Nonresonant Confocal Raman Imaging of DNA and Protein Distribution in Apoptotic Cells

Abstract

Nonresonant confocal Raman imaging has been used to map the DNA and the protein distributions in individual single human cells. The images are obtained on an improved homebuilt confocal Raman microscope. After statistical analysis, using singular value decomposition, the Raman images are reconstructed from the spectra covering the fingerprint region. The data are obtained at a step interval of approximately 250 nm and cover a field from 8- to 15- micro m square in size. Dwell times at each pixel are between 0.5 and 2 s, depending on the nature and the state of the cell under investigation. High quality nonresonant Raman images can only be obtained under these conditions using continuous wave high laser powers between 60 and 120 mW. We will present evidence that these laser powers can still safely be used to recover the chemical distributions in fixed cells. The developed Raman imaging method is used to image directly, i.e., without prior labeling, the nucleotide condensation and the protein distribution in the so-called nuclear fragments of apoptotic HeLa cells. In the control (nonapoptotic) HeLa cells, we show, for the first time by Raman microspectroscopy, the presence of the RNA in a cell nucleus.

Analysis of interaction between the apicomplexan protozoan Toxoplasma gondii and host cells using label-free Raman spectroscopy

“…RMS is a well-established analytical technique that enables label-free chemical analysis of individual cells and bacteria with sub-micrometric spatial resolution. [4][5][6][7][8] RMS was also used for time-course experiments on individual live cells revealing molecular processes not attainable with other imaging. [9][10][11][12][13] The integration of environmental chambers with inverted Raman microscopes can allow time-and spatially-resolved molecular studies of cellular processes spanning days and weeks, such as stem cell differentiation.…”

Growth substrate induced functional changes elucidated by FTIR and Raman spectroscopy in in–vitro cultured human keratinocytes

“…At present Fourier Transform Infrared Microspectrosopy (FTIRM) and Confocal Raman Microspectroscopy (CRM) have identified in-situ molecular alterations associated with cell death via apoptosis [1][2][3] or necrosis [4,5], or as a result of proliferative changes in cellular activity [6][7][8] or mitosis [9]. It has also been shown that these modalities can identify molecular changes associated with changes in the phenotype of differentiating embryonic cells [10,11].…”

Paclitaxel distribution in poly(ethylene glycol)/poly(lactide-co-glycolic acid) blends and its release visualized by coherent anti-Stokes Raman scattering microscopy

“…While this method provides chemical selectivity, the long excitation wavelength used in FT-IR microscopy offers the spatial resolution of several to tens micrometer, which does not allow the visualization of 3D distribution of drug molecules in thin polymer films or microparticles. Raman microscopy [16][17][18] provides better spatial resolution by using a shorter excitation wavelength, but the weak Raman scattering necessitates a long image acquisition time from minutes to hours. A recently developed nonlinear optical imaging technique that is based on coherent anti-Stokes Raman scattering (CARS) circumvents these difficulties.…”