High‐speed X‐ray‐induced luminescence computed tomography

X‐ray‐induced luminescence computed tomography (XLCT) is an emerging molecular imaging. Challenges in improving spatial resolution and reducing the scan time in a whole‐body field of view (FOV) still remain for practical in vivo applications. In this study, we present a novel XLCT technique capable of obtaining three‐dimensional (3D) images from a single snapshot. Specifically, a customed two‐planar‐mirror component is integrated into a cone beam XLCT imaging system to obtain multiple optical views of an object simultaneously. Furthermore, a compressive sensing based algorithm is adopted to improve the efficiency of 3D XLCT image reconstruction. Numerical simulations and experiments were conducted to validate the single snapshot X‐ray‐induced luminescence computed tomography (SS‐XLCT). The results show that the 3D distribution of the nanophosphor targets can be visualized much faster than conventional cone beam XLCT imaging method that was used in our comparisons while maintaining comparable spatial resolution as in conventional XLCT imaging. SS‐XLCT has the potential to harness the power of XLCT for rapid whole‐body in vivo molecular imaging of small animals.     

High optical-throughput spectroscopic singlet oxygen and photosensitizer luminescence dosimeter for monitoring of photodynamic therapy

We report a high light-throughput spectroscopic dosimeter system that is able to noninvasively measure luminescence signals of singlet oxygen (¹O₂) produced during photodynamic therapy (PDT) using a CW (continuous wave) light source. The system is based on a compact, fiber-coupled, high collection efficiency spectrometer (>50% transmittance) designed to maximize optical throughput but with sufficient spectral resolution (~7 nm). This is adequate to detect ¹O₂ phosphorescence in the presence of strong luminescence background in vivo. This system provides simultaneous acquisition of multiple spectral data points, allowing for more accurate determination of luminescence baseline via spectral fitting and thus the extraction of ¹O₂ phosphorescence signal based solely on spectroscopic decomposition, without the need for time-gating. Simultaneous collection of photons at different wavelengths improves the quantum efficiency of the system when compared to sequential spectral measurements such as filter-wheel or tunable-filter based systems. A prototype system was tested during in vivo PDT tumor regression experiments using benzoporphyrin derivative (BPD) photosensitizer. It was found that the treatment efficacy (tumor growth inhibition rate) correlated more strongly with ¹O₂ phosphorescence than with PS fluorescence. These results indicate that this high photon-collection efficiency spectrometer instrument may offer a viable option for real-time ¹O₂ dosimetry during PDT treatment using CW light.     

Heterologous expression of the novel α-helical hybrid peptide PR-FO in Bacillus subtilis

PR-FO is a novel α-helical hybrid antimicrobial peptide (AMP) with strong antimicrobial activities and high stability, and the potential to develop into a new generation of antimicrobial agents. In this study, the encoded gene sequence of SMT3-PR-FO was designed and transformed into B. subtilis WB800N. Fusion proteins with concentrations of 16 mg L⁻¹ (SP_amyQ) and 23 mg L⁻¹ (SP_sacB) were obtained after purification by a Ni–NTA resin column. A total of 3 mg (SP_amyQ) and 4 mg (SP_sacB) of PR-FO with a purity of 90% was obtained from 1 L fermentation cultures. Recombinant PR-FO exhibited high inhibition activities against both gram-negative bacteria and gram-positive bacteria, and low haemolytic activity against human red blood cells. These results indicated that the rSMT3-PR-FO could be expressed under the guidance of SP_amyQ and SP_sacB, and the maltose-induced expression strategy might be a safe and efficient method for the soluble peptides production in B. subtilis.

     

Enhanced spatial resolution for snapshot hyperspectral imaging of blood perfusion and melanin information within human tissue

We report a reconstruction method to achieve high spatial resolution for hyperspectral imaging of chromophore features in skin in vivo. The method utilizes an established structure‐adaptive normalized convolution algorithm to reconstruct high spatial resolution of hyperspectral images from snapshot low‐resolution hyperspectral image sequences captured by a snapshot spectral camera. The reconstructed images at chromophore‐sensitive wavebands are used to map the skin features of interest. We demonstrate the method experimentally by mapping the blood perfusion and melanin features (moles) on the facial skin. The method relaxes the constrains of the relatively low spatial resolution in the snapshot hyperspectral camera, making it more usable in imaging applications.     

Deep-skin multiphoton microscopy in vivo excited at 1600 nm: A comparative investigation with silicone oil and deuterium dioxide immersion

Multiphoton microscopy (MPM) excited at the 1700-nm window has enabled deep-tissue penetration in biological tissue, especially brain. MPM of skin may also benefit from this deep-penetration capability. Skin is a layered structure with varying refractive index (from 1.34 to 1.5). Consequently, proper immersion medium should be selected when imaging with high numerical aperture objective lens. To provide guidelines for immersion medium selection for skin MPM, here we demonstrate comparative experimental investigation of deep-skin MPM excited at 1600 nm in vivo, using both silicone oil and deuterium dioxide (D₂O) immersion. We specifically characterize imaging depths, signal levels and spatial resolution. Our results show that both immersion media give similar performance in imaging depth and spatial resolution, while signal levels are slightly better with silicone oil immersion. We also demonstrate that local injection of fluorescent beads into the skin is a viable technique for spatial resolution characterization in vivo.      

Optimization of spatial resolution and scattering effects for biomedical fluorescence imaging by using sub-regions of the shortwave infrared spectrum

We evaluated the impact of light-scattering effects on spatial resolution in different shortwave infrared (SWIR) sub-regions by analyzing two SWIR emissive phantoms made of polydimethylsiloxane (PDMS)-gold nanoclusters (Au NCs) composite covered with mice skin, or capillary tubes filled with Au NCs or IRDye 800CW at different depth in intralipids and finally, after administration of the Au NCs intravenously in mice. Our findings highlighted the benefit of working at the highest tested spectral range of the SWIR region with a 50% enhancement of spatial resolution measured in artificial model when moving from NIR-II (1000-1300 nm) to NIR-IIa (1300-1450 nm) region, and a 25% reduction of the scattering from the skin determined by point spread function analysis from the NIR-II to NIR-IIb region (1500-1700 nm). We also confirmed that a series of Monte Carlo restoration of images significantly improved the spatial resolution in vivo in mice in deep tissues both in the NIR-II and NIR-IIa spectral windows.     

The effect of tube focal spot size and acquisition mode on task-based image quality performance of a GE revolution HD dual energy CT scanner

PurposeTo assess the task-based performance of images obtained under different focal spot size and acquisition mode on a dual-energy CT scanner.MethodsAxial CT image series of the Catphan phantom were obtained using a tube focus at different sizes. Acquisitions were performed in standard single-energy, high resolution (HR) and dual-energy modes. Images were reconstructed using conventional and high definition (HD) kernels. Task-based transfer function at the 50% level (TTF_50%) for teflon, delrin, low density polyethylene (LDPE) and acrylic, as well as image noise and noise texture, were assessed across all focal spots and acquisition modes using Noise Power Spectrum (NPS) analysis. A non-prewhitening mathematical observer model was used to calculate detectability index ($d_{\mathit{NPW}}^{\prime }$).ResultsTTF_50% degraded with increasing focal spot size. TTF_50% ranged from 0.67 mm⁻¹ for teflon to 0.25 mm⁻¹ for acrylic. For standard kernel, image noise and NPS-determined average spatial frequency were 8.3 HU and 0.29 mm⁻¹, respectively in single-energy, 12.0 HU and 0.37 mm⁻¹ in HR, and 7.9 HU and 0.26 mm⁻¹ in dual-energy mode. For standard kernel, $d_{\mathit{NPW}}^{\prime }$ was 61 in single-energy and HR mode and reduced to 56 in dual-energy mode.ConclusionsThe task-based image quality assessment metrics have shown that spatial resolution is higher for higher image contrast materials and detectability is higher in the standard single-energy mode compared to HR and dual-energy mode. The results of the current study provide CT operators the required knowledge to characterize their CT system towards the optimization of its clinical performance.     

Three-dimensional spatial reconstruction of coronary arteries based on fusion of intravascular optical coherence tomography and coronary angiography

We present a three-dimensional (3D) spatial reconstruction of coronary arteries based on fusion of intravascular optical coherence tomography (IVOCT) and digital subtraction angiography (DSA). Centerline of vessel in DSA images is exacted by multi-scale filtering, adaptive segmentation, morphology thinning and Dijkstra's shortest path algorithm. We apply the cross-correction between lumen shapes of IVOCT and DSA images and match their stenosis positions to realize co-registration. By matching the location and tangent direction of the vessel centerline of DSA images and segmented lumen coordinates of IVOCT along pullback path, 3D spatial models of vessel lumen are reconstructed. Using 1121 distinct positions selected from eight vessels, the correlation coefficient between 3D IVOCT model and DSA image in measuring lumen radius is 0.94% and 97.7% of the positions fall within the limit of agreement by Bland–Altman analysis, which means that the 3D spatial reconstruction IVOCT models and DSA images have high matching level.     

A novel in vivo Cerenkov luminescence image‐guided surgery on primary and metastatic colorectal cancer

Intraoperative Cerenkov luminescence imaging (CLI) can effectively improve the performance of tumor surgery. Nevertheless, the existing approaches are still unsatisfying to the clinical demands of open surgery. This study develops a novel intraoperative in vivo CLI approach to investigate the potential and value of Cerenkov luminescence (CL) image‐guided surgery. A system characterized with high sensitivity (19.61 kBq mL^{?1 18}F‐FDG) and desirable spatial resolution (88.34 μm) is developed. CL image‐guided surgery is performed on colorectal cancer (CRC) models of mice and swine. Tumor surgery is guided by the static CL images, and the resection quality is evaluated quantitatively and contrasted with other imaging modalities exemplified by bioluminescence imaging (BLI). The in vivo results demonstrated the effectiveness of the proposed intraoperative CLI approach for removing primary and metastatic CRC. Safety of performing in vivo CL image‐guided surgery is verified as well through radiation measurements of related staffs. Overall, the developed intraoperative in vivo CLI approach can efficiently improve the cancer treatment.     

Inside Cover: Sparse‐graph manifold learning method for bioluminescence tomography (J. Biophotonics 4/2020)

This study proposed a Sparse‐Graph Manifold Learning (SGML) method to balance the sparseness and morphology preserving for bioluminescence tomography reconstruction. It inherits the benefits of non‐convex sparsity constraint and dynamic Laplacian graph model. The results of numerical simulations and in vivo experiments demonstrate that the proposed method yields accurate and robust results in terms of tumor spatial location and morphology recovery. Further details can be found in the article by Hongbo Guo, Ling Gao, Jingjing Yu, et al. ( e201960218 )

     

Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis

Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SP_Nuc) by that of L. lactis protein Usp45 (SP_Usp) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SP_Usp than when directed by SP_Nuc. This SP_Usp effect on Nuc secretion is not due to a better antifolding activity, since SP_Usp:Nuc precursor proteins display enzymatic activity in vitro, while SP_Nuc:Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895–1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SP_Usp and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.     

High spatio-temporal resolution measurement of A1R and A2AR interactions combined with Iem-spFRET and E-FRET methods

A₁R-A_2AR heterodimers regulate striatal glutamatergic neurotransmission. However, few researches about kinetics have been reported. Here, we combined Iem-spFRET and E-FRET to investigate the kinetics of A₁R and A_2AR interaction. Iem-spFRET obtains the energy transfer efficiency of the whole cell. E-FRET gets energy transfer efficiency with high spatial resolution, whereas, it was prone to biases because background was easily selected due to manual operation. To study the interaction with high spatio-temporal resolution, Iem-spFRET was used to correct the deviation of E-FRET. In this paper, A₁R and A_2AR interaction was monitored, and the changes of FRET efficiency of the whole or/and partial cell membrane were described. The results showed that activation of A₁R or A_2AR leads to rapid aggregation, inhibition of A₁R or A_2AR leads to slow segregation, and the interaction is reversible. These results demonstrated that combination of Iem-spFRET and E-FRET could measure A₁R and A_2AR interaction with high spatio-temporal resolution.     

M-BISON: Microarray-based integration of data sources using networks

Background  

The accurate detection of differentially expressed (DE) genes has become a central task in microarray analysis. Unfortunately, the noise level and experimental variability of microarrays can be limiting. While a number of existing methods partially overcome these limitations by incorporating biological knowledge in the form of gene groups, these methods sacrifice gene-level resolution. This loss of precision can be inappropriate, especially if the desired output is a ranked list of individual genes. To address this shortcoming, we developed M-BISON (Microarray-Based Integration of data SOurces using Networks), a formal probabilistic model that integrates background biological knowledge with microarray data to predict individual DE genes.     

LUMIMESCENCE IN PELAGIA NOCTILUCA

1. Ca and K condition the irritability of Pelagia both in regard to rhythmical contractions and general luminescence. If either ion is omitted from the solution conduction of stimuli for pulsations and luminescence does not occur, although local responses still persist. 2. When Mg is omitted from the solution, Pelagia shows hyper-irritability with respect to rhythmical contraction and general luminescence. This is referable to the unantagonized action of K and Ca ions. 3. Exposure to the carbon arc suppresses general luminescence, the effect depending upon the quantity of light i.e. intensity x time of exposure. 4. The luminescent material secreted by Pelagia is inactive in sea water, but when put into salt solutions is activated by some of them. The efficiency of the salts, measured by brightness of light, is in the following order: MgSO₄, K₂SO₄, Na₃ citrate, KCl, BaCl₂, SrCl₂, CaCl₂, and LiCl while NaCl and MgCl₂ act as inhibitors. 5. Acidity inhibits the reaction, alkalinity promotes it. NH₄OH in concentrations 0.27 N to 0.9 N causes luminescence for 10 minutes at 20°. 6. The average temperature coefficient for the reaction of the luminescent substance when activated by ammonia or MgSO₄ is 2.18 for a temperature interval of 10°C. 7. The luminescence reaction cannot be the result of cytolysis, because (a) raising the temperature of sea water in which luminous material is immersed does not cause luminescence, although sufficient to produce cytolysis. (b) The salt solutions used in our experiments to cause luminescence, do not act cytolytically on cells in general.     

A 3D-printed Apparatus for Imaging Multiple Rats Simultaneously

CT (computerized tomography) is a necessary imaging modality for cancer staging and disease monitoring. Rodent models of cancer are commonly studied prior to human clinical trials, but CT in rodents can be difficult due to their small size and constant movement, which necessitates general anesthesia. Because microCT equipment is not always available, clinical CT may be a viable alternative. Limitations of microCT and clinical CT include biosecurity, anesthesia to limit image distortion due to motion, and cost. To address several of these constraints, we created a 3D-printed apparatus that accommodated simultaneous imaging of as many as 9 rats under gas anesthesia. Rats were anesthetized in series and placed in a 3 × 3 arrangement. To assess differences in attenuation between individual chambers and rows or columns in the device, we first imaged a standardized phantom plug as a control. We hypothesized that attenuation of specific rat organs would not be affected regardless of the location or position in the 3D-printed device. Four organs—liver, kidney, femur, and brain—were evaluated in 9 rats. For both the phantom and kidneys, statistically significant, but clinically negligible, effects on attenuation were noted between rows but not between columns. We attribute this finding to the absence of a top layer of the apparatus, which thus created asymmetric attenuation and beam hardening through the device. This apparatus allowed us to successfully image 9 rats simultaneously in a clinical CT machine, with negligible effects on attenuation. Planned improvements in this apparatus include completely enclosed versions for biosecure imaging.

Preclinical studies routinely require the use of advanced diagnostic imaging techniques to evaluate animal models of human disease.²⁵ Longitudinal imaging studies with cohorts that are large enough to permit valid conclusions may be limited by cost and time factors. In addition, research institutions frequently use human equipment, necessitating strict pathogen exclusion measures.¹⁶ Although microCT (computerized tomography) has been developed and used for high anatomic resolution of small animals, the equipment may be unavailable for use or difficult to access.²³ In addition, the high resolution (typically 50 to 100 um³ voxel spacing) afforded by this equipment may not be necessary for a given experiment. A clinical CT machine (typical spatial resolution, 0.5 to 1.0 mm³) may be an appropriate alternative in some situations. To overcome cost and time constraints, several multi-animal imaging devices have been created; however, they rely on injectable anesthesia.^10,33 This reliance can be challenging with regard to accurate monitoring of anesthesia in stacked or scaffold-style imaging devices, difficulty in redosing (if needed), and prolonged recovery times. Furthermore, the use of injectable anesthetics for CT has been associated with risk of corneal ulceration.³² In contrast, inhalant anesthesia achieves rapid induction, is easy to adjust to maintain appropriate anesthetic depth, and allows a quick recovery.⁶The majority of imaging devices described to date have been limited to use with mice.^10,33 Rats are the second most frequently used mammal in biomedical research and their popularity is growing due to advances in genome editing techniques, larger animal size, and physiologic parameters more similar to humans.⁶ Improving the convenience and cost of imaging rats has the potential to advance preclinical study outcomes by facilitating longitudinal translational studies.To address the challenges just described, we created a 3D-printed imaging device that would allow simultaneous imaging of multiple rats under inhalation anesthesia. After creating the device, we tested whether animals in that configuration could be scanned accurately without poor image quality or loss of detail. In any stacked or rotary device, the penetrating X-ray beam has to pass through multiple animals in various locations and scaffold materials. We tested whether our device would alter the attenuation of the X-ray beam and thus artifactually alter the image itself. A CT image is created by measuring the attenuation coefficient of various tissues; the attenuation is proportional to the tissue''s electron density. Stated simply, attenuation of the X-ray beam is greater in tissues that have greater density. The densities are measured by using Hounsfield units (HU), where 0 HU equals water density. Tissues with a greater electron density than water have a positive HU value and appear brighter, and tissues with a lower electron density than water have a negative HU value and seem darker.⁵ We hypothesized that the position of a rat in the apparatus would not affect the attenuation associated with specific organs (that is brain, liver, bone, and kidney). We also hypothesized that row and column would not affect the attenuation of a phantom control or organs.     

One- and Two-Dimensional lH NMR Study of the Substance P Fragment ARG-PRO-LYS-PRO

Abstract

The Substance P fragment Arg¹Pro²-Lys³-Pro⁴ (SP_1–4) has been extensively investigated by means of proton nuclear magnetic resonance at 400 MHz. The combined application of different 2D techniques and a comparison of SP_1–4 with its derivative SPM-amide allowed the complete and unambiguous assignment of the proton NMR spectrum. Conformational data obtained from the different NMR parameters are compared with theoretical calculations. The results suggest that SP_1–4 exists, at the chosen experimental conditions, as a stretched molecule.