The overall goal of the following procedure is to obtain super resolution fluorescence images that elucidate important aspects of vesicle organization at a resolution otherwise unattainable with fluorescence microscopy. This is accomplished by first transecting hippocampal neurons with DNA encoding, photo convertible chimeras of vesicle cargo. In the second step, sparsely sampled raw images are collected with the super resolution microscopy system.
Next, the raw images are processed to produce a super resolution image. In the final step, the data is quantified to elucidate the organization of the vesicles. Ultimately, line profiles of puncta intensity can be used to compare vesicle separation to vesicle width, thereby determining whether the vesicles are close enough to generate a cluster Using this procedure.
Single color photo activated localization microscopy or palm can be used to study the organization of organelles in most types of cultured cells. To launch the super resolution imaging system for image acquisition begin by turning on the power to the arc lamp. Next, flip the system PC and component switches on the power remote.
Switch to on after the microscope control tablet comes on, turn on the computer and launch the software. Expand the boot status window to verify that the system is loading normally, and choose the start system option in the login dialogue. To position the sample for visualization, open the front and top panels on the laser safety cabinet and tilt the transmitted light arm to access the stage and the objectives.
Next, put oil on the alpha plan APO Chromat 100 x total internal reflection, fluorescence or turf objective. Then choose a low magnification objective for the initial focusing step. Now mount the sample onto the stage and raise the objective toward the sample.
Monitoring the lens position using the X, Y, Z function of the control tablet. Stop when the lens is in the ballpark of focus. To visualize the sample and fine tune the focus.
Lower the light arm and fully close the access panels. Then in the locate tab, click the ocular expansion symbol To bring up a schematic of the light path, choose trans on and verify that the schematic shows transmitted light from the bulb impinging on the sample. Look into the oculars and bring the cells into focus using the control tablet as a guide.
Then to identify the cells expressing the DRA two, choose reflected light and the filter appropriate for viewing green emission from den two. Scan the cover slip using low intensity reflected light until a healthy bright cell is found, and then to set up the software to acquire the palm data. Switch to the acquisition tab, and use the experiment manager to load the appropriate experimental palm acquisition configuration.
When the pop-up menu appears with a query about switching on lasers, click yes Then to focus the image of the cell at the camera plane. Choose only the 488 track. Adjust the acquisition parameters such as the E-M-C-C-D gain, and then choose continuous acquisition.
Next, adjust the display to the desired brightness and move the objective until the image on the screen is in focus. Then to collect a conventional wide field image, click the snap button and save the image. To set up the turf based illumination on the 488 track, choose turf on the epi turf button.
To optimize the turf based illumination, choose continuous acquisition and adjust the turf angle lighter until the background suddenly darkens and the specimen can be focused in just one plane. Switch to the 561 track with the 405 nanometer laser off. Set the E-M-C-C-D gain at about 200 and the exposure time at about 50 milliseconds.
Choose the emission filter that transmits in the red and set the laser intensity at about 40%Double check the turf angle for the 561 track. To reduce background, illuminate the sample until the background fades. Using the 561 track with only the 561 laser activated.
To collect the palm data, turn on the 405 nanometer laser for the 561 track and adjust the intensity of the 405 laser to about 0.01%To monitor the computed palm image during acquisition, expand the online processing options menu and check online processing palm to process with default parameters. Click start experiment to begin acquisition during the data collection, visually monitor photo conversion, increasing the intensity of the 405 nanometer photo converting laser. When denture two photo conversion begins to diminish.
When photo conversion no longer increases following a laser intensity increase, click finish current step. To end the experiment, save the file. To process the saved images, click on the processing tab, expand the method menu, and expand the palm sub menu.
Choose palm again from the four sub options. Select the appropriate image so that it appears in the viewing window. Expand the method parameters and click select to input the image, expand the settings field and choose default to execute peak finding and localization with the default options.
Press apply in the processing tab to compute the palm image, which will appear in a separate tab in the viewing window to filter out any poorly sampled components in the data. Use the nyquist sampling theorem and the remove palm outliers tool to delete peaks in regions where the means spacing between peaks, lowercase D is too large to resolve a vesicle of diameter, capital D with the palm image in the viewing window, click select to input the image and click apply. The new image will appear in a separate tab in the viewing window to filter out any poorly localized flora.Fours.
Go to the PAL filter tab, confine the accepted flora. Four localization precisions to a sigma of less than 35 nanometers. To choose a display mode for the palm image, go to the PAL rendering tab, select a pixel resolution, and the desired display mode.
In this image, a representative palm end product of the imaging and processing of a neuronal dense core vesicle or DCV is shown in red. The lateral coordinates of the localized flora fours in white are shown using the Centro display mode and the super resolution image of the associated vesicle is shown using the Gaussian display mode. In this second figure, analogous widefield in green and palm in red images of the soma and proximal processes of an eight days in vitro hippocampal neuron expressing tpa, a danger two are displayed.
Important features of the images include the extensive one-to-one correspondence and overlap in yellow between the puncta and the conventional and palm images. The significantly smaller size of the palm puncta and the occasional resolution of a single widefield puncta into multiple palm Punta. Here, a palm image of D cvs along part of one process of a second hippocampal neuron expressing tpa.
DENDRA two is shown. Note, the small diameter and homogeneous appearance of the puncta and the infrequent observation of closely opposed puncta, which represent putative DCV clusters. In this final figure, a simple quantitative method for determining if two or more cvs are close enough to comprise a cluster is outlined.
Line profiles of the puncta intensities are generated as shown in the graphs, which can then be used to quantify DCV separation and DCV width. If the separation significantly exceeds the width, the D cvs do not contact each other. Whereas if the separation is approximately the same as the width, the CVS may be in contact based on these criteria, the cvs in this image were classified as not in contact, whereas the cvs in this image were classified as in contact Following this procedure.
Other methods, like two color palm can be used to study the relative localization of distinct structures.
