The overall goal of this procedure is to accurately image endosomes in three dimensional space over time or in four dimensions. This is accomplished by first dissecting snake muscles and arranging the tissue in the imaging dish. The second step is to stain the tissue with vital dyes to stimulate endosome formation.
Next, the preparation is imaged in four dimensions for one or more neuromuscular junctions. The final step is careful data analysis to characterize the behavior of macro endosomes. Ultimately, the presence and movement of endosomes over time within the presynaptic space can be demonstrated by four dimensional fluorescence imaging.
The main advantage of this technique is that the snake abdominal muscle is only a single fiber thick, which allows for high quality images to be generated using standard epi fluorescence microscopy Tissue preparations from the garter snake will be used in this protocol. Reptilian tissue should be kept at low temperatures, so it remains physiological for longer times and with less bacterial contamination. For lysosomal vital dye staining, the dye is dissolved one to 5, 000 in cold reptilian ringers solution.
To get a final concentration of 0.2 micromolar, add the dye to the tissue and incubate at four degrees Celsius for 15 minutes. After 15 minutes, wash the tissue several times with cold ringers for all FM 1 43 staining. Pre mount the sample in an imaging dish with magnetic pins prior to staining.
For the purpose of this video, only FM 1 43 staining using potassium chloride stimulation will be demonstrated. The dye diluted one to 500 in high potassium chloride ringers to get a final concentration of seven micromolar is added to the tissue, and then the tissue is incubated at four degrees Celsius for a maximum of 30 to 60 seconds. Subsequently, quickly washed three times with cold ringers for about one minute per rinse.
All tissues should be imaged as soon as possible after being stained and washed with cold ringer solution. If an inverted microscope is used, utilize an imaging chamber whose bottom contains a thin round cover slip. Typically, the imaging chamber should have a thin stainless steel bottom with a 25 millimeter diameter.
Number one thickness glass cover slip at the center to configure the preparation for imaging. Orient it using magnetic pins so that the muscle is centered as best as possible within the imaging dish and laying as flat as possible. Cover the preparation with a small volume of ringer solution.
Mount the dish, placing it as close to the center of the stage as possible. Choose an appropriate objective to obtain the highest possible 3D resolution. A 63 x water objective will be used for all the experiments in this demonstration.
To begin this procedure, open the imaging window in slide book 5.0. Several menus are contained here in tabs. Choose the 63 x water objective using the second tab Z.Set the Z stack interval.
In this example, a Z step of 1.5 microns is used for live imaging. Select the time-lapse frame rate. An optimal rate is one that is just adequate to smoothly resolve changes with time, such as movement interactions or fusion events.
For example, 10 time points at 32nd intervals for a total time interval of five minutes. Next, select the total depth of field while displaying the terminal live manually focused to the top of the focus of interest, and then go a little bit above it. Select set top.
Slowly focus through the sample until everything is out of focus. Again, the number of planes and total travel values displayed will indicate the depth of field selected in the capture window. Select the used top and bottom positions radio button to transfer the parameters just selected For image capture, start image acquisition.
Complete all live imaging for a particular preparation. Retain all raw data files while digital storage is usually not a problem. Processing time is significant even with fast personal computers or workstation.
For this reason, crop images into a region of interest ensure that the entire region of interest is in the cropped window during the entire time. Course D can involve image stacks using appropriate software and the correct point spread function, confirm that resolution is improved and that no artifact has been generated by the deconvolution algorithm. Use an interpolation algorithm to expand Z axis.
Apparent resolution, a six x expansion from an actual 1.5 micron image plane separation to an apparent 0.25 micron separation is suggested. Perform contrast brightness, noise filtering, photo bleaching, and other typical image processing adjustments. If desired.
Image manipulation standards dictate that for most scientific work, it is appropriate that the same corrections be applied to all images both within a stack and at different time points. The consequence of a particular adjustment should be assessed among all images. Experiment with various filtering and image enhancement techniques.
For example, lelos and filtering is useful to increase contrast of small structures above background. The brightness of pixels at the center of a running window is enhanced while brightness of surrounding areas is reduced. Note that some filtering methods do not work well in combination.
Apply drift correction if there is substantial movement of the preparation over time. Such movement is common in living muscle, even with drugs added to suppress action potentials and synaptic potentials. A pixel registration algorithm aligns the time lapse images and can reduce, but usually not completely eliminate such motion.
In this study, four D live imaging of snake neuromuscular terminals was used to determine if macro endosomes or me move towards the plasma membrane and quickly disappear, suggesting that their number decreases because some of them access cyto. The data is displayed as 3D volume views at six time points with a frame interval of 60 seconds. To illustrate the Z depth of a single buton, an me can be observed moving away from the Y axis indicated by the red dashed line over several frames before disappearing between time points five and six, that the time required for disappearance was less than the frame interval is consistent with exocytosis just before disappearance.
The ME appears to be located at the batons plasma membrane. The same raw dataset is shown next as a movie at a lower magnification. The sequence is briefly paused at the beginning of each 24 3D rotation steps about the Y axis to draw attention to the soon to be disappearing me indicated by the red arrow.
Note that the ME is visible approaches the edge of the buton disappears and does not return in all viewing perspectives. Because of lower Z axis resolution compared to XY resolution, the shape of the ME appears to lengthen and shorten depending on the viewing perspective. Electron microscopy studies have shown that endosomes in motor terminals are small in size near the diffraction limit of light.
Hence, fusion of these endosomes cannot be absolutely distinguished from close spatial association at light level. In this study, Mees were labeled with FM 1 43 indicated in green and acidic endosomes or AEs were labeled with a lysosomal. Vital dye indicated in red data from one time point of a four D movie are displayed as 3D volume views in three orientations.
The top panels one through three show the conventional view from above the XY plane. The middle panels four through six show a view perpendicular to the XY plane and in the direction of the large red arrow. The bottom panels seven through nine show a view mutually perpendicular to both views above in the direction of the large blue arrow.
An me is marked by a green arrow and two AEs are marked by red arrows. Structures that fluoresced in both colors appearing yellow were occasionally noted in four D image records. In this example, an endosome containing both dyes is marked by a yellow arrow in panels three, six, and nine.
The overlap of red and green is equally complete in all three orthogonal projections indicating a putative fused em me ae. This movie shows the same dataset configured to rotate on the Y axis at one frame per second. The putative fused MEAE remains yellow from all perspectives.
Digital deconvolution is a useful tool to enhance resolution of 3D as well as four D images. A comparison between raw data and devolved data is shown here. For one typical snake nerve terminal stained during stimulation with FM 1 43 Once mastered, this technique can be done in 30 to 60 minutes if performed properly.
Following this procedure, other methods such as immunohistochemistry can be performed in order to answer additional questions such as colocalization of macro endosomes with presynaptic markers.
