High Throughput Multichannel Fluorescence Microscopy with Microlens Arrays
TL;DR: In this paper, a multi-wavelength emitting laser light source is configured to receive and expand input light emitted from the light source, and a dichroic mirror was configured to reflect the expanded input light.
Abstract: A microscope includes a multi-wavelength emitting laser light source. A microscope objective is configured to receive and expand input light emitted from the light source, and a dichroic mirror is configured to reflect the expanded input light. A micro lens array with a plurality of micro lenses splits the reflected and expanded input light onto a fluorescence producing sample. A lens collectively captures the fluorescence for each micro lens in the plurality of micro lenses, and a camera receives the fluorescence from the lens and produces an image of the sample based on the received fluorescence.
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TL;DR: A mobile phone microscope that uses the internal flash or sunlight as the illumination source, thereby reducing complexity whilst maintaining functionality and performance is presented.
Abstract: Mobile phone microscopes are a natural platform for point-of-care imaging, but current solutions require an externally powered illumination source, thereby adding bulk and cost. We present a mobile phone microscope that uses the internal flash or sunlight as the illumination source, thereby reducing complexity whilst maintaining functionality and performance. The microscope is capable of both brightfield and darkfield imaging modes, enabling microscopic visualisation of samples ranging from plant to mammalian cells. We describe the microscope design principles, assembly process, and demonstrate its imaging capabilities through the visualisation of unlabelled cell nuclei to observing the motility of cattle sperm and zooplankton.
68 citations
TL;DR: A novel form of LSFM is presented that uses incoherent extended focusing to produce divergence free light-sheets with near diffraction-limited resolution and uniform intensity distribution along the propagation direction.
Abstract: Light-sheet fluorescence microscopy (LSFM) affords highly parallelized 3D imaging with optical sectioning capability and minimal light exposure. However, using Gaussian beams for light-sheet generation results in a trade-off between beam waist thickness and the area over which the beam can approximate a light-sheet. Novel techniques for LSFM are disclosed that uses extended focusing and/or laser line focuses to produce divergence free light-sheets with near diffraction-limited resolution and uniform intensity distribution.
63 citations
TL;DR: A conceptually different super-resolution approach which circumvents limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes and relies on markers with super-linear dependence of the emission on the excitation power.
Abstract: Sub-diffraction microscopy enables bio-imaging with unprecedented clarity. However, most super-resolution methods require complex, costly purpose-built systems, involve image post-processing and struggle with sub-diffraction imaging in 3D. Here, we realize a conceptually different super-resolution approach which circumvents these limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes. We refer to it as super-linear excitation-emission (SEE) microscopy, as it relies on markers with super-linear dependence of the emission on the excitation power. Super-linear markers proposed here are upconversion nanoparticles of NaYF4, doped with 20% Yb and unconventionally high 8% Tm, which are conveniently excited in the near-infrared biological window. We develop a computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. Imaging of colominic acid-coated upconversion nanoparticles endocytosed by neuronal cells, at resolutions twice better than the diffraction limit both in lateral and axial directions, illustrates the applicability of SEE microscopy for sub-cellular biology.
48 citations
TL;DR: In this paper, a self-assembled bi-dimensional array of microspheres is used to realize multi-focus parallel detection scheme for FCS, which enables FCS experiments at low picomolar concentrations with a drastic reduction in apparatus cost and alignment constraints, ideal for microfluidic chip integration.
Abstract: Fluorescence sensing and fluorescence correlation spectroscopy (FCS) are powerful methods to detect and characterize single molecules; yet, their use has been restricted by expensive and complex optical apparatus. Here, we present a simple integrated design using a self-assembled bi-dimensional array of microspheres to realize multi-focus parallel detection scheme for FCS. We simultaneously illuminate and collect the fluorescence from several tens of microspheres, which all generate their own photonic nanojet to efficiently excite the molecules and collect the fluorescence emission. Each photonic nanojet contributes to the global detection volume, reaching FCS detection volumes of several tens of femtoliters while preserving the fluorescence excitation and collection efficiencies. The microspheres photonic nanojets array enables FCS experiments at low picomolar concentrations with a drastic reduction in apparatus cost and alignment constraints, ideal for microfluidic chip integration.
38 citations
TL;DR: This work uses both a coherent and an incoherent imaging model to develop algorithms for joint retrieval of the 3D super-resolved fluorescent and complex-field distributions of the sample and reconstructs images that resolve features beyond the physical diffraction-limit set by the system's objective.
Abstract: High-content biological microscopy targets high-resolution imaging across large fields-of-view, often achieved by computational imaging approaches. Previously, we demonstrated 2D multimodal high-content microscopy via structured illumination microscopy (SIM) with resolution >2× the diffraction limit, using speckle illumination from Scotch tape. In this work, we extend the method to 3D by leveraging the fact that the speckle illumination is in fact a 3D structured pattern. We use both a coherent and an incoherent imaging model to develop algorithms for joint retrieval of the 3D super-resolved fluorescent and complex-field distributions of the sample. Our reconstructed images resolve features beyond the physical diffraction-limit set by the system’s objective and demonstrate 3D multimodal imaging with ∼0.6×0.6×6 μm 3 resolution over a volume of ∼314×500×24 μm 3.
29 citations
References
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TL;DR: This work developed a method that, based on the Fourier Shift Theorem, computes all possible translations between pairs of 3D images, yielding the best overlap in terms of the cross-correlation measure and subsequently finds the globally optimal configuration of the whole group of3D images.
Abstract: Motivation: Modern anatomical and developmental studies often require high-resolution imaging of large specimens in three dimensions (3D). Confocal microscopy produces high-resolution 3D images, but is limited by a relatively small field of view compared with the size of large biological specimens. Therefore, motorized stages that move the sample are used to create a tiled scan of the whole specimen. The physical coordinates provided by the microscope stage are not precise enough to allow direct reconstruction (Stitching) of the whole image from individual image stacks.
Results: To optimally stitch a large collection of 3D confocal images, we developed a method that, based on the Fourier Shift Theorem, computes all possible translations between pairs of 3D images, yielding the best overlap in terms of the cross-correlation measure and subsequently finds the globally optimal configuration of the whole group of 3D images. This method avoids the propagation of errors by consecutive registration steps. Additionally, to compensate the brightness differences between tiles, we apply a smooth, non-linear intensity transition between the overlapping images. Our stitching approach is fast, works on 2D and 3D images, and for small image sets does not require prior knowledge about the tile configuration.
Availability: The implementation of this method is available as an ImageJ plugin distributed as a part of the Fiji project (FijiisjustImageJ: http://pacific.mpi-cbg.de/).
Contact: tomancak@mpi-cbg.de
1,989 citations
TL;DR: Fluorescence microscopy is making the transition to a quantitative and high-throughput technology to enable these techniques to be applied to functional genomics experiments.
Abstract: Fluorescence microscopy is a powerful tool to assay biological processes in intact living cells. Now, fluorescence microscopy is becoming a quantitative and high-throughput technology that can be applied to functional genomics experiments and can provide data for systems-biology approaches. In this post-genomic era, we need to define gene function on a genome-wide scale for model organisms and humans. The fundamental unit of biological processes is the cell. Among the most powerful tools to assay such processes in the physiological context of intact living cells are fluorescence microscopy and related imaging techniques. To enable these techniques to be applied to functional genomics experiments, fluorescence microscopy is making the transition to a quantitative and high-throughput technology.
422 citations
TL;DR: Several lines of mice are presented that overcome limitations of the initial lines, and an adaptation of the method for use in adeno-associated viral vectors is reported, providing technical advice about how best to image Brainbow-expressing tissue.
Abstract: In the transgenic multicolor labeling strategy called ‘Brainbow’, Cre-loxP recombination is used to create a stochastic choice of expression among fluorescent proteins, resulting in the indelible marking of mouse neurons with multiple distinct colors. This method has been adapted to non-neuronal cells in mice and to neurons in fish and flies, but its full potential has yet to be realized in the mouse brain. Here we present several lines of mice that overcome limitations of the initial lines, and we report an adaptation of the method for use in adeno-associated viral vectors. We also provide technical advice about how best to image Brainbow-expressing tissue.
353 citations
TL;DR: This review describes how cellular imaging technologies contribute to the drug discovery process and addresses both high-content and high-throughput needs.
Abstract: Traditional screening paradigms often focus on single targets. To facilitate drug discovery in the more complex physiological environment of a cell or organism, powerful cellular imaging systems have been developed. The emergence of these detection technologies allows the quantitative analysis of cellular events and visualization of relevant cellular phenotypes. Cellular imaging facilitates the integration of complex biology into the screening process, and addresses both high-content and high-throughput needs. This review describes how cellular imaging technologies contribute to the drug discovery process.
335 citations
TL;DR: This review aims at describing the current capabilities and limits of the technology as well as highlighting necessary developments that are required to exploit fully the potential of high-content screening and analysis.
Abstract: The term "high-content screening" has become synonymous with imaging screens using automated microscopes and automated image analysis. The term was coined a little over 10 years ago. Since then the technology has evolved considerably and has established itself firmly in the drug discovery and development industry. Both the instruments and the software controlling the instruments and analyzing the data have come to maturity, so the full benefits of high-content screening can now be realized. Those benefits are the capability of carrying out phenotypic multiparametric cellular assays in an unbiased, fully automated, and quantitative fashion. Automated microscopes and automated image analysis are being applied at all stages of the drug discovery and development pipeline. All major pharmaceutical companies have adopted the technology and it is in the process of being embraced broadly by the academic community. This review aims at describing the current capabilities and limits of the technology as well as highlighting necessary developments that are required to exploit fully the potential of high-content screening and analysis.
164 citations