The overall goal of this protocol is to observe the development of Tribolium castaneum embryos noninvasively over very long periods of time. Light sheet based fluorescence microscopy helps to answer morphogenesis related questions in the emerging insect model organism Tribolium castaneum addressing cell migration and proliferation patterns from the early blastoderm to the onset of muscular movement. The main advantage of this method is the critical factors such as photobleaching and phototoxicity are kept to a minimum even when the embryos are observed for several days.
To mount an embryo into the sample chamber using an Agarose Column first fill a one millimeter syringe with distilled water and use a small rubber hose to attach the syringe to a glass capillary. Fill the capillary half way with distilled water then use a paint brush to transfer an embryo to the inner side of the glass capillary opening, and stick the capillary into the liquid agarose slowly drawing a few microliters of the agarose along with the embryo into the capillary. When the embryo is in place there should be a few microliters of agarose both above and below the embryo.
When the agarose has solidified shorten the capillary to a length of about five millimeters. The remaining fragment should be completely filled with agarose and the embryo should be found within the second quartile. Then insert the steel cylinder with the pin into the sample chamber, and stick the capillary fragment on top of the pin, The Agarose Column should should protrude a few millimeters from the region of the capillary fragment that contains the embryo.
To mount the embryo into the sample chamber using an Agarose Hemisphere first draw 200 microliters of liquid Agarose into a one millimeter syringe, and use the syringe to fill the steel pipe with the sample chamber with Agarose from the top of the pipe until a hemisphere forms at the bottom opening. When the Agarose has solidified pick up an embryo with the tip of a paint brush and rearrange the embryo so that the anterior end becomes accessible. Dip the Agarose Hemisphere into liquid Agarose to cover the hemisphere with a thin film and place the embryo with its anterior end upright on the pole of the Agarose Hemisphere.
Turn the steel pipe and use the brush to correct the position of the embryo as necessary. Ideally less than half of the embryo surface should be covered in agarose and the flanks should be as steep as possible. Too much agarose on the embryo's surface leads to a mechanic constriction and a reduced gas and ion exchange, which has a dramatic effect on the embryo's survival rate.
Then slowly insert the steel pipe into the sample chamber. To mount the embryo into the sample chamber using a Cobweb Holder first select an embryo with the paint brush and set the paint brush aside. Next cover the slotted hole of the cobweb holder with five to eight microliters of liquid agarose then remove most of the agarose until only a thin agarose film remains.
Place the embryo on to the agarose film and adjust its position carefully moving the embryo to the x-axis center of the slotted hole and aligning its elongated anterior posterior axis with the elongated axis of the slotted hole. Make sure to place the embryo onto the agarose film within 10-15 seconds after its application that is while the agarose is still liquid or else it will not attach properly. Then slowly insert the Cobweb holder into the sample chamber positioning the holder so that it does not interfere with the excitation and emission light.
For live imaging assays first configure the time-lapse, then in the transmission light mode position the embryo along the X and Y axis into the center of the field of view without exposing the embryo to the laser. Rotate the embryo 360 degrees in 90 degree steps to examine the tissue for any damage that could have occurred during the mounting process. In the florescence mode define the Z-Stack for each direction adding a 25 to 50 micrometer spacial buffer in front of and behind the embryo and define the Z spacing.
For long term imaging ensure that the sample chamber is filled with sufficient imaging buffer and cover the sample chamber opening. Then start the imaging process, monitoring the correct acquisition along all of the directions in all of the channels for all of the embryos. At the end of the imaging period remove the sample holder from the sample chamber and transfer the embryo to an object slide.
If an agarose column was used use a sharp blade to extract the embryos from the surrounding agarose. If an agarose hemisphere was used transfer the hemisphere with a flat side to the object slide. For the cobweb holder method use a paint brush to gently dismount the embryo from the agarose film.
Then incubate the embryo loaded object slide in a small glass dish in a saturated humidity atmosphere under standard conditions until the larvae hatch. To process the data of the observed embryo open the files of interest in the appropriate imaging analysis software and rotate the Z-Stacks to align the anterior posterior axis of the embryo with the Y axis as necessary. Crop the Z-Stacks along the X and Y axis so that only a minimal background remains then calculate the Z Maximum Projection for each Z-Stack.
Then depending on the transgenic line and imaging modalities make the appropriate dynamic intensity adjustments of the time-stacks to correct for any photobleaching or flora for expression fluctuations as necessary. Using a transgenic line that expresses a Histone 2B M-Emerald fusion protein under the control of the alpha-tubublin one promoter 66 hours of embryonic development at room temperature were recorded from germ band retraction to dorsal closure. This enhancer trap-line can be used to visualize neuronal clusters within the head appendages and the dynamics of cirrosa migration during dorsal closure.
Imaging a similar transgenic line which carries the same trans-gene but at a different genomic location the optical sections of the transition from gastrulation to germ band elongation show the internal cell arrangement of the emerging germ band. Using the glia-blue transgenic line the acquisition of Z-Stacks along multiple directions via sample rotation allows the observation of glial cell reorganization along and around the ventral nerve cord as well as their proliferation dynamics of the dorsal side of the head lobes in the same embryo. Small florescent dyes can also be used for the specific labeling of intercellular structures to highlight certain embryonic features such as the cirrhosis gar, the posterior ventral cirrhosis cells, or the serosa amnion germ band tissue tri-layer that emerges during the serosa window closure.
Dual color florescent dye staining allows the further visualization of two inter-cellular structures in the same embryo. For example the actin cytoskeleton and the nuclear envelope. Once the recording is finished it's important for the quality control of the experiment to retrieve the embryos and raise them to healthy fertile adults.
This procedure can be combined with other techniques such as Parental RNA Difference to analyze knocked down phenotypes.
