Flux or speed? Examining speckle contrast imaging of vascular flows.

Speckle contrast imaging enables rapid mapping of relative blood flow distributions using camera detection of back-scattered laser light. However, speckle derived flow measures deviate from direct measurements of erythrocyte speeds by 47 ± 15% (n = 13 mice) in vessels of various calibers. Alternatively, deviations with estimates of volumetric flux are on average 91 ± 43%. We highlight and attempt to alleviate this discrepancy by accounting for the effects of multiple dynamic scattering with speckle imaging of microfluidic channels of varying sizes and then with red blood cell (RBC) tracking correlated speckle imaging of vascular flows in the cerebral cortex. By revisiting the governing dynamic light scattering models, we test the ability to predict the degree of multiple dynamic scattering across vessels in order to correct for the observed discrepancies between relative RBC speeds and multi-exposure speckle imaging estimates of inverse correlation times. The analysis reveals that traditional speckle contrast imagery of vascular flows is neither a measure of volumetric flux nor particle speed, but rather the product of speed and vessel diameter. The corrected speckle estimates of the relative RBC speeds have an average 10 ± 3% deviation in vivo with those obtained from RBC tracking.

Molecular profiling of individual tumor cells by hyperspectral microscopic imaging.

We developed a hyperspectral microscopic imaging (HMI) platform that can precisely identify and quantify 10 molecular markers in individual cancer cells in a single pass. The exploitation of an improved separation of circulating tumor cells and the application of HMI provided an opportunity (1) to identify molecular changes in these cells, (2) to recognize the coexpression of these markers, (3) to pose an important opportunity for noninvasive diagnosis, and (4) to use targeted therapy. We balanced the intensity of 10 fluorochromes bound to 10 different antibodies, each specific to a particular tumor marker, so that the intensity of each fluorochrome can be discerned from overlapping emissions. Using 2 touch preps from each primary breast cancer, the average molecular marker intensities of 25 tumor cells gave a representative molecular signature for the tumor despite some cellular heterogeneity. The intensities determined by the HMI correlate well with the conventional 0-3+ analysis by experts in cellular pathology. Because additional multiplexes can be developed using the same fluorochromes but different antibodies, this analysis allows quantification of many molecular markers on a population of tumor cells. HMI can be automated completely, and eventually, it could allow the standardization of protein biomarkers and improve reproducibility among clinical pathology laboratories.

Selective early cardiolipin peroxidation after traumatic brain injury: an oxidative lipidomics analysis.

OBJECTIVE: Enhanced lipid peroxidation is well established in traumatic brain injury. However, its molecular targets, identity of peroxidized phospholipid species, and their signaling role have not been deciphered. METHODS: Using controlled cortical impact as a model of traumatic brain injury, we employed a newly developed oxidative lipidomics approach to qualitatively and quantitatively characterize the lipid peroxidation response. RESULTS: Electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry analysis of rat cortical mitochondrial/synaptosomal fractions demonstrated the presence of highly oxidizable molecular species containing C(22:6) fatty acid residues in all major classes of phospholipids. However, the pattern of phospholipid oxidation at 3 hours after injury displayed a nonrandom character independent of abundance of oxidizable species and included only one mitochondria-specific phospholipid, cardiolipin (CL). This selective CL peroxidation was followed at 24 hours by peroxidation of other phospholipids, most prominently phosphatidylserine, but also phosphatidylcholine and phosphatidylethanolamine. CL oxidation preceded appearance of biomarkers of apoptosis (caspase-3 activation, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positivity) and oxidative stress (loss of glutathione and ascorbate). INTERPRETATION: The temporal sequence combined with the recently demonstrated role of CL hydroperoxides (CL-OOH) in in vitro models of apoptosis suggest that CL-OOH may be both a key in vivo trigger of apoptotic cell death and a therapeutic target in experimental traumatic brain injury.