Nikon FRAP Protocol - IGMM Imaging Facility
FRAP protocol for the Nikon A1 Confocal *This manual describes how to enable and use the bleaching features of the confocal and only serves as a reference if you have already been trained to perform FRAP experiments by a member of facility staff. Microscope Preparation Ensure the incubation chamber is turned on at least 1hr prior to starting the experiment and that the objective lens you require is mounted on the microscope. If you are using a stage top chamber to hold the plate/dish then put this in the incubation chamber to heat up. Software setup Ask one of the imaging team to give you access to the FRAP layout. This will appear as a tab in the lower left hand corner of the screen. When you select this layout you should see some additional windows open: A1 Stimulation-‐ Allows you to select the laser line and power for the bleaching phase ND Stimulation-‐ Here you configure the time lapse element of the experiment. Scan parameters for image acquisition Usually speed of acquisition (image fame rate) is the key factor in FRAP which means image quality generally suffers. The scan parameters used will depend on the sample but a rule of thumb is to use a frame size of no more than 512 x 512 and keep the scan speed as fast as possible (1.1 pixel dwell). Ensure the “Ch Series” sequential scanning option is turned off. Parameters for bleaching A1 Stimulation window In the A1 Stimulation window, select the Synchronise lasers checkbox. This simplifies the look of this menu and assumes you will use the same laser lines on all areas designated for bleaching. HV Mode should be set to “Keep HV”. This feature selects whether the detector gain is set to zero during the non-‐ acquisition bleaching phase to avoid detector overload which can occur if too much light hits the detector. Press the laser line button(s) you wish to use for bleaching, usually 405 & set the power (=>70%) but you will need to determine what is required empirically. The Scan Speed dropdown box refers to the scan speed (frames per second) used when bleaching the ROI only. A slower scan speed will increase the bleaching rate but may be less suitable if the recovery of the protein of interest is dynamic. When you finally start bleaching, if you find that the region bleached is offset from the position of the ROI then a Manual Shift Alignment must be performed (ask a member of facility staff to help you with this). Configuring the time phases of the experiment In the ND Acquisition window, if not already present enable 3 rows in the table by pressing the check boxes in the Phase column. In the Acq/Stim column use the dropdown lists to select Acquisition, Bleaching, Acquisition in the consecutive rows. Usually the first acquisition phase is used to acquire a couple of images prior to bleaching for a before and after comparison. The duration of the bleach and second acquisition phase will need to be determined. You need to aim to bleach the fluorescence in the ROI so that the intensity drops by =>90%. Tick the Perform time measurement checkbox. This ensures the intensity statistics within the ROI’s are recorded throughout the experiment. You may wish to save the experiment automatically to disk as its acquired. This will depend on the total time lapse duration. Adding bleaching ROI’s to the image Find an area on the sample that you'd like to bleach then select the down arrow next to the ROI button found in the right margin of the live window. Select the “Simple ROI Editor” menu option. Select either the Rectangle or Ellipse ROI tool and draw the ROI onto the image. The ROI can be resized by moving to the edge of the ROI when the cursor changes to a double headed arrow. The ROI can be moved around the image when the cursor changes to a four headed arrow. To Delete a single ROI click on it then press the Delete key on the keyboard To delete all ROIs press the Clear button in the Simple ROI Editor tool panel Press Finish when you have finished editing the ROIs Ideally you should have a minimum of 3 ROIs. One assigned as the bleach region and two others, the first being a Background ROI and the second being a Reference ROI. Right click on the ROI that will be used for bleaching and select “Use as stimulation ROI”. Ensure that the Background ROI is placed on a black area of the image where there are no cells. Then right click this ROI and select “Use as background ROI”. Place the third ROI on another cell which won't be bleached, then right click on it and select “use as Reference ROI”. Note that each ROI now has a label that denotes its function, S1, R2 and B3 Now that a Stimulation ROI has been defined, the Apply Stimulation Settings button is available in the ND Stimulation menu. When this button is pressed the Run Now button becomes available. Select “Run Now” to start the FRAP experiment.
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