The overall goal of this procedure is to investigate neurotransmitter vesicle dynamics in neuroblastoma cells using pH sensitive probes and total internal reflection fluorescence microscopy or turf earth. This is accomplished by first plating cells onto glass cover slips and transecting them with genetically encoded pH sensitive fluorescent probes of vesicle fusion and recycling, which are selectively targeted to synaptic vesicles. Protein expression is expected within 24 to 48 hours.
The second step is to record vesicle dynamics by total internal reflection microscopy. This step is particularly critical for the success of the experiment and a step-by-step description on how to achieve turf configuration is provided.Next. Once the correct turf configuration is reached, the sequential images can be automatically recorded by the software in basal conditions or stimulated conditions.
The final step is to process images and extract data from the videos using commercial software or homemade algorithms. Ultimately using turf microscopy to record the changes in the fluorescent signal deriving from vesicle fusion to the plasma membrane. It is possible to obtain information on the frequency of fusion events and the mode of vesicle fusion.
The main advantage of this technique over the existing methods, like for example, electro microscopy and electrophysiology, is that it provides both special and temporary solution required to detect vesicle dynamics. Indeed, highly contrasted images obtained by this technique allow to detect signals from from single vesicles. The cheap base image acquisition analysis provides the temporary solution necessary to detect the dynamics.
This method can help to answer a key question in the nervous science field, such as understanding the role of a particular synaptic protein on bicycle dynamics, or the mechanism of action of drugs acting on the synaptic transmissions. Generally, Individuals new to this matter, we struggle with that analysis because completely automatic procedures to follow vesicle and to register the fluctuation of the PROEs Signal are not always available. We first, the idea of this method when we decided to investigate the impact of pathogenic mutant or synaptic protein on Nevo transmitter vesicle release.
The existing methods were not sufficiently comprehensive, so we decide to set up this technique in the neuroblastoma cell line SH SY five one to reach the appropriate solution. Visual demonstration of this method is critical as some key step, such as how to achieve tier four configuration and how to extract the quantitative data from Videos are critical to explain using word while it can be more understandable and persuasive. If visualized.
To perform turf imaging, set up a motorized inverted microscope, laser source, and the turf slider as outlined in the text protocol. To achieve turf illumination. Remove the glass cover with transfected cells and insert it in the appropriate imaging chamber.
Assemble the chamber and add 500 microliters of Krebs ringer solution in the center of the glass. Then add oil over the objective. Place the imaging chamber on the stage of the microscope and position the objective under the glass cover slip.
Position the safe cover over the sample in epi fluorescence mode. Focus on the cover slip and choose transfected cells placed in the chamber center under software control. Switch to turf illumination in live mode.
To set the turf configuration, check the position of the beam that emerges out of the objective on the sample cover. When the beam is positioned in the center of the objective lens, a spot is visible in the center of the turf sample cover and the cell is imaged in epi fluorescence mode. To reach the critical angle, move the focused spot in the Y direction using the angle adjustment screw on the turf slider.
When the beam converges on the sample plane at an angle larger than the critical angle, the spot disappears and a straight thin focused line is evident in the middle of the sample cover. To fine tune the turf angle, use a cell sample. Watch the fluorescence image on the video.
At this stage, an epi fluorescence like image is still visible. Gently move the screw until turf condition is achieved. Here only one optical plane of the cell is in focus, resulting in a flat image with high contrast.
To perform sample imaging, set the single channel time-lapse experiment appropriate exposure times are between 40 and 80 milliseconds. Acquire images at one or two hertz sampling frequency. Add 500 microliters of Krebs ringer solution and record cells in turf microscopy mode.
Save the time sequential images of the resting condition. Focus on the same cell and record under the same conditions of resting. After five frames, add five microliters of potassium chloride Krebs ringer solution and keep potassium chloride in the chamber.
Save the time. Sequential images of the stimulated condition. Use a sequence fluorescence intensity macro for fluorescence intensity, quantification in a region of interest, or ROI of the image over the course of the movie.
To do so, first, open the time sequential images. Go to the macro menu and select sequence fluorescence intensity. In the analysis window navigate.
To select the ROI choose one of the selection tools in the menu. To create the ROI place three ROIs in regions of the cell membrane without spots. For the background RO, I employ this background ROI to evaluate the photo bleaching and to set the threshold for fusion event analysis.
With the ROIs selected, click okay. Automatically calculate the average fluorescence intensity of each ROI over the course of the movie, export the data to a spreadsheet program for further analysis. To evaluate the photo bleaching open the fluorescence intensity rose.
Background ROIs normalize the fluorescence intensity values in each frame to the initial intensity value and average the values. Highlight the average data and create a line plot using the chart menu options. From the data analysis menu, select trendline to open the plot analysis dialogue.
Set the type of regression to exponential regression. Then select display equation on chart. In the graph window, the exponential equation appears and the parameter values are automatically assigned.
After applying the exponential correction to the intensity values as described in the text protocol, set the threshold by opening a normalized and corrected background. ROI then calculate the average fluorescent signal and its standard deviation. The average value plus three standard deviation units represents the threshold.
To select the fusion events. First, open the time sequential images with image analysis software, apply a Gaussian filter to the active image sequence. Analyze images using the tool count objects or a macro, which allows a selection of an object whose pixels have average fluorescence intensity within a defined range.
Set the intensity range manually using the threshold function apply a macro filters objects to select only objects meeting specific criteria. First apply ranges option for the aspect. Aspect reports the ratio between the major axis and the minor axis of the ellipse equivalent to the object.
Adequate values for aspect are a minimum of one and a maximum of 2.3. Then apply ranges for the diameter criterion In pixels diameter reports the average length of the diameters measured at two degrees intervals, joining two outlined points and passing through the OID of the object. Next select display objects.
Selected objects will appear superimposed to the turf microscopy image clue in the analysis only those spots that show a short transient increase in fluorescence intensity immediately followed by a marked loss of signal. Manually employ the circular selection to create an ROI of approximately one spot diameter radially around selected vesicle. With the ROI selected, calculate the average fluorescence intensity of each ROI over the course of the movie.
Export the time course of the fluorescence changes measured in each experimental ROI to A spreadsheet proceed to normalize the intensity value in each frame to the initial fluorescence intensity. After applying the exponential correction to the intensity values in each frame, as before, apply logical functions using spreadsheet or math packages to calculate the total number of fusion events, the time each fusion occurs and the amplitude of fluorescent peak. Assume the increase of fluorescence intensity exceeding the threshold as vesicle fusion to the plasma membrane and the resulting peak as a fusion event.
Calculate the peak width as the difference between the last and the first X value of each peak. Then multiply this value by one over the sampling frequency. Consider this value as the time of vesicle fusion and adhesion at the plasma membrane before vesicle, re acidification and recycling.
Next, calculate the whole cell area under the curve as a sum of values over threshold. Consider this value as net fluorescent change During the recording time due to the spontaneous or evoked synaptic activity, calculate the peak height as the difference between the maximum y value of each peak and the threshold, and consider this value as indicative of the fusion type. In whole cell analysis, the total number and the frequency of fusion events occurring in the cell are analyzed under resting conditions.
Vesicles occasionally approach the plasma membrane and fuse with it in the fluorescent intensity plot. This is reflected by an abrupt change of fluorescent intensity over the threshold after application of the secretary stimulus. Different peaks of variable fluorescence intensity suddenly appear in the fluorescence intensity profile plot.
Note the increase in peak number and height after potassium stimulation. In the single peak analysis, every single fusion event is analyzed. Two parameters are measured.
The peak width, which corresponds to the time of vesicle, adhesion, and fusion to the plasma membrane, as well as the peak height, which reflects the mechanism of peak fusion. Under potassium depolarization, the peak height and width increase, mirroring a change in the motor vesicle fusion to the plasma membrane. While attending this procedure, it's important to remember that this can technique can allow only the visualization of the event taking place at the plasma membrane or immediately Following this procedure.
Other method, like a wealthy microscopy can be performed in order to answer additional questions like the fate of internalized bicycles, proteins within the cells After it's development. This technique paved the way for researchers working in the field of cellular biology and physiology to study dynamic event occurring at the personal membrane, and such as vesical Exocytosis, cell matrix edition, and ion fluxes. After watching this video, you should have a good understanding of how to achieve a tier four configuration, record vertical dynamics, and analyze data.
