Gigapixel fluorescence microscopy with a water immersion microlens array
TL;DR: High throughput gigapixel fluorescence microscopy with a microlens array is demonstrated with a parallelized microscopy system to image samples in micro well plates and fluorescent imaging of tissue samples through coverslips is demonstrated.
Abstract: We demonstrate high throughput gigapixel fluorescence microscopy with a microlens array. We show, for the first time to the best of our knowledge, the use of a parallelized microscopy system to image samples in micro well plates. We image centimeter-scale regions of 384-well micro well plates at 1.72 μm resolution at a raw pixel throughput of 25.4 Mpx/s. Taking into account the fact that about half the well plate area consists of the plastic support region between wells, this corresponds to a sample pixel throughput of 13.2 Mpx/s, more than double that of the commercial state-of-the-art at the time of writing. Fluorescent imaging of tissue samples through coverslips is also demonstrated.
Citations
More filters
TL;DR: In this paper, a planar photonic chip is used to hold a biological sample and generate the necessary light patterns for structured illumination microscopy, which enables live-cell super-resolution imaging of subcellular structures at high speeds.
Abstract: Structured illumination microscopy (SIM) enables live-cell super-resolution imaging of subcellular structures at high speeds. At present, linear SIM uses free-space optics to illuminate the sample with the desired light patterns; however, such arrangements are prone to misalignment and add cost and complexity to the microscope. Here, we present an alternative photonic chip-based two-dimensional SIM approach (cSIM) in which the conventional glass sample slide in a microscope is replaced by a planar photonic chip that importantly both holds and illuminates the specimen. The photonic chip reduces the footprint of the light illumination path of SIM to around 4 × 4 cm2. An array of optical waveguides on the chip creates standing wave interference patterns at different angles, which illuminate the sample via evanescent fields. High-refractive-index silicon nitride waveguides allow a 2.3 times enhancement in imaging spatial resolution, exceeding the usual 2 times limit of SIM. In summary, cSIM offers a simple, stable and affordable approach for performing two-dimensional super-resolution imaging over a large field of view. The use of a photonic integrated circuit to both hold a biological sample and generate the necessary light patterns for structured illumination microscopy promises convenient super-resolution imaging.
77 citations
TL;DR: In this article, a photonic-chip-based total internal reflection fluorescence (TIRF)-SIM was proposed to reduce the complexity of the optical setup needed to acquire TIRF-SIM images.
Abstract: Structured illumination microscopy (SIM) enables live cell, super-resolution imaging at high speeds. SIM uses sophisticated optical systems to generate pre-determined excitation light patterns, and reconstruction algorithms to enhance the resolution by up to a factor of two. The optical set-up of SIM relies on delicate free-space optics to generate the light patterns, and a high numerical aperture objective lens to project the pattern on the sample. These arrangements are prone to miss-alignment, often with high costs, and with the final resolution-enhancement being limited by the numerical aperture of the collection optics. Here, we present a photonic-chip based total internal reflection fluorescence (TIRF)-SIM that greatly reduces the complexity of the optical setup needed to acquire TIRF-SIM images. This is achieved by taking out the light delivery from the microscope and transferring it to a photonic-chip. The conventional glass slide is replaced by the planar photonic chip, that both holds and illuminates the specimen. The chip is used to create a standing wave interference pattern, which illuminates the sample via evanescent fields. The phase of the interference pattern is controlled by the use of thermo-optical modulation, leaving the footprint of the light illumination path for the SIM system to around 4 by 4 cm$^2$. Furthermore, we show that by the use of the photonic-chip technology, the resolution enhancement of SIM can be increased above that of the conventional approach. In addition, by the separation of excitation and collection light paths the technology opens the possibility to use low numerical objective lenses, without sacrificing on the SIM resolution. Chip-based SIM represents a simple, stable and affordable approach, which could enable widespread penetration of the technique and might also open avenues for high throughput optical super-resolution microscopy.
76 citations
TL;DR: The advent of large field-of-view chip-based nanoscopy opens up new routes in diagnostics where high throughput is needed for the detection of non-diffuse disease, or rare events such as the early detection of cancer.
Abstract: Optical nanoscopy techniques can image intracellular structures with high specificity at sub-diffraction limited resolution, bridging the resolution gap between optical microscopy and electron microscopy. So far conventional nanoscopy lacks the ability to generate high throughput data, as the imaged region is small. Photonic chip-based nanoscopy has demonstrated the potential for imaging large areas, but at a lateral resolution of 130 nm. However, all the existing super-resolution methods provide a resolution of 100 nm or better. In this work, chip-based nanoscopy is demonstrated with a resolution of 75 nm over an extraordinarily large area of 0.5 mm × 0.5 mm, using a low magnification and high N.A. objective lens. Furthermore, the performance of chip-based nanoscopy is benchmarked by studying the localization precision and illumination homogeneity for different waveguide widths. The advent of large field-of-view chip-based nanoscopy opens up new routes in diagnostics where high throughput is needed for the detection of non-diffuse disease, or rare events such as the early detection of cancer.
39 citations
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.
34 citations
TL;DR: In this article, the authors used laterally-translated Scotch tape to generate high-resolution speckle illumination patterns across a large FOV, and custom optimization algorithms then jointly reconstruct the sample's super-resolution fluorescent and quantitative phase distributions, while digitally correcting for system imperfections.
Abstract: High-content biological microscopy targets high-resolution imaging across large fields-of-view (FOVs). Recent works have demonstrated that computational imaging can provide efficient solutions for high-content microscopy. Here, we use speckle structured illumination microscopy (SIM) as a robust and cost-effective solution for high-content fluorescence microscopy with simultaneous high-content quantitative phase (QP). This multi-modal compatibility is essential for studies requiring cross-correlative biological analysis. Our method uses laterally-translated Scotch tape to generate high-resolution speckle illumination patterns across a large FOV. Custom optimization algorithms then jointly reconstruct the sample’s super-resolution fluorescent (incoherent) and QP (coherent) distributions, while digitally correcting for system imperfections such as unknown speckle illumination patterns, system aberrations and pattern translations. Beyond previous linear SIM works, we achieve resolution gains of 4× the objective’s diffraction-limited native resolution, resulting in 700 nm fluorescence and 1.2 μm QP resolution, across a FOV of 2×2.7 mm 2, giving a space-bandwidth product (SBP) of 60 megapixels.
31 citations
References
More filters
TL;DR: In this paper, an array-based high-throughput technique that facilitates gene expression and copy number surveys of very large numbers of tumors is presented. But, it is limited to a single tumor tissue microarray.
Abstract: Many genes and signalling pathways controlling cell proliferation, death and differentiation, as well as genomic integrity, are involved in cancer development. New techniques, such as serial analysis of gene expression and cDNA microarrays, have enabled measurement of the expression of thousands of genes in a single experiment, revealing many new, potentially important cancer genes. These genome screening tools can comprehensively survey one tumor at a time; however, analysis of hundreds of specimens from patients in different stages of disease is needed to establish the diagnostic, prognostic and therapeutic importance of each of the emerging cancer gene candidates. Here we have developed an array-based high-throughput technique that facilitates gene expression and copy number surveys of very large numbers of tumors. As many as 1000 cylindrical tissue biopsies from individual tumors can be distributed in a single tumor tissue microarray. Sections of the microarray provide targets for parallel in situ detection of DNA, RNA and protein targets in each specimen on the array, and consecutive sections allow the rapid analysis of hundreds of molecular markers in the same set of specimens. Our detection of six gene amplifications as well as p53 and estrogen receptor expression in breast cancer demonstrates the power of this technique for defining new subgroups of tumors.
4,164 citations
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: In this article, the authors report on their activities in design, fabrication, characterization and system integration of refractive microlens arrays for sensors and microsystems, including neural networks and multiple pupil imaging systems for photolithography.
Abstract: We report on our activities in design, fabrication, characterization and system integration of refractive microlens arrays for sensors and microsystems. Examples for chemical analysis systems (, blood gas sensor), neural networks and multiple pupil imaging systems for photolithography (microlens and smart mask lithography) are presented.
416 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