Microscopy: Imaging far and wide
The idea of a microscope typically conjures up thoughts of a highly magnified image of a tiny sample captured in unprecedented detail. In principle, however, the physical mechanisms that allow us to see such microscopic details do not actually require the observed region or the specimen itself to be particularly small. Rather, the performance of a microscope is primarily limited by the diffraction of light as it passes through the various optical elements in the instrument.
The resolution of a microscope, , is defined as the shortest distance between two points on a specimen that can still be seen as two distinct points in the image. In 1873 Ernst Abbe showed that diffraction limited the resolution of a microscope to , where is the wavelength of the light, is the refractive index of the medium the light travels through, and is the half-angle at which the objective captures light from the sample: is also known as the "numerical aperture" of the objective.
As Abbe's equation makes clear, the resolution is independent of the magnification. Rather, it is the design of the objective that determines both the resolution of the microscope and its "field-of-view" (that is, the overall size of the sample that can be imaged). And although a variety of microscope objectives are available from commercial manufacturers, they are designed to either capture a fairly large field-of-view (typically a millimeter or so across) at low resolution, or a small field-of-view (typically a few hundred micrometers or less across) at high resolution. By contrast, to obtain a high-resolution image of a large specimen we need a combination of a large field-of-view, a long working distance (that is, the distance between the objective and the focal plane of the microscope), and a high numerical aperture: however, there are no commercial microscopes that offer this combination at present.
Now, in eLife, Gail McConnell of Strathclyde University and colleagues – Johanna Trägårdh, Rumelo Amor, John Dempster, Es Reid and Brad Amos (who is also at the MRC Laboratory for Molecular Biology) – report that they have developed an objective called the Mesolens that offers both a relatively large field-of-view and a relatively high resolution (Figure 1; McConnell et al., 2016). Using a custom-built confocal microscope equipped with the Mesolens, McConnell et al. were able to capture beautiful single-shot images of whole mouse embryos with cellular resolution, and also capture images of explants from the brains of rat embryos, again with cellular resolution.
The Mesolens was made possible by a combination of sophisticated optical design and skillful engineering, including excellent correction for the various optical aberrations that would otherwise compromise high-resolution imaging over a large field-of-view. Moreover, the Mesolens is an immersion lens, which means that the gap between the objective and the sample is filled with a medium such as oil, water or glycerol: McConnell et al. designed their lens to provide high-quality images in a variety of immersion media with different optical properties.
A confocal microscope is able to image a sample in all three dimensions by taking two-dimensional images at a range of different depths: the Mesolens allowed McConnell et al. to image volumes of up to 6 x 6 x 3 mm3 with a lateral resolution of 0.7–0.8 micrometers and an axial resolution of 7–8 micrometers (with a numerical aperture of 0.47). These figures are factors of at least two and three higher than the resolutions that can be achieved with currently available large field-of-view commercial objectives.
In addition to applications in developmental biology and embryology, including whole-embryo phenotyping and screening applications that require rapid imaging of large specimens, the ability to image samples measuring several millimeters across with near-cellular resolution will be useful in many other areas of the life sciences. In neuroscience, for example, researchers are trying to combine techniques such as tissue clearing and tissue expansion (Höckendorf et al., 2014; Marx, 2016) with large-volume light microscopy (Ji et al., 2016) to image entire mammalian brains at high resolution; the Mesolens could prove useful in these efforts. And extending beyond its application in confocal microscopy, the Mesolens could have applications in light-sheet microscopy (Keller and Ahrens, 2015), which excels in the rapid imaging of large volumes. In particular, combining the Mesolens with other techniques could reduce the need to use "optical tiling" when imaging large specimens, or to subject large specimens to sectioning and other destructive sample-preparation techniques.
The Mesolens highlights the increasingly important role of custom optical designs in improving the performance of light-based microscopes. Recent, complementary efforts in mesoscale imaging with two-photon microscopy also rely heavily on custom-built objectives. For example, the two-photon random access microscope developed by Karel Svoboda of the HHMI Janelia Research Campus and co-workers features a custom objective with a numerical aperture of 0.6 and a field-of-view of 5 millimeters, and enables imaging up to a depth of 1 millimeter (Sofroniew et al., 2016). The twin-region, panoramic two-photon microscope (Trepan2p) developed by Spencer Smith of the University of North Carolina and co-workers features a custom objective with a numerical aperture of 0.43 and a field of view of 3.5 millimeters, and has been used to image multiple cortical areas in mice (Stirman et al., 2016). Other examples of this emerging trend to develop new microscopes with custom-built optics include lattice light-sheet microscopy (Chen et al., 2014) and IsoView light-sheet microscopy (Chhetri et al., 2015).
By extending the scale of optical microscopy to several millimeters, this new generation of custom-built objectives also emphasizes the need for new wide-field detection systems that can take full advantage of the capabilities of these new lenses. To optimally sample the large field-of-view captured by the Mesolens in a wide-field microscope, gigapixel cameras will be needed: for comparison, a state-of-the-art scientific camera will typically have 4–6 megapixels. Various gigapixel designs are already under development (Brady et al., 2012; Zheng et al., 2014), and further advances in this domain will help exploit the full potential of the Mesolens in imaging large volumes at high resolution.
Multiscale gigapixel photographyNature 486:386–389.https://doi.org/10.1038/nature11150
Making biology transparentNature Biotechnology 32:1104–1105.https://doi.org/10.1038/nbt.3061
Technologies for imaging neural activity in large volumesNature Neuroscience 19:1154–1164.https://doi.org/10.1038/nn.4358
Optimizing probes to image cleared tissueNature Methods 13:205–209.https://doi.org/10.1038/nmeth.3774
0.5 gigapixel microscopy using a flatbed scannerBiomedical Optics Express 5:1–8.https://doi.org/10.1364/BOE.5.000001
Article and author information
- Version of Record published: September 23, 2016 (version 1)
© 2016, Chhetri et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
- Page views
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
- Developmental Biology
- Computational and Systems Biology
Many developmental processes depend on precise temporal control of gene expression. We have previously established a theoretical framework for regulatory strategies that can govern such high temporal precision, but experimental validation of these predictions was still lacking. Here, we use the time-dependent expression of a Wnt receptor that controls neuroblast migration in Caenorhabditis elegans as a tractable system to study a robust, cell-intrinsic timing mechanism in vivo. Single-molecule mRNA quantification showed that the expression of the receptor increases non-linearly, a dynamic that is predicted to enhance timing precision over an unregulated, linear increase in timekeeper abundance. We show that this upregulation depends on transcriptional activation, providing in vivo evidence for a model in which the timing of receptor expression is regulated through an accumulating activator that triggers expression when a specific threshold is reached. This timing mechanism acts across a cell division that occurs in the neuroblast lineage and is influenced by the asymmetry of the division. Finally, we show that positive feedback of receptor expression through the canonical Wnt pathway enhances temporal precision. We conclude that robust cell-intrinsic timing can be achieved by combining regulation and feedback of the timekeeper gene.
- Developmental Biology
- Stem Cells and Regenerative Medicine
During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation stage human embryos. Human induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF- progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.