Multi-focus microscope with HiLo algorithm for fast 3-D fluorescent imaging.
ABSTRACT: In this paper, we present a new multi-focus microscope (MFM) system based on a phase mask and HiLo algorithm, achieving high-speed (20 volumes per second), high-resolution, low-noise 3-D fluorescent imaging. During imaging, the emissions from the specimen at nine different depths are simultaneously modulated and focused to different regions on a single CCD chip, i.e., the CCD chip is subdivided into nine regions to record images from the different selected depths. Next, HiLo algorithm is applied to remove the background noises and to form clean 3-D images. To visualize larger volumes, the nine layers are scanned axially, realizing fast 3-D imaging. In the imaging experiments, a mouse kidney sample of ~ 60 × 60 × 16 ?m3 is visualized with only 10 raw images, demonstrating substantially enhanced resolution and contrast as well as suppressed background noises. The new method will find important applications in 3-D fluorescent imaging, e.g., recording fast dynamic events at multiple depths in vivo.
Project description:We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.
Project description:Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.
Project description:We present here a fast optical sectioning method for mesoscopy based on HiLo microscopy, which makes possible imaging of specimens of up to 4.4?mm?×?3?mm?×?3?mm in volume in under 17?hours (estimated for a z-stack comprising 1000 images excluding computation time) with subcellular resolution throughout. Widefield epifluorescence imaging is performed with the Mesolens using a high pixel-number camera capable of sensor-shifting to generate a 259.5 Megapixel image, and we have developed custom software to perform HiLo processing of the very large datasets. Using this method, we obtain comparable sectioning strength to confocal laser scanning microscopy (CLSM), with sections as thin as 6.8?±?0.2 ?m and raw acquisition speed of 1?minute per slice which is up to 30 times faster than CLSM on the full field of view (FOV) of the Mesolens of 4.4?mm with lateral resolution of 0.7 ?m and axial resolution of 7 ?m. We have applied this HiLo mesoscopy method to image fixed and fluorescently stained hippocampal neuronal specimens and a 5-day old zebrafish larva.
Project description:The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target (HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells.
Project description:BACKGROUND AND PURPOSE:Arterial spin-labeling (ASL) was recently introduced as a noninvasive method to evaluate cerebral hemodynamics. The purposes of this study were to assess the ability of ASL imaging to detect crossed cerebellar diaschisis (CCD) in patients with their first unilateral supratentorial hyperacute stroke and to identify imaging or clinical factors significantly associated with CCD. MATERIALS AND METHODS:We reviewed 204 consecutive patients who underwent MRI less than 8 hours after the onset of stroke symptoms. The inclusion criteria were supratentorial abnormality in diffusion-weighted images in the absence of a cerebellar or brain stem lesion, bilateral supratentorial infarction, subacute or chronic infarction, and MR angiography showing vertebrobasilar system disease. For qualitative analysis, asymmetric cerebellar hypoperfusion in ASL images was categorized into 3 grades. Quantitative analysis was performed to calculate the asymmetric index (AI). The patients' demographic and clinical features and outcomes were recorded. Univariate and multivariate analyses were also performed. RESULTS:A total of 32 patients met the inclusion criteria, and 24 (75%) presented CCD. Univariate analyses revealed more frequent arterial occlusions, higher diffusion-weighted imaging (DWI) lesion volumes and higher initial NIHSS and mRS scores in the CCD-positive group compared with the CCD-negative group (all p < .05). The presence of arterial occlusion and the initial mRS scores were related with the AI (all p < .05). Multivariate analyses revealed that arterial occlusion and the initial mRS scores were significantly associated with CCD and AI. CONCLUSION:ASL imaging could detect CCD in 75% of patients with hyperacute infarction. We found that CCD was more prevalent in patients with arterial occlusion, larger ischemic brain volumes, and higher initial NIHSS and mRS scores. In particular, vessel occlusion and initial mRS score appeared to be significantly related with CCD pathophysiology in the hyperacute stage.
Project description:Advance care planning (ACP) seeks to promote care delivery that is concordant with patients' informed wishes. Scalability and cost may be barriers to widespread ACP, and video decision aids may help address such barriers.Our primary hypothesis was that ACP documentation would increase in Hilo after ACP video implementation. Secondary hypotheses included increased use of hospice, fewer deaths in the hospital, and decreased costs in the last month of life.The city of Hilo in Hawai'i (population 43,263), which is served by one 276-bed hospital (Hilo Medical Center), one hospice (the Hospice of Hilo), and 30 primary care physicians.The intervention consisted of a single, 1- to 4-h training and access to a suite of ACP video decision aids.Prior to implementation, the rate of ACP documentation for hospitalized patients with late-stage disease was 3.2 % (11/346). After the intervention, ACP documentation was 39.9 % (1,107/2,773) (P?<?0.001). Primary care providers in the intervention had an ACP completion rate for patients over 75 years of 37.0 % (1,437/3,888) compared to control providers, who had an average of 25.6 % (10,760/42,099) (P?<?0.001). The rate of discharge from hospital to hospice for patients with late-stage disease was 5.7 % prior to the intervention and 13.8 % after the intervention (P?<?0.001). The average total insurance cost for the last month of life among Hilo patients was $3,458 (95 % CI $3,051 to 3,865) lower per patient after the intervention when compared to the control region.Implementing ACP video decision aids was associated with improved ACP documentation, greater use of hospice, and decreased costs. Decision aids that promote ACP offer a scalable and cost-efficient medium to place patients at the center of their care.
Project description:It is increasingly important to measure cell mechanical properties in three-dimensional environments. Particle tracking microrheology (PTM) can measure cellular viscoelastic properties; however, out-of-plane data can introduce artifacts into these measurements. We developed a technique that employs HiLo microscopy to reduce out-of-plane contributions. This method eliminated signals from 90% of probes 0.5 μm or further from the focal plane, while retaining all in-plane probes. We used this technique to characterize live-cell bilayers and found that there were significant, frequency-dependent changes to the extracted cell moduli when compared to conventional analysis. Our results indicate that removal of out-of-plane information is vital for accurate assessments of cell mechanical properties.
Project description:We describe a charge-coupled device (CCD) imaging system for microarrays capable of acquiring quantitative, high dynamic range images of very large fields. Illumination is supplied by an arc lamp, and filters are used to define excitation and emission bands. The system is linear down to fluorochrome densities <<1 molecule/microm2. The ratios of the illumination intensity distributions for all excitation wavelengths have a maximum deviation approximately +/-4% over the object field, so that images can be analyzed without computational corrections for the illumination pattern unless higher accuracy is desired. Custom designed detection optics produce achromatic images of the spectral region from approximately 450 to approximately 750 nm. Acquisition of a series of images of multiple fluorochromes from multiple arrays occurs under computer control. The version of the system described in detail provides images of 20 mm square areas using a 27 mm square, 2K x 2K pixel, cooled CCD chip with a well depth of approximately 10(5) electrons, and provides ratio measurements accurate to a few percent over a dynamic range in intensity >1000. Resolution referred to the sample is 10 microm, sufficient for obtaining quantitative multicolor images from >30,000 array elements in an 18 mm x 18 mm square.
Project description:Temporal focusing multiphoton microscopy (TFMPM) has the advantage of area excitation in an axial confinement of only a few microns; hence, it can offer fast three-dimensional (3D) multiphoton imaging. Herein, fast volumetric imaging via a developed digital micromirror device (DMD)-based TFMPM has been realized through the synchronization of an electron multiplying charge-coupled device (EMCCD) with a dynamic piezoelectric stage for axial scanning. The volumetric imaging rate can achieve 30 volumes per second according to the EMCCD frame rate of more than 400 frames per second, which allows for the 3D Brownian motion of one-micron fluorescent beads to be spatially observed. Furthermore, it is demonstrated that the dynamic HiLo structural multiphoton microscope can reject background noise by way of the fast volumetric imaging with high-speed DMD patterned illumination.
Project description:Candida parapsilosis is the most frequent cause of catheter-related candidemia among non-Candida albicans species. This may be related to intrinsic capabilities as adhering and forming a biofilm on abiotic surfaces such as on medical devices. As previously demonstrated, patients infected with high biofilm-producing C. parapsilosis isolates had a greater mortality risk compared to patients infected with low biofilm-producing C. parapsilosis isolates. We developed the BIOF-HILO assay, a MALDI-TOF mass spectrometry (MS)-based assay, which compares mass spectra obtained from attached and suspended isolate cells during the early (i.e., 3-h) adhesion phase of in vitro biofilm formation. The composite correlation index (CCI) analysis was used to discriminate between mass spectra differences of the two cell types, classifying all 50 C. parapsilosis clinical isolates, included in the study, after only 3-h of testing, in high or low biofilm producers. All high (n = 25) or low (n = 25) biofilm producers had, according to CCI mass spectra comparison values, higher or lower than one CCI ratios, which were obtained by dividing the CCIsuspended cells by the CCIattached cells. In conclusion, the BIOF-HILO assay allows a rapid categorization of C. parapsilosis clinical isolates in high or low biofilm producers. This information, if timely provided to physicians, may improve treatment outcomes in patients with C. parapsilosis candidemia.