Studying single living cells and chromosomes by confocal Raman microspectroscopy

Abstract

Many indirect methods have been developed to study the constitution and conformation of macromolecules inside the living cell. Direct analysis by Raman spectroscopy is an ideal complement to techniques using directly labelled fluorescent probes or of indirect labelling with mono- and polyclonal antibodies. The high information content of Raman spectra can characterize biological macromolecules both in solution and in crystals. The positions, intensities and linewidths of the Raman lines (corresponding to vibrational energy levels) in spectra of DNA-protein complexes yield information about the composition, secondary structure and interactions of these molecules, including the chemical microenvironment of molecular subgroups. The main drawback of the method is the low Raman scattering cross-section of biological macromolecules, which until now has prohibited studies at the level of the single cell with the exception of (salmon) sperm heads, in which the DNA is condensed to an exceptionally high degree. Ultraviolet-resonance Raman spectroscopy has been used to obtain single cell spectra (and F. Sureau and P. Y. Turpin, personal communication), but in this method absorption of laser light may impair the integrity of the sample. We have avoided this problem in developing a novel, highly sensitive confocal Raman microspectrometer for nonresonant Raman spectroscopy. Our instrument makes it possible to study single cells and chromosomes with a high spatial resolution (approximately less than 1 micron 3).

Carotenoids located in human lymphocyte subpopulations and natural killer cells by Raman microspectroscopy

“…Raman Measurements Raman spectra of human lymphocytes and NK cells were measured using the confocal Raman microspectrometer described previously (31)(32)(33). The main features of this instrument are summarized in Fig.…”

“…We have recently described the possibility of performing nonresonant Raman microspectroscopic studies of single cells and chromosomes with a spatial resolution of 0.45 x 0.45 x 1.3 pm3 (31)(32)(33) and the possibility of combining fluorescence activated cell sorting and Raman microspectroscopy (34) (see also Materials and Methods). Here these techniques were applied to investigate the presence and subcellular location of carotenoids in B-lymphocytes (CD19 + ), helperiinducer T-lymphocytes (CD4 + ), cytotoxicisuppressor T-lymphocytes (CD8 + 1, TyG-lymphocytes [T-cell receptor (TCR) yS+], and NK cells (CD16+) isolated from samples of human peripheral blood obtained from healthy individuals.…”

Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells

“…Raman spectroscopy examines the inelastic scattering of photons by molecular bonds, where the scattered photons can either lose part of their energy and are redshifted ("Stokes"-shifted) or gain energy and are blue-shifted ("anti-Stokes"-shifted). Raman spectroscopy has recently become more popular in the life sciences with the advent of new, more powerful and reliable laser sources and more sensitive light detectors [18]. This technique is mostly noninvasive and nondestructive at moderate photon energies and works in vitro and in vivo under a wide range of environmental conditions.…”

“…The first report in the literature of a single-cell Raman analysis was in fact conducted on individual living salmon sperm cells that were investigated within the cavity of a highpower laser system [22]. Shortly thereafter it was shown, that if combined with confocal laser microscopy, Raman spectroscopy can be used to obtain information from individual living cells even with subcellular resolution [18]. Since then, the applications of Raman spectroscopy to single cells have grown exponentially [23,24], and applications related to single cancer cells [25], human embryonic stem cells [26], and even biological particles as small as lipoproteins [27] and bacterial spores [28] have been demonstrated.…”

Combined Fourier transform infrared and Raman spectroscopic approach for identification of multidrug resistance phenotype in cancer cell lines

“…Because the Raman microspectrometer collects data from a volume of approximately a few micron cube of the sample, spectra depend very much on the site on which the laser has been focused. 28 In other words, spectra of even adjacent areas can be quite different. In order to get a good representative spectrum, 25 or more spectra from adjacent positions in a 50 Â 50 m 2 selected site, were recorded at 10-m intervals and a mean spectrum was taken as representative of the site.…”