Determination of regional bone blood flow by means of fluorescent microspheres using an automated sample-processing procedure

The determination of regional blood flow utilizing fluorescent microspheres (FMs) is an established method for numerous organs. Recent progress, in particular the automation of sample processing, has further improved this method. However, the FM method (reference sample technique), which allows repetitive measurement of regional organ blood flow, has so far not been used for the determination of blood flow in bone. The aim of the present study was to establish FM for the quantification of regional bone blood flow (RBBF). Female, anesthetized New Zealand rabbits (n = 6) received left ventricular injections of different amounts of FM at six subsequent time points. In order to examine the precision of RBBF determination, two different FM species were injected simultaneously at the sixth injection. At the end of the experiments the femoral and tibial condyles of each hind limb were removed and the fluorescence intensity in the tissue samples was measured by an automated procedure. In an in vitro study we have shown that acid digestion of the crystalline matrix has no effect on the fluorescence characteristics of FM. The determination of the number of spheres per tissue sample revealed that depending on the tissue sample size up to 3 x 10(6) spheres/injection were necessary to obtain about 400 microspheres in the individual bone samples. RBBF values of the tibial and femoral condyles did not differ at various injection intervals. The tibial blood flow values varied between 6.6 +/- 1.1 and 8.5 +/- 1.4 ml/min/100 g and were significantly higher than those of the femur (4.3 +/- 1.1 to 6.0 +/- 1.8 ml/min/100 g). The bone blood flow values obtained by simultaneous injection of two FM species correlated significantly (r = 0.96, slope = 1.06, intercept = 0.05), the mean difference was 0.39 +/- 1.11 ml/min/100 g. Our data demonstrate that the measurement of RBBF by means of FM allows a valid determination of RBBF. Copyright (C) 2003 S. Karger AG, Basel

Comparison of regional blood flow values measured by radioactive and fluorescent microspheres

Fluorescent microspheres (FM) have become an attractive alternative to radioactive microspheres (RM) for the measurement of regional blood flow (RBF). The aim of the present study was to investigate the comparability of both methods by measuring RBF with FM and RM. Eight anaesthetised pigs received simultaneous, left atrial injections of FM and RM with a diameter of 15 mum at six different time points. Blood reference samples were collected from the descending aorta. RBF was determined in tissue samples of the myocardium, spleen and kidneys of all 8 animals. After radioactivity of the tissue samples was determined, the samples were processed automatically for measuring fluorescence using a recently developed filter device (SPU). RBF was calculated with both the isotope and spectrometric data of both methods for each sample resulting in a total of 10,512 blood flow values. The comparison of the RBF values yielded high linear correlation (mean r(2) = 0.95 +/- 0.03 to 0.97 +/- 0.02) and excellent agreement (bias 5.4-6.7%, precision 9.9-16.5%) of both methods. Our results indicate the validity of MS and of the automated tissue processing technique by means of the SPU. Copyright (C) 2002 S. Karger AG, Basel

Use of fluorescent micropsheres to measure coronary flow reserve in rat animal model

Heart attacks result from reduced or blocked blood flow through major coronary arteries, resulting in permanent damage to heart muscle. Coronary blood flow (CBF) is thus important to measure in experimental animal models of heart disease. A standard method to measure CBF uses tracer microspheres (Ø = 15 µm) injected into the left ventricle that flow through coronary arteries but cannot pass through capillaries and so become trapped in heart muscle. Previously, radioactive or colored microspheres have quantified the number of tracers trapped in the muscle. Fluorescent microspheres offer a more recent and more sensitive measurement mode. However, fluorescent microspheres have not often been used to measure CBF in small animals (rats, mice) that are now the most common animal models used in heart research. This thesis aimed to develop the techniques for use of fluorescent microspheres to measure CBF in rat animal models used by the cell biology laboratories at UMDNJ-Newark. Two non-overlapping fluorescent wavelengths were chosen (yellow-green; red). Using a spectrophotometer, fluorescence intensity was calibrated for known numbers of microspheres (set via controlled dilution). CBF in two rats was measured at rest and during maximal vasodilation (adenosine) using procedures for colored microspheres. After euthanasia, hearts were removed, and blood samples and left ventricular tissue were processed using a sedimentation method for full recovery of fluorescent microspheres, which were scanned through the spectrophotometer to count fluorescence intensity. Using the predetermined calibration curve, the number of microspheres in each sample was determined; from this CBF was calculated. CBF averaged 5.9 ml/min/g at rest, which was within the normal range for rats quoted in recent literature. With maximal vasodilation, CBF increased to an average of 12.9 ml/min/g, which indicated a coronary flow reserve that was 2.2 times the resting level. The same value for coronary flow velocity reserve (2.2) was measured in 6 rats using Doppler echocardiography. The consistency of these results suggests that the procedures developed for fluorescent microspheres lead to repeatable and reliable measurement of coronary blood flow in rats

Automated Fourier space region-recognition filtering for off-axis digital holographic microscopy

Automated label-free quantitative imaging of biological samples can greatly benefit high throughput diseases diagnosis. Digital holographic microscopy (DHM) is a powerful quantitative label-free imaging tool that retrieves structural details of cellular samples non-invasively. In off-axis DHM, a proper spatial filtering window in Fourier space is crucial to the quality of reconstructed phase image. Here we describe a region-recognition approach that combines shape recognition with an iterative thresholding to extracts the optimal shape of frequency components. The region recognition technique offers fully automated adaptive filtering that can operate with a variety of samples and imaging conditions. When imaging through optically scattering biological hydrogel matrix, the technique surpasses previous histogram thresholding techniques without requiring any manual intervention. Finally, we automate the extraction of the statistical difference of optical height between malaria parasite infected and uninfected red blood cells. The method described here pave way to greater autonomy in automated DHM imaging for imaging live cell in thick cell cultures

Advances in Microtechnology for Improved Cytotoxicity Assessment

In vitro cytotoxicity testing is essential in the pharmaceutical and environmental industry to study the effects of potential harmful compounds for human health. Classical assays present several disadvantages: they are commonly based on live-death labelling, are highly time consuming and/or require skilled personnel to be performed. The current trend is to reduce the number of required cells and the time during the analysis, while increasing the screening capability and the accuracy and sensitivity of the assays, aiming single cell resolution. Microfabrication and surface engineering are enabling novel approaches for cytotoxicity assessment, offering high sensitivity and the possibility of automation in order to minimize user intervention. This review aims to overview the different microtechnology approaches available in this field, focusing on the novel developments for high-throughput, dynamic and real time screening of cytotoxic compounds.Funding support from: University of the Basque Country (PIF16/204), the funding support from Gobierno de España, Ministerio de Economía y Competitividad, with Grant No. BIO2016-80417-P (AEI/FEDER, UE) and Gobierno Vasco under grand IT1271-19

DNA SEPARATION AT A STRETCH AND MULTISTAGE MAGNETIC SEPARATION OF MICROSPHERES

This thesis consists of two parts. The first part focuses on development of a novel DNA separation technology by tethering DNA strands to a solid surface and then stretching the DNA with an electric field. The anchor is such designed that the critical force to detach a DNA is independent of its size. Because the stretching force is proportional to the DNA net charge, a gradual increase of the electric field leads to size-based removal of the DNA from the surface and thus DNA separation. This strategy may provide a convenient, low-cost, and high-speed alternative to existing methods for DNA separation, because sieving matrices are not required, separated DNA can be readily recovered, and in principle, there is no upper limit on the length of DNA that can be separated. Using this method, we have demonstrated (i) efficient separation of lambda double-stranded DNA (dsDNA) (48,502 bp) from human genomic dsDNA (>100 kbp) in a dc electric field applied between two parallel plates, (ii) separation of short single-stranded DNA (ssDNA) with less than 100 nucleotides (nt) at 10-nt resolution by tethering and stretching DNA in microfluidic channels filled with a low conductivity buffer, and (iii) separation of short ssDNA by taking the advantage of the strong yet evolving non-uniform electric field near the charged Au surface in contact with an electrolyte. The second part of my thesis focuses on development of a multistage separation technology to circumvent the challenge caused by non-specific interactions in current single-stage magnetic separation techniques. The key idea is to allow the magnetic particles (MNPs) to reversibly capture and release the targets by manipulating the hydrophobic interaction between the MNPs and the targets. This will be enabled by attaching temperature-responsive polymers to both the MNPs and the targets. Through temperature cycling, which triggers the reversible hydrophilic-to-hydrophobic phase transition of the polymers, the targets can be reversibly captured and released by the MNPs (due to hydrophobic interaction) at a higher efficiency than the non-targets which may also be captured and released by the MNPs due to non-specific interactions. The difference in the capture-and-release efficiencies of targets versus non-targets in a single cycle will be amplified by multiple separation stages, following a similar concept to the distillation process. As a proof-of-concept demonstration, we have demonstrated efficient separation of poly(N-isopropylacrylamide) (PNIPAM, a temperature responsive polymer)-functionalized polystyrene (PS) microspheres from bare PS microspheres by using PNIPAM-functionalized MNPs. The overall enrichment factor is observed to significantly increase with the number of separation stages, and reaches as high as 1.87 E+5 after 5 stages

A proposal for a coordinated effort for the determination of brainwide neuroanatomical connectivity in model organisms at a mesoscopic scale

In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is however critical both for basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brain-wide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brain-wide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open access data repository; compatibility with existing resources, and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.Comment: 41 page

Scanning-fluorescence Reader Based on Embedded System

To measure the concentration of C-reactive protein (CRP) in serum, a portable, scanning-fluorescence reader based on time-resolved fluoroimmunoassays was developed. The scanning-fluorescence reader integrates with the AD7707 converter, which performs at a high accuracy. The photosensitive diode acts as the photoelectric conversion device, an optical module based on optical fibers, which is able to concentrate the excitation light from an LED into a line-shape beam, was designed to sendand receive the optical signal. The device subsequently addresses waveform data using a gradient, smoothing, and binarization method. When the device measures the CRP fluorescence test strip, the results exhibited a good linearity (0.99998) and the CVs (coefficient of variation) were below 5%, which indicate high accuracy. At the same time the system is low cost and small size