Tumor samples were dissected into three parts: these were snap frozen in liquid nitrogen, fixed in 4% formalin, or fixed in acetic acid–formalin ethanol saline. The tumor model used is known to be very sensitive to the MTD of cisplatin, whereas nontreated tumors grow rapidly. This could result in control animals being removed from the experiment on the basis of humane end points (tumor volume > 1500 selleck chemical mm3) or in a minimal amount of measurable tumor tissue in the treated animals before the end of the experiment. Therefore, animals with slightly higher tumor volumes were included in the treatment group. Throughout the course of the experiment,
starting 3 weeks before the tumor grafting, the animals were given a purified diet to eliminate autofluorescence from chlorophyll . During the optical spectroscopy measurements, the animals were deeply anesthetized using 1.5% isoflurane mixed with oxygen. All animal procedures were approved by the Animal Ethics Committee of the Netherlands Cancer Institute. DRS and AFS measurements were performed using a portable spectroscopic system, which consists of two light sources and two spectrometers (Figure 1). For the DRS measurements, a Tungsten halogen ATM/ATR targets broadband light source (360-2500 nm) with an embedded shutter was used. For
AFS, the system was equipped with a semiconductor laser (λ = 377 nm) to induce autofluorescence. One spectrometer was used to resolve light in the visible wavelength range, i.e., 400 until 1100 nm (DU420A-BRDD; Andor Technology, Belfast, Northern Ireland), the other to resolve near-infrared light from 900 to 1700 nm (DU492A-1.7; Andor Technology). The spectrometers were controlled by a custom-made
LabVIEW software user interface (National Instruments, Austin, TX) to acquire and save the data. The calibration procedure has been described elaborately by Nachabe et al. Methane monooxygenase . A custom fiber-optic needle that can probe tissue at the needle tip was developed. The needle consisted of a 21-G (0.82 mm) outer cannula and a 22-G adjustable stylet (Figure 1B), containing four identical fibers with a core diameter of 100 μm. To minimize tissue damage, the optical fibers were retracted during needle insertion. The optical fibers were protruded after positioning the needle at the right position to establish optimal tissue contact. Two fibers were connected to the broadband light source and laser, whereas the two other fibers were connected to the spectrometers to capture diffusely scattered light and fluorescence from the tissue. Two different source-detector separations (SDSs) were used (1.5 and 0.15 mm). The spectra acquired with the 1.5-mm SDS were used for the DRS data analyses, whereas the DRS spectra measured with the 0.15-mm SDS were used to correct for absorption and scattering in the fluorescence spectra.