Laser Doppler imaging for intraoperative human brain mapping.

The identification and accurate location of centers of brain activity are vital both in neuro-surgery and brain research. This study aimed to provide a non-invasive, non-contact, accurate, rapid and user-friendly means of producing functional images intraoperatively. To this end a full field Laser Doppler imager was developed and integrated within the surgical microscope and perfusion images of the cortical surface were acquired during awake surgery whilst the patient performed a predetermined task. The regions of brain activity showed a clear signal (10-20% with respect to the baseline) related to the stimulation protocol which lead to intraoperative functional brain maps of strong statistical significance and which correlate well with the preoperative fMRI and intraoperative cortical electro-stimulation. These initial results achieved with a prototype device and wavelet based regressor analysis (the hemodynamic response function being derived from MRI applications) demonstrate the feasibility of LDI as an appropriate technique for intraoperative functional brain imaging.

Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images.

BACKGROUND: For the investigation of the molecular mechanisms involved in neurite outgrowth and differentiation, accurate and reproducible segmentation and quantification of neuronal processes are a prerequisite. To facilitate this task, we developed a semiautomatic neurite tracing technique. This article describes the design and validation of the technique. METHODS: The technique was compared to fully manual delineation. Four observers repeatedly traced selected neurites in 20 fluorescence microscopy images of cells in culture, using both methods. Accuracy and reproducibility were determined by comparing the tracings to high-resolution reference tracings, using two error measures. Labor intensiveness was measured in numbers of mouse clicks required. The significance of the results was determined by a Student t-test and by analysis of variance. RESULTS: Both methods slightly underestimated the true neurite length, but the differences were not unanimously significant. The average deviation from the true neurite centerline was a factor 2.6 smaller with the developed technique compared to fully manual tracing. Intraobserver variability in the respective measures was reduced by a factor 6.0 and 23.2. Interobserver variability was reduced by a factor 2.4 and 8.8, respectively, and labor intensiveness by a factor 3.3. CONCLUSIONS: Providing similar accuracy in measuring neurite length, significantly improved accuracy in neurite centerline extraction, and significantly improved reproducibility and reduced labor intensiveness, the developed technique may replace fully manual tracing methods.

Minimum views required to characterize cataracts when using the Scheimpflug camera.

We performed Scheimpflug slit lamp photography and computerized image analysis on 20 normal and 25 cataractous lenses using 18 slit images for each lens taken 10 degrees apart. The data gathered from the normals served as the reference to estimate the accuracy of representation of the cataracts by the least number of views (18 and less) using a Fourier interpolative algorithm. Using the error obtained with one view for the normals, our study suggests that the minimum number of views necessary for adequate characterization is two for cortical cataracts, two for nuclear cataracts, and six for posterior subcapsular cataracts. This information will be useful in longitudinal studies of cataracts, since most researchers presently use only one view, which may be adequate for normals but not for cataractous lenses. We found the Fourier interpolative algorithm useful in estimating the minimum views required for the current method of analyzing Scheimpflug images, and it can be easily applied to other similar images.

Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.

Effect of phorbol esters on rat lacrimal gland protein secretion.

To identify a role for protein kinase C in lacrimal gland protein secretion, we incubated rat exorbital lacrimal gland acini in the ester 4-beta-phorbol 12, 13 dibutyrate (beta-phorbol dibutyrate), its inactive isomer 4-alpha-phorbol 12, 13 dibutyrate (alpha-phorbol dibutyrate), and the diacylglycerol analog 1,2-oleoyl acetylglycerol (OAG). We determined protein secretion by measuring the activity of peroxidase, a protein secreted by lacrimal gland acini. beta-phorbol dibutyrate, but not alpha-phorbol dibutyrate, stimulated peroxidase secretion in a concentration-dependent manner with 3 X 10(-8) M producing maximal secretion. OAG (10(-6) M) also stimulated peroxidase secretion. To determine whether muscarinic and alpha 1-adrenergic agonists activate protein kinase C, we added beta-phorbol dibutyrate (10(-7) M) simultaneously with carbachol (10(-5) M) or phenylephrine (10(-4) M); under both conditions, secretion was less than additive. Protein secretion in the presence of beta-phorbol dibutyrate (10(-7) M) and vasoactive intestinal peptide (VIP) (10(-8) M), the latter that acts through cAMP, was additive, and when the beta-phorbol dibutyrate but not the VIP concentration was decreased to 10(-8) M, secretion was potentiated. We conclude that muscarinic and alpha 1-adrenergic agonists, but not VIP, stimulated lacrimal gland protein secretion by activating protein kinase C.

Role of cyclic AMP and Ca2+ in potentiation of rat lacrimal gland protein secretion.

Addition of a cholinergic agonist carbachol and vasoactive intestinal peptide (VIP) to dispersed rat exorbital lacrimal gland acini produces protein secretion, measured by secretion of the enzyme peroxidase, that was statistically significantly greater than additive (potentiated). To determine where in stimulus-secretion coupling these secretagogues interact to potentiate secretion, rat exorbital gland acini were incubated simultaneously with cyclic AMP- and Ca2+-dependent agonists and protein secretion, cyclic AMP level, or Ca2+ concentration measured. As a measure of protein secretion, the supernatant obtained after centrifugation of acini was analyzed for peroxidase, a protein secreted by rat lacrimal glands. Interaction did not occur at the receptor level, because peroxidase secretion also was potentiated by simultaneous addition of carbachol and forskolin, which activates the catalytic subunit of adenyl cyclase. A potentiated increase in the cyclic AMP level did not potentiate protein secretion, because the level was the same with VIP as with carbachol and VIP added together at concentrations that potentiated peroxidase secretion. A potentiated increase in free intracellular [Ca2+] did not potentiate protein secretion, because [Ca2+] was greater with carbachol than with carbachol and VIP added together at concentrations that potentiated peroxidase secretion. We conclude that cholinergic- and VIP-dependent pathways interact to potentiate lacrimal gland protein secretion after the rise of intracellular cyclic AMP or Ca2+.