In order to perform the reconstructions of developing organs within the Drosophila melanogaster, we need to determine the forward model that best describes propagation of light within the pupa itself. The procedure begins with selecting a white pre pupa and gluing it at the end of a capillary tube such that the poppa's anterior posterior axis is oriented parallel to the tube. A small capillary tube is filled with some fluorescent dye and inserted in the pupil case.
After careful axis adjusting, the pupa is rotated 360 degrees. Images are acquired at different angles. The acquired fluorescent trans illumination data are used to determine the forward model that will be used to image organ development.
Hi, my name is Claudia and I'm at the Center for Assistance Biology at Massachusetts General Hospital, Harvard Medical School. I'm Ula Petou from the Paramount Lab in Harvard Medical School, And I am Daniel Rozanski from the Institute for Biological and Medical Imaging at the Technical University of Munich and Heliman Center Munich. Today we'll show you a procedure for three dimensional reconstruction of whole body in Oph Melano Casta.
We use this procedure in our lab for in vivo visualization of developing theof organs during pupil status. So let's get started Before beginning. Please note that there isn't accompanying video that should be viewed prior to this one, where some technical aspects of optical projection tomography are explained in greater detail than they are here.
In addition, the other video explains imaging of prepared tissues or organs of larger animals. For all the experiments covered in this video, forge drosophila transgenes will be used. They are EL A gal four expressed in the salivary glands, apteris gal four, which is expressed in the larval precursor tissues of the adult wing and thorax, the transgene that makes fluorescent protein.
In response to gal four called U-A-S-G-F-P and white 1118, the not fluorescent control males carrying the UAS transgenes are crossed to virgin females carrying the GAL four transgenes to achieve tissue. Specific expression of GFP crosses are set up from eight to 10 virgin females and males on standard fly media with a sprinkle of baker's yeast. On the surface, the vials are housed at 25 degrees Celsius in a humidified incubator under a 12 hour light dark cycle.
Two days after the initiation of the cross, the parents are transferred to a fresh vial and five to six days later, PUI will start populating the vial white PrepU. I are selected for GFP expression under a fluorescent dissecting scope. Selection of white pre pui ensures proper staging of the animals since they remain white for about one hour.
After pupil ation, then they become pigmented or brownish. GFP expressing pui are gently collected from the vials using a wet paintbrush and placed in a drop of water or PBS in a Petri dish, buy must be wiped clean of autofluorescent materials. Using the paintbrush, they should now be ready for tomographic imaging.
If the PrepU I need to continue aging to arrive at the correct developmental stage, they can be left in a Petri dish with a wet paper towel or in a clean food vial. This will ensure sufficient humidity and proper development for imaging. The PrepU must be adhered to the interior of an 800 micron diameter glass capillary tube.
Paint the posterior end of the pupil with super glue orient the glued surface of the pre pupa into the tube such that the fly's anterior posterior access is parallel with the tube. The anterior part of the animal will overhang the capillary tube. The capillary tube is now fixed onto the rotational stage of the microscope.
In a vertical position, adjust the tilting axis of the rotational stage so that the rotational axis of the tube is in parallel with the pixel columns of the imaging CCD. With the position of the fly tweaked, proceed with imaging. In this example, animals with fluorescent salivary glands are to be imaged to oph expressing GFP in their salivary glands can be generated from a cross between EAG four and U-A-S-G-F-P stocks.
We begin with a mounted pre pupa illuminate the pre pupa with an excitation beam and collect the GFP fluorescent signal. In transillumination, rotate the PrepU 360 degrees along its vertical axis and acquire the trans illumination images. Each image corresponds to 10 degrees of rotation.
The total acquisition time depends on the amount of fluorescence and on the intensity of the excitation beam. Typical times are in the order of one minute it. In this example, a DSO pupa expressing GFP in the wing imaginal discs will be imaged.
This pupa can be collected from a vial of a cross between Aus GAL four and U-A-S-G-F-P stocks. The pre pupa is mounted as previously described. However, since the imaging time for this experiment is about eight hours, it is critical that the environment remains humid and at room temperature To avoid dehydration and death of the animal, a shutter is placed over the excitation beam to prevent the quenching of floral fours in the tissue of interest, as well as to ensure that the animal is not burnt by the laser beam as before, illuminate with the excitation beam and collect the GFP fluorescent signal by trans illumination.
The pre pupa is completely rotated along its vertical axis. During data collection, the rotation takes only one minute and is repeated at different time intervals. To create a time lapse series of images of the wings during development, data can be acquired for at least six hours at a rate of 100 images per hour.
It is vital that the total light exposure is controlled with this shutter in order to prevent harm or photo bleaching. This movie shows images acquired between the PrepU stage and pupil head aversion. When reconstructing the three dimensional image, there is a strong forward scattering that needs to be accounted for, which cannot be approximated by diffusion.
So to make the reconstruction a forward scattering model of light propagation must be determined For simplicity, we neglect absorption. The amount of scattering is tested experimentally. To do this, a foreign object with standardized fluorescence is inserted into the non fluorescent white 1118 pre pupa, which is then scanned to generate the forward model data begin by filling a 100 micron silica capillary tube with PS 5.5 fluorescent dye.
Using capillary action, push the capillary tube into the pupa at a 45 degree angle to the anterior axis, mount the PrepU in a capillary tube and position it appropriately on the rotating stage as previously described. Now illuminate the pre pupa with an excitation laser beam with a low numerical aperture lens and collect fluorescent trans illumination images of the pre pupa. As previously described, MATLAB software is used to fit the data into the explicit theoretical forward model To ease the reconstruction process, the firm e approximation to the transport equation is selected as it describes the forward scattering regime and also fits the experimental data.
Having determined the correct forward model, we can reconstruct the salivary glands and obtain the time lapse tomographic reconstructions. If you plan to use a free space tomography, configuration algorithms for index matching can also be set up. First, we show a 3D reconstruction of the salivary glands early in development.
For comparison, histological staining of the salivary glands shows that the reconstructions correlate well with the histology. This time lapse movie of development of the wing imaginal discs was acquired from a single live specimen. For comparison histological staining of the wing disc at the same time points shows that the reconstructions correlate well with the histology.
We have just shown you how to build a system to image three-dimensional structures within oph in vivo, and over time. This allows to follow us, the morphogenesis in Drosophila, and to follow the development of the wings over six consecutive hours. When doing this procedure, it's crucial to choose the correct forward model for light propagation within drosophila.
This method can be used in combination with histological techniques to shed light in organ development during oph pupil stages, and it can be modified for the study of different organisms of similar size. So that's it. Thanks for watching and good luck with your experiments.
