N.M. Sijtsema

Nitric oxide flow tagging in unseeded air

A scheme for molecular tagging velocimetry is presented that can be used in air flows without any kind of seeding. The method is based on the local and instantaneous creation of nitric oxide (NO) molecules from N(2) and O(2) in the waist region of a focused ArF excimer laser beam. This NO distribution is advected by the flow and can be visualized any time later by laser-induced fluorescence in the gamma bands. The creation of NO is confirmed by use of an excitation spectrum. Two examples of the application of the new scheme for air-flow velocimetry are given in which single laser pulses are used for creation and visualization of NO.

CT-based delineation of organs at risk in the head and neck region: DAHANCA, EORTC, GORTEC, HKNPCSG, NCIC CTG, NCRI, NRG Oncology and TROG consensus guidelines

PURPOSEnThe objective of this project was to define consensus guidelines for delineating organs at risk (OARs) for head and neck radiotherapy for routine daily practice and for research purposes.nnnMETHODSnConsensus guidelines were formulated based on in-depth discussions of a panel of European, North American, Asian and Australian radiation oncologists.nnnRESULTSnTwenty-five OARs in the head and neck region were defined with a concise description of their main anatomic boundaries. The Supplemental material provides an atlas of the consensus guidelines, projected on 1mm axial slices. The atlas can also be obtained in DICOM-RT format on request.nnnCONCLUSIONnConsensus guidelines for head and neck OAR delineation were defined, aiming to decrease interobserver variability among clinicians and radiotherapy centers.

Confocal Direct Imaging Raman Microscope: Design and Applications in Biology

A confocal direct imaging Raman microscope (CDIRM) based on two synchronized scanning mirrors, a monochromator, and two charge-coupled device (CCD) cameras has been developed. With this system it is possible to make both Raman spectra of a small measurement volume and images of a larger sample area in one specific Raman band. The spatial resolution of the system was determined for two limiting situations: a small sphere and a thin layer. The image of a 0.282 μm sphere appeared to have a full width at half-maximum (FWHM) of 1.2 μm in the axial and 0.37 μm in the lateral direction, whereas the image of a 275 nm layer showed an FWHM of 1.4 μm in the axial direction. Confocal Raman images were made of the DNA and protein distribution in polytene chromosomes with a relatively weak Raman signal [0.1 photons/(second·pixel)]. Further, a three-dimensional Raman image of the drug distribution in a phthalocyanine-incubated fixed cell is presented. These examples show that the CDIRM can be used to image samples with a weak Raman signal and that three-dimensional images of the distribution of specific molecules in a sample can be made.

Identifying patients who may benefit from adaptive radiotherapy : Does the literature on anatomic and dosimetric changes in head and neck organs at risk during radiotherapy provide information to help?

In the last decade, many efforts have been made to characterize anatomic changes of head and neck organs at risk (OARs) and the dosimetric consequences during radiotherapy. This review was undertaken to provide an overview of the magnitude and frequency of these effects, and to investigate whether we could find criteria to identify head and neck cancer patients who may benefit from adaptive radiotherapy (ART). Possible relationships between anatomic and dosimetric changes and outcome were explicitly considered. A literature search according to PRISMA guidelines was performed in MEDLINE and EMBASE for studies concerning anatomic or dosimetric changes of head and neck OARs during radiotherapy. Fifty-one eligible studies were found. The majority of papers reported on parotid gland (PG) anatomic and dosimetric changes. In some patients, PG mean dose differences between planning CT and repeat CT scans up to 10 Gy were reported. In other studies, only minor dosimetric effects (i.e. <1 Gy difference in PG mean dose) were observed as a result of significant anatomic changes. Only a few studies reported on the clinical relevance of anatomic and dosimetric changes in terms of complications or quality of life. Numerous potential selection criteria for anatomic and dosimetric changes during radiotherapy were found and listed. The heterogeneity between studies prevented unambiguous conclusions on how to identify patients who may benefit from ART in head and neck cancer. Potential pre-treatment selection criteria identified from this review include tumour location (nasopharyngeal carcinoma), age, body mass index, planned dose to the parotid glands, the initial parotid gland volume, and the overlap volume of the parotid glands with the target volume. These criteria should be further explored in well-designed and well-powered prospective studies, in which possible relationships between anatomic and dosimetric changes and outcome need to be established.

Air photolysis and recombination tracking: A new molecular tagging velocimetry scheme

A new scheme for molecular tagging velocimetry in unseeded airflows is presented. The method, called air photolysis and recombination tracking, is based on the photoinduced formation of nitric oxide (NO) in the waist region of a focused ArF excimer laser beam. The distribution of the formed NO molecules is imaged by planar laser-induced fluorescence in the gamma band, using a frequency-doubled dye laser beam. The role of N-2(+) ions in the NO formation process is discussed, and the lifetime of the NO molecules was determined to be at least 10 ms. The new method has been applied to a laminar and a pulsed airflow and a premixed methane/air flame. Velocities could be determined with an accuracy of 5% in the airflows (on a single-shot basis) and 13% in the flame. By the use of averaging over many laser pulses, velocities as low as 1 cm/s could be measured.

Applications of Micro-Raman Imaging in Biomedical Research

Recent results are presented of the application of imaging micro-Raman spectrometers in cellular biophysics and biomedical research. Various micro-Raman spectrometers have been developed that are now routinely applied in these fields. Results are presented that were obtained with a linescan Raman microspectrometer and with a Raman imaging microscope. Applications of Raman linescan spectrometry concern the investigation of polytene chromosomes obtained from the salivary gland of Chironomus thummi thummi. The distribution of DNA and proteins was investigated in bands and interbands. In a second example of Raman linescan spectrometry, bone implants were investigated. These bone implants were coated with thin layers of materials that improve biocompatibility. The density, crystallinity and protein distribution can be investigated. Information from Raman imaging may help in selecting the proper materials for maximum biocompatibility. Raman imaging microscopy is used whenever two-dimensional spatial information is required on the distribution of molecules or molecular components. Raman images are presented of filipine and phenylalanine in human eye-lenses in and around radial shades. Raman imaging is a particularly important tool for the study of the distribution of non-fluorescent drugs inside living cells. This is illustrated for the non-fluorescent drug cobalt octacarboxyphthalocyanine.

Site-directed mutagenesis of Met243, a residue of myeloperoxidase involved in binding of the prosthetic group

Abstractu2002The optical absorbance spectrum of reduced myeloperoxidase is red-shifted with respect to that of other haemoproteins, and has the Soret band at 472u2009nm and the α band at 636u2009nm. The origin of the red shift is poorly understood, but the interaction of the protein matrix with the chromophore is thought to play an important role. Met243 is one of the three residues in close proximity to the prosthetic group of the enzyme, and we have examined the effect of a Met243Gln mutation on the spectroscopic properties and catalytic activity of the enzyme. The mutation has a large effect on the position of the Soret band in the optical absorbance spectrum of the reduced mutated enzyme, which shifts from 472u2009nm to 445u2009nm. The alkaline pyridine haemochrome spectrum is greatly affected and similar to that of protohaem. The mutation also drastically affects the resonance Raman (RR) spectrum, which is indicative of an iron porphyrin-like chromophore. The mutant enzyme is unable to peroxidise chloride to hypochlorous acid. We conclude that there are two factors involved which account for the red-shifted Soret band. One of them is the interaction of Met243 with the prosthetic group via a special sulfonium linkage. The other factor which contributes is the presence of ester linkages between hydroxylated methyl groups on the haem and glutamate and aspartate residues, respectively. The results, combined with those of previous studies, now give us a comprehensive picture of the various factors which contribute to the unusual optical properties of myeloperoxidase.

Confocal Raman microspectroscopy of the activation of single neutrophilic granulocytes.

Abstract Confocal Raman micro-spectroscopy has been applied to investigate the activation process of single, living neutrophilic granulocytes. Both resting cells as well as activated cells were measured. The activation of cells was performed with phorbol-12-myristate-13-acetate activator and Escherichia Coli bacteria. Raman microspectroscopy combines a high spatial resolution inside a single, living cell with detailed material information. Using this approach it can be concluded that activation of the cells with phorbol-12-myristate-13-acetate causes a change in the redox state of cytochrome b558. This protein is a part of the NADPH-oxidase complex that neutrophilic granulocytes employ to generate O2–, superoxide anion. Additionally a change in the redox state of myeloperoxidase can be observed. Myeloperoxidase is known to react with O2–. Activation of the cells with bacteria gives rise to corresponding changes in the Raman spectra. From this single cell study it can be concluded that the enzymes cytochrome b558 and myeloperoxidase are present inside the cytoplasm of the living cell, while participating in the redox processes. Activation causes an intra-cellular release of oxygen metabolites. Activation with bacteria of neutrophilic granulocytes from a patient with chronic granulomatous disease, that contain no cytochrome b558, led to typical changes in the redox state of myeloperoxidase. This indicates that in the bacterium/neutrophilic granulocyte system oxygen metabolites are generated that are capable of reacting with MPO.

Resonance Raman microspectroscopy of myeloperoxidase and cytochrome b558 in human neutrophilic granulocytes

With (resonance) Raman microscospectroscopy, it is possible to investigate the chemical constitution of a very small volume (0.5 fl) in a living cell. We have measured resonance Raman spectra in the cytoplasm of living normal, myeloperoxidase (MPO)-deficient, and cytochrome b558-deficient neutrophils and in isolated specific and azurophilic granule fractions, using an excitation wavelength of 413.1 nm. Similar experiments were performed after reduction of the redox centers by the addition of sodium dithionite. The specific and azurophilic granules in both redox states appeared to have clearly distinguishable Raman spectra when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b558-deficient neutrophils showed Raman spectra similar to that of the isolated MPO. The spectra of the specific granules and the MPO-deficient neutrophils corresponded very well to published cytochrome b558 spectra. The resonance Raman spectrum of the cytoplasmic region of normal neutrophilic granulocytes could be fitted with a combination of the spectra of the specific and azurophilic granules, which shows that the Raman signal of neutrophilic granulocytes mainly originates from MPO and cytochrome b558, at an excitation wavelength of 413.1 nm.

Systematic review of the role of a belly board device in radiotherapy delivery in patients with pelvic malignancies

PURPOSEnThis review analyses the literature concerning the influence of the patient position (supine, prone and prone on a belly board device (BB) on the irradiated small-bowel-volume (SB-V)) and the resulting morbidity of radiation therapy (RT) in pelvic malignancies.nnnMETHODSnA literature search was performed in MEDLINE, web of science and Scopus.nnnRESULTSnForty-six full papers were found, of which 33 met the eligibility criteria. Fifteen articles focussed on the irradiated SB-V using dose volume histograms (DVHs). Twenty-seven articles studied the patient setup in different patient positions. This review showed that a prone treatment position can result in a lower irradiated SB-V as compared to a supine position, but a more significant reduction of the SB-V can be reached by the additional use of a BB in prone position, for both 3D-CRT and IMRT treatment plans. This reduction of the irradiated SB-V might result in a reduced GI-morbidity. The patient position did not influence the required PTV margins for prostate and rectum.nnnCONCLUSIONSnThe irradiated SB-V can be maximally reduced by the use of a prone treatment position combined with a BB for both 3D-CRT and IMRT, which might individually result in a reduction of GI-morbidity.