In this video, we will demonstrate novel techniques that allow us to image pube of Drosophila Melanogaster without compromising the structural integrity of the puer. By keeping the pupil case intact, we minimize the impact of imaging on the development and viability of the pupa. We use a resonant scanning 8, 000 hertz confocal microscope for imaging.
Importantly, the very fast scan speed and high sensitivity of this microscope greatly reduces bleaching, allowing for extended periods of observation. With this method, we've been able to visualize a variety of cell biological processes, including the behavior, fop, podia, morphogenesis, phagocytosis, and mitosis. With this technique, we can observe events in situ for extended periods of time without damaging the organism.
The observations gained from these methods can be used to understand a variety of biological phenomena at this relatively inaccessible stage of drosophila development, the Cheese people, the appropriate stage, we examine them while still in the vial looking for key indicators at developmental stage to study cell divisions, we look for pipa that have averted their head, but still don't show green packets and meconium and the dorsal abdomen. Individuals are loosened by touching the peep case with a moist paintbrush transferring water to dissolve the salivary glue. Pipa are then gently nudged free, picked up with the paintbrush and transfer to a Petri dish containing water.
The people are thoroughly washed in the water to clean the case of food or debris. We then line them up, dorsal side up to make sure they're of the correct stage. Handle them carefully.
They can easily die if even a small hole is made in the pipe. Select individuals are carefully placed and glass bottom Petri dish and orient it so that the feature of interest is closest to the cover slip and as parallel to the surface as possible. To observe the pupil wing, we prop the pupa against the support, such as a thin length, the modeling clay or dental wax, ensuring that they do not move after orientation.
We then invite the dish to make sure that the feature of interest is visible and well oriented. Typically, mount multiple pipa at one time guard against the possibility that one is not expressing the fluorescent protein or is not at a desired stage. Once the pipa are all mounted and oriented properly, we touch the base of each pipa with a paintbrush moistened with diod dilin glycol, a non-toxic mountain that matches the refractive index of oil of pure or a water of dilute with water.
This serves a dual purpose of reducing diffraction from the pupil case and reducing refraction of immersion Lenses are used Once we have properly mounted and arranged the pupi. We move to the laser scanning confocal microscope. We use an inverted like a SP five equipped with a resonance scanner.
The dish is mounted on the microscope and we locate the puput using the microscope optics. Once the pupa is found, adjust focus so that the feature of interest in this case, the wing is visible. We identify the wing by the presence of a large tracheal element running through the middle of the wing.
When the region of interest is identified and the microscope is focused, we to the 20 x dry objective. After reconfirming focus, we switch the microscope to confocal mode. We adjust the parameters of the confocal to minimize light exposure.
Light intensity is kept below 10%transmission. To maximize the brightness, we open the pinhole to 130 microns for the 20 x lens with azuma four and set the gain to 900 volts. With these settings, we can image for hours without affecting the length of the mitotic interval or the health of the animal.
Next, we set the number of Z sections and the frequency of image capture to match the characteristics of the biological phenomenon we are trying to observe, for example, one minute imaging intervals sufficiently capture all important features of cell division. But three second intervals are important to observe the dynamics of phylo podia. Focus up and down through the epithelium until you find your desired feature.
If necessary, use the microscope and XYZT mode to collect Z stacks through time. The duration of movies is also an important parameter. We have made videos as long as 10 hours.
When imaging multiple fluorescent molecules simultaneously, we try to make the total imaging duration shorter. In principle, each fluorescent tag causes greater production of reactive oxygen species. In practice, we've observed no lethality associated with imaging triply labeled lines for one hour or less.
If the target tissue or the process is smaller than about five microns, you'll likely need to use an immersion lens. Keep in mind that these lenses focus the laser to a finer point, thereby increasing the light intensity, and you must adjust the imaging parameters accordingly, Movies are transferred from the confocal microscope to a computer running the freeway image analysis program, Fiji. With Fiji, we can open measure and manipulate features in the multidimensional images generated by the confocal.
This method of imaging has allowed us to study the position of the nuclei with respect to the cell cycle. In the pupil wing, we collected a Z stack from the top of the epithelium to the bottom most observable nuclei at one minute intervals. In these movies, we only observed divisions occurring in the apical confocal sections shown here on the left, the more basal sections shown on the right reveal the return of newly divided nuclei from the superficial layer, but do not contain other features of mitosis.
These data show that the rapidly dividing pupil wing epithelium is similar to the larval wing disc and to other MedOne. Epithelial in that nuclei move to the apical surface of the epithelium during mitosis. Despite this commonality, the pupil wing epithelium is relatively unst stratified with most nuclei very close to the top layer.
Previous methods of imaging drosophila during early stages of pupil development have been hampered, largely due to attempts to remove or penetrate the pupil case. We describe here a novel approach that allows us to visualize adult cells during development without affecting viability of the organism. Our method is qualitatively different from others in that it keeps the pupil case fully intact.
The combination of confocal microscopy with resonant scanning allows us to visualize developing tissues within 20 microns of the pupil case. The main limitations in resolving depth are the working distance of the lens and scattering from the case in tissue. However, our method allows us to resolve features of the epithelial cells from the apex to the base.
We have used this technique to observe mitosis, phylo, podia, morphogenesis, and phagocytosis. In addition to this, we have studied as many as three distinct fluorescent tags in one sample. We believe that the approaches described here opened the early period of pupil development up to the study of the outermost epithelial layers.
When combined with the power of geal genetics, this accessibility may further improve our understanding of adult development.
