An FT-IR alternative to the Pap smear The Papanicolaou smear test, or "Pap smear", is commonly used to screen for preinvasive human cervical cancer. Despite its widespread acceptance, the Pap smear has a sufficiently high false negative rate (20%) to prompt Donald McNaughton and colleagues at Monash University (Australia) and the University of Melbourne (Australia) to investigate the use of FT-IR spectroscopy as an alternative means of detecting cervical cancer. The method is based on observing changes in tissue biochemistry that must precede any morphological or symptomatic manifestations of the cancer. The researchers studied exfoliated cervical cells from 272 patients. Six spectra were re-
corded for each patient, and these were visually sorted into two types based on their profiles. Type 1 spectra exhibited a profile characteristic of normal epithelial cells, with intense glyocogen bands and a pronounced symmetric phosphate stretch. Type 2 spectra exhibited features suggestive of dysplastic or malignant transformation, with pronounced symmetric and asymmetric phosphate modes and a reduction in glycogen band intensity. Principal component analysis (PCA) using a reduced data matrix resulted in a score plot that showed general correlation with the visually categorized spectra. Strong correlation between spectra visually classified as abnormal and an abnormal biopsy indicates that visual inspection or subsequent PCA of cervical sample spectra is comparable to the Pap smear.
PM-NSOM of rhodamine 110 Near-field scanning optical microscopy (NSOM) beats the diffraction limit by providing two things smaller than the wavelength of light used—the size of the source and the distance from the source to the sample. Because visible light is used, contrast mechanisms such as fluorescence and refractive index changes are the most common. Paul F. Barbara and co-workers at the University of Minnesota have designed a new form of polarization-modulation (PM) NSOM that allows detailed characterization of optically anisotropic samples through simultaneous collection of images that give the magnitude and direction of the optical anisotropy. The linearly polarized light coupled into the NSOM probe is rotated through 180° at 2 kHz, with 2L combination of an electrooptic modulator and a quarter-wave plate. The anisotropic optical properties of the sample produce a polarization-dependent modulation in the intensity of near-field light coupled through the sample to the detector Using lock-in detection two images are recorded as the amplitude and phase of the polarization-dependent response For PM-NSOM imaging of samples that absorb the probe wavelength ("resonant PM-NSOM"), the amplitude image provides a measure of the local anisotropy of the extinction coefficient, and the phase image indicates the transition dipole orientation. These effects are demonstrated by imaging rhodamine 110 with the 514-nm line of an argon ion laser.
Spectra of malignant HeLa cells also display spectra similar to type 2 spectra in the 1300-950 cm-1 region. (Biospectroscopy 1996,2,143-53)
Representative spectra from type 1, type 2, and HeLa cells. (Adapted with permission of John Wiley & Sons.)
New strategy for assigning MS protein fragments One way to probe the structure and behavior of macromolecules such as polypeptides is with soft ionization MS techniques. However, identifying the fragment ions in the spectra can be a nightmare. For example, Christopher M. Dobson and colleagues from the University of Oxford (U.K.) and Bruker Instruments have looked at fragment ions produced during the collisioninduced dissociation (CID) of the well-characterized protein hen lysozyme. Exhaustive computer searching for the peak at 584.4 yields 96 fragments that have calculated m/z within 1000 ppm of this Vcduc unci 30 within 50 To overcome this problem, the researchers studied the CID-MS of uniformly isotopically labeled proteins that are normally prepared for NMR studies and used atom counting to determine elements of the ion fragment's empirical formula. Using the high resolving power Resonant PM-NSOM amplitude (top) and phase (bottom) images of rhodamine 110 crystals recorded at 514 nm.
PM-NSOM can also be used to image nonabsorbing samples ("nonresonant PMNSOM") provided that the refractive index of the sample is anisotropic in the near field. Samples can be imaged with shot-noise limited S/N and spatial resolution better than 100 nm. (J. .hys. Chem. 1996,100,13794-803)
CID-FTMS spectrum of hen lysozyme and distribution of natural abundance isotope peaks from six fragment ions.
Analytical Chemistry News & Features, October 1, 1996 595 A
