Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.
The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.
We present here a novel time-lapse imaging assay to monitor cell behavior in chicken embryonic slice culture. This assay enables high resolution imaging of living tissue for up to 70 hours, although time frames between 24-48 hours are easier to capture. The use of a high NA oil objective and relatively short intervals between time-points enables image acquisition at high resolution, as well as allowing us to monitor cell behavior that often occurs rapidly, and can be easily missed during conventional time-lapse imaging using confocal microscopy.
The major advantage of this assay is the ability to image cells over long periods of time. The use of wide-field6 instead of confocal laser scanning7 microscopy is critical for this. Confocal microscopy has traditionally offered several advantages over wide-field microscopy, including the ability to take optical sections and eliminate out of focus information directly7, but the use of a pinhole leads to a loss of light to the detector8, excluding a significant amount of information, and necessitating longer exposure times. Wide-field microscopy, on the other hand, uses full field illumination and all the light passing through the objective is sent to the detector. This coupled with a high quantum efficiency cooled coupled device (CCD) camera ensures imaging with a high signal to noise ratio compared to confocal microscopy9, with very fast exposure times. In our application, we must image for long periods, record 3D stacks and ultimately resolve small—near-diffraction limited structures. Although confocal microscopy prevents out-of-focus light from ever reaching the detector and thus significantly reduces background in thick, densely-labeled samples7, 8, it only achieves a usable signal-to-noise ratio with much larger light input than wide-field microscopy10. For very light-sensitive sparsely-labeled samples like our electroporated spinal cord slices, therefore, wide-field microscopy performs better than confocal microscopy. When combined with image restoration by deconvolution, which removes out of focus information and improves contrast, wide-field is then particularly good for detection of small, dim objects11. While not appropriate for every tissue imaging experiment, this approach has been very effective for our live cell imaging application.
The choice of fluorescent protein is an important factor that can influence cell survival. We find that the best results are obtained from constructs that use green fluorescent protein (GFP)12 as a marker. We are currently assessing a range of red fluorescent proteins that can be effectively used in combination with GFP for dual channel time-lapses. There are a variety of such proteins available13. Although many proteins are stable as fusions with a fluorescent protein, some proteins may be rendered unstable by fusion, resulting in reduced cell viability. In such cases, the use of constructs containing an internal ribosome entry site (IRES) is useful to separate the protein of interest from the fluorescent protein.
When imaging spinal cord slices, care must be taken to minimize exposure to fluorescent light. The microscope eyepieces must be used only with transmitted light (brightfield) to locate and position slices. Fluorescent images should always be acquired using the microscope software using minimal exposure times. These should be kept in the range of 5-50 ms and the use of neutral density filters should be experimented with. Although longer exposure times may initially produce clearer images, the phototoxic effects of fluorescent light exposure may lead to cell death. The first 5-10 μm of tissue that is closest to the coverslip should not be imaged as this region contains tissue that may have been damaged during slicing.
Although the examples presented here are of embryos electroporated at HH stage 10 and imaged 6-7 hours later, this method can be used for embryos up to HH stage 18. At later stages, the spinal cord tissue is much larger and so is difficult to slice by hand. In these cases, embedding the embryos in low melting point agarose and sectioning with a vibratome may yield better results. Even though we present this assay as a method for imaging cells in the developing spinal cord, this approach has also been modified to image other embryonic tissues, including the sensory placodes3.
The authors have nothing to disclose.
Reagent | Company | Catalogue number | Comments |
WillCo Dish | WillCo Wells | GWst-3522 | |
poly-l-lysine | Sigma | P8970 | 0.1% in Tris |
Borosilicate glass capillaries | Harvard Instruments | GC100-10 | 1.0mm outer diameter, 0.58mm inner diameter |
Electrodes (Genetrodes, in ovo, L-shaped | BTX | 10-001850-01 | 5mm gold plated |
ECM 830 square pulse generator | BTX | 45-0002 | for in ovo electroopration |
Fast Green | Sigma | F7258 | |
Type I rat collagen | Sigma | C3857 | |
L-15 medium (powder) | Sigma | 2.76 g/L in distilled water for 5x solution |
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Sodium bicarbonate | Sigma | S8761 | 7.5% solution |
Neurobasal medium | Invitrogen | 12348-017 | without phenol red |
Glutamax | Invitrogen | A1286001 | 100x solution |
B-27 supplement | Invitrogen | 17504-044 | |
Gentamicin | Invitrogen | 15710049 | 10 mg/ml |
L15 medium (liquid 1x) | Invitrogen | 11415-049 | |
Microknife | Altomed | A10102 | 15 degree incline |
Stainless steel headless pins | Watkins and Doncaster | E6871 | A1 size |
Sylgard 184 elastomer | VWR | 634165S | |
DeltaVision Core microscope system | Applied Precision | ||
Coolsnap HQ2 CCD camera | Photometrics |