Signal intensity analysis and compensation for dual-channel wide-field fluorescence microscope

Quantitative analysis of cochlear signal intensity on three-dimensional and contrast-enhanced fluid-attenuated inversion recovery images in patients with Meniere's disease: Correlation with the pure tone audiometry test

Wide-field fluorescence microscopy (WFFM) is widely adopted in biomedical studies. However, axial resolution in most WFFM is poor due to the absence of optical-sectioning capability. To achieve wide-field optical-sectioning, several methods have been proposed, most of which need at least two images to reconstruct one optical sectioning image. So, the frame rate of current wide-field optical sectioning microscopy is no more than half of that of conventional WFFM, which may not meet the speed requirement of fast biodynamic studies. We introduce a novel high-speed, wide-field optical sectioning method based on local contrast weighting function and two-dimensional Hilbert-Huang transform, in which only one structured image is required to reconstruct an optical sectioning image. In this way, the loss of temporal resolution in conventional wide-field optical sectioning microscopy is compensated. We validated this method with the imaging of mouse brain slices.

Purpose and Background− Reportedly, magnetic resonance imaging (MRI) detects mammographically and sonographically (US) occult incidental lesions in 10-29% of patients with a malignant primary breast lesion. We evaluated diagnostic performance of the BI-RADS reporting system and utility of T2- and diffusion weighted imaging (DWI) for MRI detected additional lesions. Methods− This prospective study protocol included 3.0T structural breast MRI with T2-weighted imaging and DWI performed according to EUSOMA guidelines in 112 consecutive patients with primary breast lesions (mean age 57.0±12.7 years, range 38-80 years). Breast lesions with one or more suspective feature according to the BI-RADS lexigon were biopsied. T2 signal intensity (SI) and DWI findings were assessed. Results− Altogether 33 (29.5%) patients had 36 primarily MRI detected incidental additional lesions. In addition, 36 sographically or mammographically detected lesions were histopathologically confirmed and a total of 8 lesions were followed up. Of incidental lesions, 16 (44.4%) proved to be malignant. Mean size was 0.82±0.29 cm (range 0.5 cm to 1.5 cm) and 1.5±1.61 cm (range 0.5 cm to 5.0 cm) for 29 mass lesions and 7 NMLE lesions, respectively. In mass lesions, traditional morphological or kinetic features were not correlated to malignancy except for fast initial enhancement (P=0.003). The BI-RADS classification produced 100% sensitivity, 40% specificity, 53.3% PPV, 100% NPV and 51.7% overall accuracy. Both the low T2 SI (P=0.05) and low ADC values (P<0.001) significantly correlated with malignancy yeilding improved 86.7%/90.5% specificity and 65.0%/90.5% negative predictive value, respectively (McNemar's test, P<0.01). Combining low T2 SI, ADC values or morphologic or kinetic features did not improve the results. Conclusion− A single suspective morphologic or kinetic MRI feature is the most sensitive parameter to discover an incidental breast malignancy, as some incidental lesions present with only a few suspicious MRI feature. Specificity of MRI is improved when T2 SI or DWI are addressed. Citation Format: Arponen O, Sudah M, Masarwah A, Sutela A, Vanninen R. Incidentally detected enhancing lesions found on preoperative breast MRI: Analysis of T2 signal intensity and apparent diffusion coefficient significantly improve classification. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-02-14.

Quantitative analysis of T2 signal intensities in Alzheimer's Disease

The objective of this study was to evaluate the diagnostic accuracy of chemical-shift (CS) magnetic resonance (MR) imaging in the differential diagnosis of adenoma and nonadenoma adrenal masses. We enrolled 36 patients (9 men, 27 women, mean age 51.3+14.4 years) with unilateral (n=31) or bilateral (n=5) adrenal masses incidentally discovered during imaging examinations [ultrasound (US), computed tomography (CT)] performed for other indications. A total of 41 adrenal lesions were evaluated (mean diameter 3.0+2.2 cm). Histology (n=19), biopsy (n=3) or clinical-imaging follow-up (n=19) demonstrated 29 adenomas, five pheochromocytomas, three cysts and four carcinomas. MR imaging was performed using the following breath-hold sequences: T1-fast field echo (FFE) [repetition time (TR)/echo time (TE)=236/4.6 ms], T2-turbo spin echo-single shot (TSE-SSh) (TR/TE=831/80 ms), T1-DUAL-FFE (TR=236, double TE=4.6/2.3 ms in phase and out of phase) and T1-FFE after gadolinium-DTPA (Gd). Axial and coronal imaging planes were used, with a slice thickness of 3-5 mm. MR images were qualitatively assessed for signal intensity of the adrenal mass relative to the liver on T1, T2, CS and T1-Gd scans; diagnostic criteria for adenomas were isointensity or hypointensity on both T1 and T2 scans, out-of-phase CS signal loss and mild transient enhancement after Gd. Analysis of T1-T2 signal intensity showed diagnostic accuracy, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 80%, 72%, 100%, 100% and 60%, respectively. In contrast, analysis of CS and T1-Gd signal intensity showed diagnostic accuracy, sensitivity, specificity, PPV and NPV for both sequences of 93%, 90% (p<0.05 vs. T1-T2 analysis), 100%, 100% and 80% (p<0.05 vs. T1-T2 analysis), respectively. CS MR imaging significantly improves characterization of adrenal masses compared with conventional T1-T2-weighted images, providing accuracy similar to that of the T1 sequence after Gd. Therefore, the CS sequence is strongly recommended for MR study of adrenal masses, and its use might obviate the need for Gd administration.

A combination of genetically-encoded calcium indicators and micro-optics has enabled monitoring of large-scale dynamics of neuronal activity from behaving animals. In these studies, wide-field microscopy is often used to visualize neural activity. However, this method lacks optical sectioning capability, and therefore its axial resolution is generally poor. At present, it is unclear whether wide-field microscopy can visualize activity of densely packed small neurons at cellular resolution. To examine the applicability of wide-field microscopy for small-sized neurons, we recorded calcium activity of dentate granule cells having a small soma diameter of approximately 10 micrometers. Using a combination of high numerical aperture (0.8) objective lens and independent component analysis-based image segmentation technique, activity of putative single granule cell activity was separated from wide-field calcium imaging data. The result encourages wider application of wide-field microscopy in in vivo neurophysiology.

In addition to ultrasound, magnetic resonance imaging (MRI) is considered a suitable, non-invasive technique to assess the type and extent of vascular malformations. The distinction between low- and high-flow lesions is crucial because it determines appropriate patient treatment. To distinguish high-flow from low-flow lesions on the basis of the enhancement pattern on MIP images acquired from dynamic time-resolved MR angiography (MRA) and compare it with previously described MR-based methods. We examined 25 consecutive patients with previously diagnosed vascular malformations. Next, each malformation was classified as "high-flow" or "low-flow" using the following criteria: (i) findings on T1-weighted (T1W) and T2-weighted (T2W) imaging (signal voids, signal intensity); (ii) the time interval between the start of arterial enhancement and the onset of lesion enhancement (artery-lesion time); (iii) the time of maximum lesion enhancement; and (iv) analysis of the slope of the enhancement curve. Of the 25 patients, seven had high-flow and 18 had low-flow malformations. Signal voids on spin-echo T1W images were observed only in four of seven high-flow malformations and in two of 18 low-flow malformations. Analysis of signal intensity on T2W images showed increased signal intensity in 17 of 18 low-flow malformations, and in two of seven high-flow lesions. Calculation of the artery-lesion time, maximum enhancement time, and slope revealed significant differences between the high- and low-flow groups. In conclusion, the slope of the enhancement curve appears to be useful in distinguishing between high- and low-flow vascular malformations. Standardization of MR image evaluation criteria is essential.

5095 Background: Pixel-by-pixel analysis of signal intensity (SI) using dynamic contrast enhanced MRI (DCE- MRI) in cervical cancer has been reported to predict treatment outcome. Low enhancement correlates with treatment failure presumably due to poor blood supply/hypoxia. However, within a heterogeneous tumor, the relative volume and degree of low-enhancement regions that correlate with failure is uncertain. This project analyzed the threshold of low-enhancement pixels within the tumor to optimize the predictive power for treatment outcome. Methods: 101 patients with advanced cervical cancer underwent DCE-MRI early during radiation/chemotherapy (2 weeks after therapy start). The SI of each pixel was calculated from the time-intensity curve of the DCE-MRI at the plateau phase. The SI of all pixels within the tumor was plotted as a pixel SI spectrum. The lowest 2.0th through 40th percentiles of the pixel spectrum were used to define the low-enhancement volume, and each percentile category was correlated with tumor recurrence using logistic regression analysis. Results: The significance levels to predict tumor recurrence with different relative volume thresholds are shown in the Table. Prediction was most significant when thresholds of 3.5th to 7.0th percentiles were used to define the low-enhancement tumor volume. The predictive power decreased when the lowest percentile was defined as >7.5th or <3.0th percentiles of the pixel spectrum. Conclusion: Based on this limited data, the best predictive value is obtained with pixel thresholds at the 3.5th through 7th percentiles of the heterogeneous tumor spectrum. Pixels in this range may reflect tumor cells with poor blood supply/hypoxia and less response to treatment. Higher percentiles may include cell populations with variable blood supply, and lower percentiles than the 3.0th may represent partial volume effects at the tumor margin. These findings require further study. No significant financial relationships to disclose.

Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-alpha-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-alpha-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular microtubules characteristic of the parasite cytoskeleton. To understand this result, we analyzed sources of noise in the LSCM and identified illumination fluctuations on time scales from microseconds to hours that introduce significant amounts of noise. We confirmed that weakly fluorescent structures could not be imaged in LSCM by using fluorescent bead standards. By contrast, wide-field microscopy (WFM) did visualize weak fluorescent standards and the individual microtubules of the parasite cytoskeleton. We therefore measured the fluorescence per unit length of microtubule by using WFM and used this information to estimate the tubulin content of the conoid (a structure important for T. gondii infection) and in the mitotic spindle pole. The conoid contains sufficient tubulin for approximately 10 microtubule segments of 0.5-microm length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains approximately 1 microtubule per chromosome. This analysis expands the understanding of structures used for invasion and intracellular proliferation by an important human pathogen and shows the advantage of WFM combined with image deconvolution over LSCM for quantitative studies of weakly fluorescent structures in moderately thin living cells.

The aim of this study was to establish dynamic contrast-enhanced perfusion in peripheral nerves for determination of blood-nerve permeability (K) and nerve blood volume (NBV) in peripheral neuropathies as compared with healthy controls. The study was approved by the institutional ethics committee, and written informed consent was obtained from all participants. Forty-three controls (24 women, 19 men; age, 48.7 ± 17.5 years) and 59 patients with peripheral neuropathy (28 women, 31 men; age, 52.7 ± 12.4 years) were examined by a standard protocol including a T1-weighted dynamic contrast-enhanced sequence (time of repetition/time of echo, 4.91/1.64; 10 slices; resolution 0.8 × 0.6 × 3.0 mm3). Time - signal intensity analysis was performed by normalizing to pre-bolus arrival and calculating the mean contrast uptake (MCU) for each patient. Further analyses were performed by customized software to calculate K trans and NBV. Statistical analysis included 2-sided Student's t tests of controls versus patients, receiver operating characteristic analysis, and subgroup analysis of patients according to etiologies of neuropathy. Time-signal intensity analysis showed significantly increased contrast uptake in patients as compared with controls (MCU, 1.29 ± 0.15 vs 1.18 ± 0.08; P < 0.001). This was caused mainly by an increase in K trans (0.046 ± 0.025 vs 0.026 ± 0.016 min(-1); P < 0.001) and less by an increase in NBV (3.9 ± 2.6 vs 3.0 ± 1.9 mL/100 mL; P = 0.12). This trend was true for all etiologies except entrapment neuropathies. Excluding these, receiver operating characteristic analysis found an area under the curve of 0.78 (95% confidence interval, 0.69-0.89) for MCU and 0.77 (95% confidence interval, 0.65-0.90) for K to discriminate neuropathy from control. Dynamic contrast-enhanced perfusion is a feasible technique to assess K trans and NBV in peripheral nerves and may be used in future investigations on peripheral neuropathies.

Bleomycin has been used in the clinic as a chemotherapeutic agent for the treatment of several neoplasms, including non-Hodgkins lymphomas, squamous cell carcinomas, and testicular tumors. The effectiveness of bleomycin is believed to be derived from its ability to bind and oxidatively cleave DNA in the presence of a iron cofactor in vivo. A substantial amount of data on BLM has been collected, there is little information concerning the effects of bleomycin in living cells. In order to obtain data pertinent to the effects of BLM in intact cells, we have exploited the intrinsic fluorescence property of bleomycin to monitor the uptake of the drug in mammalian cells. We employed two light microscopy techniques, a wide-field and three-photon excitation (760 nm) fluorescence microscopy. Treatment of HeLa cells with bleomycin resulted in rapid to localization within the cells. In addition data collected from the wide field experiments, three-photon excitation of BLM which considerably reduced the phototoxic effect compared with UV light excitation in the wide-field microscopy indicated co-localization of the drug to regions of the cytoplasm occupied by the endoplasmic reticulum probe, DiOC<SUB>5</SUB>. The data clearly indicates that the cellular uptake of bleomycin after one minute includes the nucleus as well as in cytoplasm. Contrary to previous studies, which indicate chromosomal DNA as the target of bleomycin, the current findings suggest that the drug is distributed to many areas within the cell, including the endoplasmic reticulum, an organelle that is known to contain ribonucleic acids.

Wide-field microscopy, where full images are obtained simultaneously, is limited by the power available from speckle-free light sources. Currently, the vast majority of wide-field microscopes use either mercury arc lamps, or LEDs as the illumination source. The power available from these sources limits wide-field fluorescent microscopy to tens of microseconds temporal resolution. Lasers, while capable of producing high power and short pulses, have high spatial coherence. This leads to the formation of laser speckle that makes such sources unsuitable for wide-field imaging applications. Random Raman lasers offer the best of both worlds by producing laser-like intensities, short, nanosecond-scale, pulses, and low spatial coherence, speckle-free, output. These qualities combine to make random Raman lasers 4 orders of magnitude brighter than traditional wide-field microscopy light sources. Furthermore, the unique properties of random Raman lasers make possible the entirely new possibilities of wide-field fluorescence lifetime imaging or wide-field Raman microscopy. We will introduce the relevant physics that give rise to the unique properties of random Raman lasing, and demonstrate early proof of principle results demonstrating random Raman lasing emission being used as an imaging light source. Finally, we will discuss future directions and elucidate the benefits of using random Raman lasers as a wide-field microscopy light source.

To compare the quality of cardiac images acquired with two-and four-shot echo-planar imaging (EPI) with that of images acquired with single-shot EPI. Single-, two-, and four-shot EPI was performed to obtain axial images that traversed both cardiac ventricles. The subjects were 10 healthy men whose ages ranged from 22 to 38 years (mean, 27 years). The multiple-shot EPI data were acquired in snapshot (sequential) and cardiac-gated (non-sequential) modes. Image quality was compared by evaluation of the depiction of cardiac structures and analysis of signal intensity, contrast, and signal homogeneity. Multiple-shot EPI depicted the coronary and internal mammary arteries better than did single-shot EPI (P < .05). Intraventricular signal intensity was more homogeneous on multiple-shot images (P < .005). Cardiac-gated acquisition resulted in less image blurring and allowed acquisition of more phases per RR interval. Multiple-shot EPI rendered higher quality images than did single-shot EPI.

Variations of ground-wave signal intensity at standard broadcast frequencies appear to be more closely related to changes in tempetature than to any other single commonly observed meteorological measurement. Results presented here were obtained from an analysis of signal intensities and weather conditions over six paths between 30° and 45° north latitude. It was found that (1) the intensities tend to decrease markedly from their peak values when the temperature becomes high, (2) the amount of such decrease is approximately proportional to the path length in wavelengths, and (3) the temperature at which the peak value occurs varies with frequency. Sample curves of experimental data are presented. General relationships deduced from all paths are combined in a nomographic chart showing intensities relative to the peak value for various frequencies, path lengths, and temperatures.

To evaluate the feasibility to detect and delineate malignant breast lesions in human patients by chemical exchange saturation transfer (CEST) as an MR imaging technique without the need for contrast agent administration. Six female patients referred for pre-surgical staging due to histologically confirmed breast cancer were examined with MR at 3 T. The routine breast protocol included T (2w), STIR, T (1w)-DCE and contrast-enhanced T (1w) imaging with SPAIR fat suppression. For CEST imaging, a 3D RF-spoiled gradient echo (GRE) sequence with an optimized saturation pulse train was applied. To assess the diagnostic value of the technique, CEST effects observed between frequency offsets of 1.2 to 1.8 ppm from the bulk water resonance were compared to pharmacokinetic parameter maps (k (ep)) obtained by DCE-MRI. In 3 of 6 patients, regions with high CEST signal intensity correlated well with tumor areas as determined by DCE-MRI. Analysis of signal intensities from ROIs in tumor, fibroglandular, adipose, and muscle tissue revealed significantly higher CEST values in tumor tissue compared to fibroglandular tissue. The detection of lesions was equally well possible with DCE-MRI and CEST-MRI. In the three other patients, the tumor regions could not be delineated based on the CEST image due to artifacts, which were most likely caused by a high content of fat tissue within the ROIs. The results of this initial feasibility study indicate a significant potential of CEST-MRI to discriminate cancer from fibroglandular tissue in the human breast by a CEST contrast generated by endogenous solute molecules.