Solid State High Throughput Screening Microscopy

  1. M. Ashraf
  2. S. Mohanan
  3. B Sim
  4. A. Tam
  5. D. Brousseau
  6. S. Thibault
  7. A. Corbett
  8. G. Bub

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

We introduce a solid state high throughput screening (ssHTS) imaging modality that uses a novel Newtonian telescope design to image multiple spatially separated samples without moving parts or robotics. Conventional high-throughput imaging modalities either require movement of the sample to the focal plane of the imaging system 1–3 or movement of the imaging system itself 4,5 , or use a wide-field approach to capture several samples in one frame. Schemes which move the sample or the imaging system can be mechanically complex and are inherently slow, while wide-field imaging systems have poor light collection efficiency and resolution compared to systems that image a single sample at a given time point. Our proposed ssHTS system uses a large parabolic reflector and an imaging lenses positioned at their focal distances above each sample. A fast LED array sequentially illuminate samples to generate images that are captured with a single camera placed at the focal point of the reflector. This optical configuration allows each sample to completely fill a sensors field of view. Since each LED illuminates a single sample and LED switch times are very fast, images from spatially separated samples can be captured at rates limited only by the camera’s frame rate. The system is demonstrated by imaging cardiac monolayer and Caenorhabditis elegans ( C. elegans ) preparations.

Article activity feed

  1. eLife

    ###Reviewer #3:

    General assessment

    The authors present a cool new idea: using a large parabolic reflector in combination with a macroscopic lens array and rapidly modulated LED array to enable fast image multiplexing between spatially separated samples. I believe that there may be interesting applications that would benefit from this capability, although the authors have not clearly demonstrated one. The paper is short, and light on discussion, details, and data.

    Major comments

    1. The manuscript does not discuss several standard, key topics for any new microscope paper: "objective" numerical aperture, image resolution, optical aberration (other than distortion, which is discussed), and camera sensor size.

    2. Why was an array of low-performance singlet lenses used? With that selection, the image quality cannot be good. Can the system not be paired with an array of objectives or higher performance multielement lenses?

    3. Fluorescence imaging is not discussed or demonstrated but would obviously increase the impact of the microscope. At least some discussion would be helpful.

    4. Actual HTS applications are almost always implemented in microtiter plates (e.g. a 96-well plate) to reduce reagent costs and enable automated pipetting, etc. I do not believe anyone would implement HTS in thousands of petri dishes. The paper would be strengthened substantially by a demonstration of simultaneous recording from all (or a large subset) of the wells in a 96-well plate. It's not clear whether this is possible due to the blind spot in the center of the parabolic mirror's field of view that is blocked by the camera.

    5. One of the primary motivations for this approach is given in the first paragraph as: "wide-field imaging systems [which capture multiple samples in one frame] have poor light collection efficiency and resolution compared to systems that image a single sample at a given time point." With a f = 100 mm singlet lens, the light collection efficiency of the demonstrated microscope is also low (estimated NA = 0.12) and the resolution is unimpressive with the high-aberration lens and 1x magnification. They demonstrated only trans-illumination applications (e.g. phase contrast), where light collection efficiency is not important. I believe a fancy photography lens mounted directly on a many-megapixel camera set to image all or part of a microtiter plate could likely outperform their system in throughput and simplicity, at least for the demonstrated applications of cardiomyocytes and C. elegans.

  2. eLife

    ###Reviewer #2:

    Astronomers have spent centuries learning how to image the night sky with limited sensor hardware. Ashraf et al present an ingenious adaptation of a technology developed for telescopes-parabolic reflectors-for imaging biological samples. In principle, the approach seems like it could be incredibly useful across a wide range of applications where multiple samples must be imaged in tandem. By placing multiple samples under a single parabolic reflector, multiplexing of samples and imaging hardware can be accomplished without sample-handling robots or moving cameras. The authors highlight two applications: cardiac cells in culture and free-moving nematodes.

    The authors explain the theory behind their technique in a clear and convincing way. However, the biggest challenge in most imaging projects is making the theory work in practice. In its current form, the manuscript falls far short of demonstrating the practical usefulness of parabolic mirrors for imaging biological samples. The authors include only a small amount of image data-for the nematode work, this consists of eight images collected from two plate regions. Data of this scope cannot provide readers or reviewers with sufficient evidence with which to evaluate the quality of the technique.

    1. The images shown-are they typical or are they the best possible images that can be collected from the device? The authors do not provide any quantitative evaluation of the quality of their images, in absolute terms or relative to existing methods, with which to understand the practical performance of parabolic mirrors. The authors should estimate the spatial resolution and dynamic range that can be obtained in practice with the devices, and evaluate how such image quality metrics vary across the entire field of view. Does performance degrade towards the edge of the mirror? Does performance degrade over time, as devices become de-calibrated with use?

    2. The manuscript is additionally weakened by the absence of a non-trivial measurement made with the device. Pilot experiments are included, demonstrating that images can be collected. However, no evidence is provided to show that these images can be used to compare samples and draw biological conclusions from them. A more convincing proof-of-principle would involve the measurement of some non-trivial biological difference between samples measured with the device, either confirming previous work or discovering something new.

    3. The authors highlight the comparative simplicity of their method: it eliminates the need for motorized samples or cameras. However, this simplicity must come at some: for example a substantially increased use of space or perhaps an increase in delicate calibration required, or equipment price. If a 0.25 meter mirror is required to measure four C. elegans plates, how large a mirror would be required to measure 16 plates-the number that can typically be measured using a flatbed scanner? The authors could also expand greatly on other practical issues: for example, is a dedicated imaging table required to align mirrors and samples? Readers would benefit from a clearer evaluation of the practical trade-offs in deploying parabolic mirrors in a laboratory setting relative to other imaging approaches.

  3. eLife

    ###Reviewer #1:

    Ashraf and colleagues describe an approach to perform high throughput screening imaging without moving parts. The setup is original and offers to experimentalists the flexibility to record quasi-simultaneously stacks of images of multiple samples at the full field of resolution of the camera. The optical aberration inherent to the use of a parabolic mirror are mostly overcome by collimating light from the objective lens. The images require a post-processing in two steps for taking into account the image stretching on the detector and the variation in magnification due to the variation of the distance between the mirror and the image. Two applications illustrate the potential of the solid-state HTS.

    To my opinion, the following points need to be clarified:

    1. How homogeneous is the field of illumination with a single LED? Especially for a large field of illumination, a non-homogeneous illumination would compromise the quantifications.

    2. The accuracy of this ssHTS is related to the robustness at keeping the distance F2 constant between samples. In other words, how sensitive is the image acquisition to the potential variation in the F2 distance between samples as well as within a single large field of view?

    3. The magnification Mc must be explained.

    4. Is the post-processing compensation applied only in the y-direction?

    Assuming that such publication aims to disseminate the use of an ssHTS setup to a wide scientific community, I find the description of the setup as well as the applied image post-processing rather succinct, even with the 3D printing and source codes information.

  4. eLife

    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on April 18, 2020, follows.

    Summary

    The reviewers all recognised the originality of your solution to perform high throughput imaging without moving parts. They do have some serious reservations, primarily regarding the evaluation of the quality and utility of the technique and in addition to the other points raised, and consider it essential that you address the following:

    1. The standard topics for any new microscope paper: "objective" numerical aperture, image resolution, optical aberration, and camera sensor size, together with the specific aspects related to this technique, including dependence on homogeneous illumination, and sensitivity to maintenance of F2 distance.

    2. A substantial expansion of the scope of the data presented, to provide readers with sufficient evidence with which to evaluate the quality of the technique, including proof of principal with a 96-well plate assay.

    3. A direct quantitative comparison with existing HTS imaging solutions.

  5. Version published to 10.1101/602011v1 on bioRxiv