been recognized as a non destructive, label free, highly sensitive and specific analytical method with many potential useful applications in different fields of biomedical research and in particular cancer research and diagnosis. Although many technological improvements have been made to facilitate biomedical applications of this powerful analytical technique, it has not yet properly come into the scientific background of many potential end users. Therefore, to achieve those fundamental objectives an interdisciplinary approach is needed with basic scientists, spectroscopists, biologists and clinicians who must effectively communicate and understand each other's requirements and challenges. In this review we aim at illustrating some principles of Fourier transform (FT) Infrared (IR) vibrational spectroscopy and microscopy (microFT-IR) as a useful method to interrogate molecules in specimen by mid-IR radiation. Penetrating into basics of molecular vibrations might help us to understand whether, when and how complementary information obtained by microFT-IR could become useful in our research and/or diagnostic activities. MicroFT-IR techniques allowing to acquire information about the molecular composition and structure of a sample within a micrometric scale in a matter of seconds will be illustrated as well as some limitations will be discussed. How biochemical, structural, and dynamical information about the systems can be obtained by bench top microFT-IR instrumentation will be also presented together with some methods to treat and interpret IR spectral data and applicative examples. The mid-IR absorbance spectrum is one of the most information-rich and concise way to represent the whole "… omics" of a cell and, as such, fits all the characteristics for the development of a clinically useful biomarker.

Keywords: Molecular vibrations; cancer biomarker; cancer diagnosis; infrared microspectroscopy; infrared radiation; mid-infrared absorbance spectroscopy; pre-clinical drug screening; synchrotron radiation; unsupervised multivariate analysis; vibrational spectroscopy.

Figure 1

The basis of infrared (IR) vibrational absorbance spectroscopy. a. The classical harmonic oscillator model, b. Some examples illustratint the relationships among atomic masses, bond strength and a particular vibrational mode (stretching) of some chemical groups, c. Fundamental vibrational modes of a molecule of free water detectable at specific frequency values within the mid-IR region of the electromagnetic spectrum.

Figure 2

The schematic layout of components in an microFT-IR apparatus. The external IR beam is generally provided by Synchrotron Radiation and a dedicated beamline is required to extract IR light from the storage ring of a Synchrotron and to collimate IR light to the experimental area where the microFT-IR apparatus is usually located several meters from the exit port. The description of microFT-IR apparatus and the functioning of single components are in the text.

Figure 3

Fourier transform infrared microspectroscopy (microFT-IR) on individual cell. a. The FT-IR absorbance spectrum of an individual human formalin-fixed and air-dried monocyte deposited on ZnSe window within the sampling interval of wavenumbers 3750-800 cm⁻¹. The selected cell (36× magnification) was restricted by slits within a 15 μm×15 μm sample area. This was alternatively illuminated with IR light from a broadband internal source (Globar, blue trace) and with beam of IR light from Synchrotron Radiation (SR, red trace) focused on the sample, respectively. To obtain acceptable signal to noise ratio (S/N) values a number of 128 scans was cumulated (velocity 20 kHz, spectral resolution 4 cm⁻¹, total acquisition time 60 s) on a nitrogen cooled single element MCT detector with a detecting area of 50×50 μm². The background (ZnSe) spectrum was collected from a 15 μm×15 μm area external to the sample. Atmosphere water vapour and CO2 were compensated and the baseline was corrected using appropriate algorithms in the software. The superimposed second derivative spectra have been calculated by a generalized Savitzky-Golay smoothing algorithm on 9 points. The contribution of sub-bands in the markers peaks of amide I and amide II within the interval 1690-1480 cm⁻¹ of SR IR spectrum has been highlighted in the framed box. b. The mapping of an individual formalin-fixed and air-dried cell by SR microFT-IR. The selected area was raster-scanned by collecting a series of spectra from 18×15 points grid (10 μm×10 μm area, 2 μm step, 256 scans within the interval 4000-600 cm⁻¹, scanner velocity 20 kHz, spectral sensitivity 8 cm⁻¹, one background area every five sample spectra) on a single channel 100 μm×100 μm MCT detector. The spectra were assembled and integrals were calculated within the interval of wavenumbers between 3000 and 2800 cm⁻¹ (symmetric and antisymmetric stretching of CH3 and CH2 groups in lipid molecules) and between 1690 and 1480 cm⁻¹ (amide I and amide II modes in proteins), allowing to obtain the two dimensional distribution of lipid and proteins, respectively. The intensity scale of false colors extends from the lowest absorbance (blue) to the highest absorbance (red-violet) values in the two dimensional contour maps superimposed to the underlying object, respectively.

Figure 4

Transflectance and Attenuated Total Reflectance (ATR) techniques.

Figure 5

The unsupervised recognition of spontaneous apoptosis in the spectra of MEG-01 leukemic blasts, a. Hierarchical Cluster Analysis (HCA), was applied to several spectra of individual MEG-01 blasts that had been obtained by SR microFT-IR absorbance spectroscopy performed at the beamline B22 Infrared Microspectroscopy end station of Diamond Light Source (probing area: 15 μm×15 μm either in the sample and in the background; IR source: Synchrotron Radiation; detector: MCT with a 50 μm² detecting area; IR sampling interval: 4000-700 cm⁻⁻¹, number of scans: 128; scanner velocity 40 kHz; spectral sensitivity 4 cm⁻⁻¹). HCA was performed by a standard method and Ward's algorithm applied to the interval of frequencies 1780-1480 cm⁻⁻¹ in the spectra pre-treated with vector normalization and second derivative calculated with the Savitzky-Golay smoothing algorithm on 9 points, b. The two spectral patterns representative of apoptotic (red trace) and G0-G1 viable (blue trace) MEG-01 cells were obtained by averaging parental spectra clustered by HCA in each of the two final groups, respectively. Typical IR signatures of cell apoptosis are the shift of amide I and amide II peaks towards lower wavenum-bers and the increased absorbance at ∼1740 cm⁻⁻¹ assigned to the C=O in phospholipid. Independent validation of IR data was obtained by the analysis of apoptotic events carried out in parallel samples by complementary techniques (data not shown).