Direct observation by microscopes is a classic method in the field of developmental biology to investigate processes that lead to the growth and formation of plant and animal organisms. Since this scientific field spans a multitude of different processes, developmental biology can come from a multitude of backgrounds, such as genetics, cancer or neuroscience, and focus on embryology, regeneration, reproduction or evolutionary developmental biology (“evo-devo”).
Driven by the respective research topic, the variety of model systems is in the high numbers and still growing. Classic model systems include widely-used organisms such as C. elegans, Drosophila melanogaster, Xenopus laevis, zebrafish, chicken and mouse as well as Arabidopsis, Zea mays, Oryza sativa or Tobacco BY-2 cells in plant biology, just to name a few. New model organisms are constantly established, among them a growing number of annelid, cnidarian and insect species such as sea urchin, Hydra, Tribolium and squid.
Formerly, model systems were sorted into two main groups: The first group consisted of dedicated experimental model systems such as chicken or Xenopus laevis that is still being tightly connected to the Spemann-Mangold organizer which led to the Nobel Prize in 1935 for the discovery of the effect now known as embryonic induction. The other group comprised model systems that were already genetically accessible, such as Drosophila melanogaster which was the basis of the extensive studies of important genes and pathways that granted the Nobel Prize to Christiane Nüsslein-Volhard in 1995. With newly developed genetic tools, particularly the CRISPR/Cas9 system, more organisms will become more readily available for genetic manipulation, furthering and diversifying this field of research even more. Studies into these very fundamental biological processes help us to better understand human genetic defects that are the causes for cancer, metabolic diseases, developmental defects or neonatal mortality, just to name a few prominent examples.
Being such a heterogenous field of work, the common denominator and challenge for microscopy in developmental biology remains the need to closely follow biological processes over prolonged periods of time while avoiding any environmental or light-induced artefacts caused by phototoxicity and photobleaching effects in fluorescence microscopy. The observed specimen or structure can dramatically change in size over time, or you need to work with several different organisms and samples to address your specific scientific questions, demanding high flexibility of the microscopy systems in terms of sample space and volume that needs to be captured. In developmental biology a single microscope will probably not be suitable for all your imaging tasks so the swift and easy transfer of research samples including the resulting imaging data between different microscope systems within a complete portfolio is desirable.
Since their invention by the American entomologist Horatio S. Greenough and the Carl Zeiss workshop in Jena in 1892, stereo microscopes quickly became essential tools in biology to inspect large specimens with a more vivid, stereoscopic image and without the need for slide preparation.
The original working principle was later evolved by Ernst Abbe into a new type which was based on the telescopic principle and featured one instead of two objective lenses, the Common Main Objective (CMO). This type provides many advantages over the Greenough-type particularly for illumination, drawing and micro-photography purposes while still maintaining the excellent visual representation of the specimen, easy handling, and good ergonomics when performing time-intensive repetitive tasks.
Modern stereo microscopes can be based on either of these principles and feature various modes of illumination, manual or motorized zoom options, and integrated digital imaging capabilities, depending on the application demands and cost efficiency. Stereoscopic microscopy is an indispensable tool for mechanical manipulation (X. laevis, chick), sorting (Drosophila with morphological markers) and general sample preparation. Even more advanced stereo zoom microscopes are fully-featured imaging systems for fluorescence microscopy applications with a particularly high numerical aperture and stepping motors that automatically position the moveable lenses precisely for every imaging task while zooming. The applications for these systems range from routine screenings to demanding multidimensional fluorescence imaging.
Investigation of embryogenesis requires a microscope that provides perfect environmental conditions for the embryo when studying morphogenesis and therefore cellular behavior, cell proliferation and potentially investigation of cell-lineages, or by capturing specific genetic activity. To reliably follow cell divisions and tissue movement the whole organism needs to be captured at sufficient temporal and spatial resolution over the course of hours or even days. Apart from the amount of information that needs to be collected via fluorescence signal, phototoxic damage must be avoided as much as possible to interfere with the natural sequence of events.
Development of Light Sheet Fluorescence Microscopy (LSFM, sometimes termed as Selective Plane Illumination Microscopy, SPIM) has been greatly driven by these specific needs of embryonic studies and serves this application perfectly. The outstanding characteristic of LSFM is a perpendicular arrangement of excitation and detection beam paths. With this setup, only the focal plane is illuminated by a sheet of light resulting in an optical section. The full field of view is acquired by a digital wide-field camera system operating at high speed. Initial setups enabled scientists for the first time to follow embryonic development of Drosophila and zebrafish or Arabidopsis root growth over prolonged periods of time. The unique geometry of light sheet fluorescence microscopes supports the investigation of plant root growth and development in its natural environment and orientation.
To investigate the underlying principles of developmental processes, the investigation of spatio-temporal gene expression can be one valuable source of research. Labeling the proteins of interest with fluorescent proteins of different emission spectra is a widely used tool to visualize the activity and interaction of specific genes over time. The expression levels of the fluorescent proteins however need to be kept at the lowest possible level to avoid unwanted influence on the natural processes inside the cells. While the context and therefore an overview of the whole sample is required, it is as well often essential to resolve the localization of the signal to subcellular level.
The laser scanning microscope (LSM), often named confocal microscope, usually is the best choice for such experiments. It excels with its capability to combine instant optical sectioning for multiple fluorescent labels at high image quality, offering a great flexibility when it comes to microscope stands (upright, inverse, fixed stage) and objective lenses that are ideally suited for the desired field of view, working distance and resolution. The point scanning system includes the option to photo-manipulate the sample and activate fluorescent labels by uncaging or photoconversion to follow individual cell lines only at a specific time of development. Confocal imaging can be challenging when capturing 3D over long periods of time, since its acquisition speed is limited by its point by point scanning approach. Furthermore, valuable fluorescent signal must be discarded at a pinhole at the conjugate image plane to achieve the desired optical sectioning. With the emergence of new detection technologies such as Airyscan, some of the inherent drawbacks of confocal microscopy can be avoided or significantly attenuated.
Superresolution fluorescence microscopy with methods such as SIM (Structured Illumination Microscopy) or SMLM ( e.g. PALM, dSTORM or PAINT) brings investigations in developmental processes down to the molecular level. One of the often shown examples of this type of research is the Bruchpilot protein in the Drosophila neuromuscular junction. Quantitative superresolution imaging with single-molecule resolution allows the investigation of nanoscopic organization as well as insights into ultra-fast molecular dynamics within a single cell.
In many studies of developmental biology, a single dataset is not sufficient to draw scientific conclusions. To understand developmental processes, especially when phenotypic differences of wild-type and genetically modified mutants are studied, multiple datasets need to be collected as a reliable basis for research statistics. Apart from the number of individual embryos that need to be studied, the reproduction of the same experimental conditions can be a challenge and might lead to undesired artefacts in the results.
Previously, automated microscopy systems often were limited in regards to optical quality, experimental features, incubation options, or reproducible and fast image acquisition of raw data. Modern automated microscopy systems by ZEISS are based on high-end research components, take over repetitive tasks and greatly reduce the amount of time spend on the imaging procedure. They perform multiple runs of complex experiments and even allow for parallel acquisition of a high number of samples. At the same time, usability is greatly increased and therefore the human factor that can influence experimental results is efficiently reduced, even when the experiments are performed by unexperienced users. Thus you can collect reproducible and reliable results from the first day of your study.
Even if your samples are comparatively large in size sometimes you want to study finest details of developmental processes at the subcellular level. With electron microscopy systems you scan the surface of your fixed specimen sample at highest resolution, resulting in a 2D datafile that you easily navigate and zoom into to reveal the smallest subcellular details. However, while your organism is naturally three dimensional, these electron microscopy techniques are inherently limited to 2D surface imaging.
To overcome the latter limitation, various technologies have emerged over the past few years. These include automated array tomography workflows with integrated sample preparation and large-area imaging as well as 3D volume reconstruction with ATUMtome, enabling ultra-high-resolution datasets of whole sections while preserving the samples for further investigation and archiving. On the other hand, if sample preservation is not a necessary requirement, you can achieve even higher resolved 3D reconstruction of your most demanding specimen by using Serial Block-Face SEM (SBF-SEM) imaging with the integrated Gatan 3View microtome and Focal Charge Compensation.
Stereo and zoom microscopes from ZEISS are the indispensable tools of your developmental biology lab. Enjoy a vivid and clear stereoscopic image of your large specimen during sample preparation and investigation. Choose from various illumination, digital imaging and zoom options. Keep your specimen always in view with large object fields and extended working distances. Investigate your model organism with fluorescence microscopy and create brilliant optical sections with Apotome.2 and Axio Zoom.V16
ZEISS Celldiscoverer 7 is a boxed automated microscope which combines many characteristics that are ideal for automated acquisition of embryogenesis studies. It provides a stable environmental control so that the parallel data collection of many embryos will be done under the same stable conditions that are easily reproduced for a sequence of experiments. The optics of ZEISS Celldiscoverer 7 are optimized for water immersion and offer high resolution over a wide range of magnifications, so any sample can be imaged at best quality. Besides a wide range of wide-field contrast and fluorescence imaging configurations you can even choose to integrate confocal microscopy with LSM 900 and Airyscan 2. Automated recognition of the sample carrier and autofocus save your valuable time and give you the complete developmental processes in the embryos while minimizing the amount of light needed to expose the delicate embryos.
The ZEISS LSM 9 family of confocal microscopy systems with Airyscan 2 and Multiplex mode uniquely overcomes the obstacles of the traditional confocal method. The Airyscan 2 detector collects more photons, increasing sensitivity, speed and resolution, while at the same time preserving the main advantages of a confocal system such as multicolor imaging, direct optical sectioning and enhanced imaging depth. When the emission signals of the fluorophores in your specimen overlap or the tissue itself responds with autofluorescence, the spectral imaging and signal separation capabilities of LSM 980 collect all spectral data with a single scan. Furthermore LSM 980 can be upgraded to a multiphoton NLO system with up to 12 NDD detectors for deep tissue imaging in upright, inverse or fixed stage configuration.
Imagine you had access to an imaging system that could deliver optical sections of large samples, with virtually no phototoxicity or bleaching and with high temporal resolution. That is exactly what Lightsheet 7 from ZEISS does. The unique Multiview light sheet fluorescence microscope allows you to record the development of large, living samples and gently image them to deliver exceptionally high information content. It is also fast: Lightsheet 7 is your microscope for optical sections at high speed. Acquire images of your whole sample volume at sub-cellular resolution – in a fraction of the time it takes using other techniques.
Superresolution microscopy with ZEISS Elyra 7 enables insights for your developmental studies on the cell culture level with the possibility to investigate extremely small cellular structures and molecular processes. Elyra 7 with Lattice SIM provides better penetration into tissues than traditional SIM systems. The new Apotome mode for superfast optical sectioning and 3D imaging covers a wide application range for your developmental biology lab.
Serial block face imaging is a fast and convenient method to perform 3D imaging with nanometer resolution. With 3View you use an ultramicrotome inside the SEM chamber to repeatedly cut and image your resin embedded cell and tissue samples. Combine ZEISS Sigma and GeminiSEM with 3View to produce thousands of serial images in a single day. Block face imaging delivers perfectly aligned images in the shortest possible time and in the most convenient way. FE-SEMs from ZEISS with 3View are ideally suited to acquire high resolution 3D data with TEM like quality. The unique Gemini column delivers crisp images in large fields of view with nanometer resolution. Block face imaging of hundreds of microns of your sample in one go reduces overlap and saves stitching time.