Infrared Spectroscopic Studies of Cells and Tissues: Triple Helix Proteins as a Potential Biomarker for Tumors

In this work, the infrared (IR) spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection. Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM) of tissues. An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders. © 2013 Stelling et al

A short-pulse facility for time and angle-resolved photoe-mission experiments at the synchrotron light source DELTA

At the synchrotron light source DELTA (TU Dortmund), a short-pulsefacility is under commissioning. The U250 undulator of the storage ringis used to generate coherent sub-ps pulses at higher harmonics of aseeding laser. The VUV beamline BL5 operated by a group from PGI-6 (FZ Julich) ̈ guides those pulses into an end-station that is optimizedfor angle and spin-resolved photoemission spectroscopy experiments.A part of the seeding laser pulse is brought into the end-station via aseparate beamline and can be used as a pump beam for pump-probeexperiments. With those time and angle-resolved photoemission exper-iments that are now feasible with this unique setup we will, in the nearfuture, study magnetization dynamics of thin ferromagnetic films onmetal surfaces.The modifications of the photoemission setup that were necessaryto conduct time-resolved experiments and the current status of theshort-pulse facility will be described

The ATR IR signal decreases with decreasing dilutions of cells.

<p>Shown is a box plot for serial dilutions of a glioblastoma cell suspension. As the dilution number decreases, both the area of the 1230 cm⁻¹ band (top) and its height (bottom) appear to decrease as well. This indicates that the ATR IR signal from the cells might depend upon the density of cellular material within the beam path of the instrument. It may be the case that compression with the instrument anvil allows the beam path to be set, reducing variability in absorbance between identical measurements. The number of measurements for the 100% dilution was 12 spectra, for the 50% and 25% dilutions six spectra, and for the 12.5% dilution was four spectra. Measurements were performed over the course of three days.</p

Plot of the band heights and positions for meningiomas, glioblastomas, and normal tissue.

<p>The band height is plotted against the band position for the amide III band (top) and the proline band (bottom) for meningiomas (+, n = 13), glioblastomas (x, n = 7), and normal mouse tissue (o, n = 5 individuals, 3 measurements per individual). One spectrum was taken for the tumor measurements, and three spectra were taken for each mouse. The amide III values appear to have less variance than the proline values for normal tissue. The amide III values (top) for most of the tumors appear to be different from those seem in normal tissue. The proline values (bottom) for many meningiomas are quite high compared to the glioblastoma and the normal tissue values.</p

Band heights, positions, and deviations for rat retinal cells, human brain tumor cells, and normal mouse brain tissue.

<p>The heights and positions of the ECM bands are reasonably consistent over a range of biologically identical prepared samples. The retinal cells from the R28 cell lineage were all measured on the same day, but from different passages of the genetically identical cell line. Two measurements were performed per passage. The tumor cells were from a human glioblastoma cell culture. Measurements for these rapidly proliferating cells were performed over three days. For the cell suspensions, the average and standard deviation was calculated with all values treated as independent, identical measurements. The mouse spectra were collected from five animals over the course of two days. Three measurements were performed for each mouse. Shown is mean of means and standard deviation for the five animals. These results indicate this less intense amide III signal is above the signal to noise for the measurement. A larger number of N is required to determine if identical experiments produce values that are not statistically different from each other. Sh, shoulder.</p

Corrected triple helix region in a retinal cell culture.

<p>The R28 rat retinal cells show a broad, weak collagen band whose height is consistent for the same cell line over multiple passages. All spectra were collected on the same day over four different passages. Two spectra could be taken per passage.</p

Corrected triple helix region in a human tumor cell culture.

<p>Fast growing human glioblastoma cells. These spectra were taken over the course of three days, with two to five measurement per cell suspension.</p

Methodology for ATR infrared measurements of cells and tissues.

<p>Left: (A) Picture of a typical cell suspension (circled). (B) The portable ATR FT-IR spectromenter by Bruker. An arrow is pointing to the ATR diamond crystal. For measurements, a 10 µl aliquot of the cell suspension is pipetted on to the crystal. (C) A CaF₂ coverslip is placed on top of the 10 µl droplet, and (D) the anvil of the instrument is lowered so that the cell suspension will achieve good contact with the crystal optics. Right: (A) Shown in the boxed inset is a freshly extracted mouse brain, with a typically sized mouse brain piece on the scalpel (circled). After the brain was cut into small pieces, one was (B) placed on the ART crystal, and (C) the anvil was lowered and the ATR spectrum taken. While spectra could be obtained from tissue without lowering the anvil, the resulting spectra had inadequate signal to noise (data not shown). Pathology results (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058332#pone-0058332-g002" target="_blank">figure 2C</a>) indicated that the damage sustained by the tissue piece from the anvil is minimal, if only one or two spectra are taken with the anvil down.</p