The overall goal of this procedure is to image C Allergan's development using fluorescence light sheet microscopy. This is accomplished by first setting up the light sheet based microscope composed of an upright microscope and a small set of optomechanical elements to generate a light sheet. The second step of the procedure is to mount the embryo.
The last steps are to adjust the light sheet onto the embryo and record its development. The results can show the dynamics of fluorescently labeled proteins during CL egan's development via the analysis of 3D and time lapse videos. The main advantages of lightship microscopy over other existing methods, such as confocal microscopy, is that it allows faster imaging with lower phototoxicity.
Third, this method can provide insight into seal against development. It can also be applied to other system such as roil or zebrafish. Demonstrating the procedure will be clear.
Shales an engineer from my laboratory and Pauline Linac, an engineer from the laboratory of van. This section describes the setup assembly, and it can be left assembled for all experiments. Once the lasers, filters, telescope and periscope have all been positioned on the optical table, secure the cylindrical lens to generate the light sheet.
A different lens is adjusted for focus. The focal point of the illumination objective should coincide with the object focal point of the detection objective. Now, project the beam onto the wall or screen and move the illumination objective along the beam axis until the contours of the projection are sharp.
Include a quad line emission filter and E-M-C-C-D camera in the microscope setup. Fix the second manual translation stage to the first in a perpendicular manner. Screw the assembly to the existing microscope stage.
After removing the set cylindrical lens illumination objective, position the assembly onto the microscope and put back the illumination set onto the stages. Attach the qve holder at the perpendicular intersection of the illumination path and the detection path. Now, check the light sheet.
Fill a glass vete with fluorescein solution and place it in the holder on the stages. The light sheet should be visible. It should be horizontal and centered with respect to the detection objective lens.
Next, remove the cylindrical lens. Optimize the light sheet with 3D translation of the illumination objective, and acquire an image to measure the thickness of the light sheet. This depends on the aperture of the objective lens.
Then be sure to replace the cylindrical lens. First, cut a piece of one millimeter thick glass, 10 millimeters by 20 millimeters. Use a diamond marking pen.
Next, add 20 microliters of poly L lysine to one side, and once dry, juxtapose it to a second slide. In a fixed position, add a drop of 5%agar and smooth it with the weight of a third slide set on top. After the agar dries, remove the third slide and cut the agar pad three millimeters from the edge of the two remaining slides over the slide without poly L lysine.
Then remove the fixed slide, leaving an overhang of agar. Coat the agar with 20 microliters of poly L lysine and allow it to dry. Now put GR worms in a watch glass filled with M nine medium under a dissection microscope.
Cut the worms with a scalpel level to the vulva, thus releasing the eggs. Then identify the embryos that are of the stage of interest and collect them with a micro capillary pipette. Transfer the embryos to the agar overhang off the prepared slide just next to the edge of the slide, not on the border of the agar.
Then align the embryos with a micro capillary pipette while removing the liquid. By aspiration, the embryos will stick to the polyol lysine. It is very important to position well the embryos to obtain a good recording, and this requires a capillary with the right apture.
If too small, it'll be difficult to release the embryos. If too big, it'll be difficult to tightly control the leaked flow and to align the embryos, Cover the slide with M nine medium in a Petri dish and confirm that the embryos are still attached to the agar using magnification. Now, fix the prepared slide with embryos to the sample holder that fits into a vete.
The holder is an in-house made contraption that is designed to fit to a vete. Next, secure the vete to a Pizo electric stage and under brightfield illumination, locate an embryo. Now start up the software package controlling the acoustic optic tuneable.
Filter the camera and the stage. This software here was written in-house, but micromanager freeware can also be used. First, select the laser line.
Next, adjust the stage along the x axis until the light sheet is at its brightest. Then adjust the other two dimensions, Y and Z until the signal to noise is at its best. This may need to be repeated for each embryo.
This key to optimize the signal to ratio to obtain a good recording ly translating the of a free stage, supporting of the and elimination objective when I just relationship to obtain an ous simulation of the embryo and the best contrast. Now acquire time lapse images set the laser power exposure, time gain, and imaging interval according to the fluorescence level of the embryo and the speed of the biological process analyzed. For Zack.
Imaging set the distance between the slices and their start and end positions in c elgan expressing a histone GFP construct. Two hour time-lapse imaging was made with 20 slice stacks taken every 37 seconds. Cell divisions were clearly visible in a strain expressing tubulin fused to GFP and his stone fused to m cherry Recordings were taken for 16 minutes every 105 seconds.
3D renderings are shown at three time points. The mitotic spindles and condensed chromosomes are clearly visible and easily tracked through cell divisions. In a third strain APO lipoprotein vit two fused to GFP was imaged with mCherry labeling of the cell membrane.
Over 13 minutes, 10 slice stacks were taken every 27 seconds. Fast moving lipoprotein particles were easy to follow. Here we use the light sheet formed by more complex methods to produce.
The light sheet might be implemented to improve further the spatial resolution.
