Wide field-of-view Talbot grid-based microscopy for multicolor fluorescence imaging.
TL;DR: A microscopic slide-imaging system that can achieve multicolor, wide FOV, fluorescence imaging based on the Talbot effect, which was used to nimage green fluorescent beads, double-stained human breast cancer SK-BR-3 cells, Giardia lamblia cysts, and the Cryptosporidium parvum oocysts.
Abstract: The capability to perform multicolor, wide field-of-view (FOV) fluorescence microscopy imaging is important in screening and pathology applications. We developed a microscopic slide-imaging system that can achieve multicolor, wide FOV, fluorescence imaging based on the Talbot effect. In this system, a light-spot grid generated by the Talbot effect illuminates the sample. By tilting the excitation beam, the Talbot-focused spot scans across the sample. The images are reconstructed by collecting the fluorescence emissions that correspond to each focused spot with a relay optics arrangement. The prototype system achieved an FOV of 12 × 10 mm^2 at an acquisition time as fast as 23 s for one fluorescence channel. The resolution is fundamentally limited by spot size, with a demonstrated full-width at half-maximum spot diameter of 1.2 μm. The prototype was used to nimage green fluorescent beads, double-stained human breast cancer SK-BR-3 cells, Giardia lamblia cysts, and the Cryptosporidium parvum oocysts. This imaging method is scalable and simple for implementation of high-speed wide FOV fluorescence microscopy.
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TL;DR: An optical lens system for 3D imaging of objects up to 6 mm wide and 3 mm thick with depth resolution of only a few microns instead of the tens of microns currently attained, allowing sub-cellular detail to be resolved throughout the volume.
Abstract: Current optical microscope objectives of low magnification have low numerical aperture and therefore have too little depth resolution and discrimination to perform well in confocal and nonlinear microscopy. This is a serious limitation in important areas, including the phenotypic screening of human genes in transgenic mice by study of embryos undergoing advanced organogenesis. We have built an optical lens system for 3D imaging of objects up to 6 mm wide and 3 mm thick with depth resolution of only a few microns instead of the tens of microns currently attained, allowing sub-cellular detail to be resolved throughout the volume. We present this lens, called the Mesolens, with performance data and images from biological specimens including confocal images of whole fixed and intact fluorescently-stained 12.5-day old mouse embryos.
117 citations
Cites background from "Wide field-of-view Talbot grid-base..."
...This usually involves placing the specimen in direct contact with the imaging sensor (Pang et al., 2013), which, because of the requirement for immersion and mounting of specimens in high-index material, is impractical for thick tissue imaging....
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...The work of Pang et al. overcomes this limitation (Pang et al., 2013), and makes possible imaging of specimens spatially separated from the sensor with fluorescence contrast and an exceptionally wide field of view....
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TL;DR: A wide FOV microscopy system that uses a closed-circuit-television lens for image relay and a flatbed scanner for data acquisition is reported, showing that such an imaging system is capable of capturing a 10 mm × 7.5 mm FOV image with 0.78 µm resolution.
Abstract: The capability to perform high-resolution, wide field-of-view (FOV) microscopy imaging is highly sought after in biomedical applications. In this paper, we report a wide FOV microscopy system that uses a closed-circuit-television (CCTV) lens for image relay and a flatbed scanner for data acquisition. We show that such an imaging system is capable of capturing a 10 mm × 7.5 mm FOV image with 0.78 µm resolution, resulting in more than 0.5 billion pixels across the entire image. The resolution and field curve of the proposed system were characterized by imaging a USAF resolution target and a hole-array target. To demonstrate its application, 0.5 gigapixel images of histology slides were acquired using this system.
58 citations
TL;DR: A lens-free fluorescence imaging device using a composite filter composed of an interference filter and an absorption filter, each applied to one side of a fiber optic plate (FOP) that is compatible with super-resolution bright-field imaging techniques is reported.
Abstract: We report a lens-free fluorescence imaging device using a composite filter composed of an interference filter and an absorption filter, each applied to one side of a fiber optic plate (FOP). The transmission of angled excitation light through the interference filter is absorbed by the absorption filter. The auto-fluorescence of the absorption filter is reduced by the reflection from the interference filter of normally incident excitation light. As a result, high-performance rejection of excitation light is achieved in a lens-free device. The FOP provides a flat, hard imaging device surface that does not degrade the spatial resolution. We demonstrate excitation rejection of approximately 108:1 at a wavelength of 450 nm in a fabricated lens-free device. The resolution of fluorescence imaging is approximately 12 µm. Time-lapse imaging of cells containing green fluorescent protein was performed in a 5-µm thin-film chamber. The small dimensions of the device allow observation of cell culturing in a CO2 incubator. We also demonstrate that the proposed lens-free filter is compatible with super-resolution bright-field imaging techniques. These features open a way to develop a high-performance, dual-mode, lens-free imaging device that is expected to be a powerful tool for many applications, such as imaging of labeled cells and point-of-care assay.
41 citations
Cites background from "Wide field-of-view Talbot grid-base..."
...Another way to improve spatial resolution is to use a patterned excitation light [10,26,43]....
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Patent•
08 Mar 2012TL;DR: In this article, a phase gradient generating device was used to generate a light field incident the Talbot element. And then, a light detector captured time varying data associated with light altered by an object located at the distance from the Talboid element.
Abstract: Talbot imaging systems comprising a Talbot element, a phase gradient generating device, a light detector, and a processor. The Talbot element repeats a Talbot image at a distance from the Talbot element. The phase gradient generating device scans the Talbot image at a plane at the distance from the Talbot element by incrementally changing a phase gradient of a light field incident the Talbot element. As the Talbot image is scanned, the light detector captures time varying data associated with light altered by an object located at the distance from the Talbot element. The processor reconstructs an image of the object based on the time-varying light data.
38 citations
TL;DR: An imaging scheme that integrates multilayer sample modeling, ptychography-inspired recovery procedures, and lensless single-pixel detection to tackle the loss of high frequency components in the detection path is reported.
Abstract: A critical challenge for fluorescence imaging is the loss of high frequency components in the detection path. Such a loss can be related to the limited numerical aperture of the detection optics, aberrations of the lens, and tissue turbidity. In this paper, we report an imaging scheme that integrates multilayer sample modeling, ptychography-inspired recovery procedures, and lensless single-pixel detection to tackle this challenge. In the reported scheme, we directly placed a 3D sample on top of a single-pixel detector. We then used a known mask to generate speckle patterns in 3D and scanned this known mask to different positions for sample illumination. The sample was then modeled as multiple layers and the captured 1D fluorescence signals were used to recover multiple sample images along the z axis. The reported scheme may find applications in 3D fluorescence sectioning, time-resolved and spectrum-resolved imaging. It may also find applications in deep-tissue fluorescence imaging using the memory effect.
37 citations
References
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TL;DR: Each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the thyl gene), for example, all retinal ganglion cells or many cortical neurons were XFP positive in some lines, whereas only a few ganglions or only layer 5 cortical pyramids were labeled in others.
2,929 citations
01 Jan 2000
2,498 citations
TL;DR: In this article, a setup consisting of three transmission gratings can efficiently yield quantitative differential phase-contrast images with conventional X-ray tubes, which can be scaled up to large fields of view.
Abstract: X-ray radiographic absorption imaging is an invaluable tool in medical diagnostics and materials science. For biological tissue samples, polymers or fibre composites, however, the use of conventional X-ray radiography is limited due to their weak absorption. This is resolved at highly brilliant X-ray synchrotron or micro-focus sources by using phase-sensitive imaging methods to improve the contrast1,2. However, the requirements of the illuminating radiation mean that hard-X-ray phase-sensitive imaging has until now been impractical with more readily available X-ray sources, such as X-ray tubes. In this letter, we report how a setup consisting of three transmission gratings can efficiently yield quantitative differential phase-contrast images with conventional X-ray tubes. In contrast with existing techniques, the method requires no spatial or temporal coherence, is mechanically robust, and can be scaled up to large fields of view. Our method provides all the benefits of contrast-enhanced phase-sensitive imaging, but is also fully compatible with conventional absorption radiography. It is applicable to X-ray medical imaging, industrial non-destructive testing, and to other low-brilliance radiation, such as neutrons or atoms.
1,789 citations
TL;DR: This study carried out a genome-wide phenotypic profiling of each of the ∼21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival.
Abstract: Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the approximately 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.
812 citations
TL;DR: A bimolecular fluorescence complementation approach for the simultaneous visualization of multiple protein interactions in the same cell based on complementation between fragments of fluorescent proteins with different spectral characteristics is described.
Abstract: The specificity of biological regulatory mechanisms relies on selective interactions between different proteins in different cell types and in response to different extracellular signals. We describe a bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. This approach is based on complementation between fragments of fluorescent proteins with different spectral characteristics. We have identified 12 bimolecular fluorescent complexes that correspond to 7 different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency of the bZIP domains of Fos and Jun or the subcellular sites of interactions between these domains. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners.
777 citations