Number: _____11__________________ WORK INSTRUCTION BREAKDOWN SHEET Operation: Laser Scanning Confocal Microscope Operations Instrument: Olympus Fluoview FV10i Laser Scanning Confocal Microscope IMPORTANT STEPS KEY POINTS REASONS WHY A logical segment of the operation when something happens to advance the work. Anything in a step that might— Make or break the job Injure the worker Be a Cultural Consideration Make the work easier to do, i.e. “knack”, “trick”, special timing, bit of special information Reasons for each key point Sample Stage Preparation Gather necessary resources. If using small cylindrical sample holders, use the setup to the right. Notice the different types of “sample locks” (which hold the samples firmly in place) and “sample sites” (where the sample is held on the stage during imaging). Gather necessary resources. If using glass slides, use the setup to the right. Notice the “oil deposition site” (where oil is deposited before using the 60x Objective) and the “stage key” (used to align to microscope when inserting stage). Place sample site on the stage (small cylindrical samples). For small cylindrical sample holders, place the sample onto the sample
Before moving on to using the Observation Window, it should be noted: Look to the window at the right indicated by the green square. In this area the Operator can select the cannels that the image is being displayed on. By turning these channels on and off, very different images and features can be observed. When the Operator is ready to move on, click “NEXT”. Try turning off the phase contrast first. If most of the image is in phase contrast, a new sett of dyes needs to be selected that will cause the sample to fluoresce. DO NOT CLICK “Start” or the acquisition process start all over. Observation Window Layout The image directly above is the image map created during the previous steps. In this window the Operator can navigate around the sample to find points of interest. This is done by double clicking anywhere on the image to focus the lasers at that point. Use the channel buttons above the image to adjust which wavelength to be observed. Use the “Zoom” bar on the side to identify important features first. Use the container selection tool, located at the very top of the window, to change the container. This avoids wasting time using the laser to look at the image; it is a slow process and it can damage the sample. This changes the area of the slide to be observed. Using the “Repeat” button. At 1X the machine rasters at a standard speed and The Repeat button determines the rate at
yields much clearer images. At 4X the image is generated much quicker, but the resolution is poor. which the microscope rasters an image. Raster speed does not affect the final image; however, it does influence the rate of photobleaching of the sample. A quicker rastering speed means more passes by the laser over the area of interest. More passes means more excitation which inevitably leads to photobleaching, resulting in an unimagable section of the sample. Select the channels that the image is being displayed on by using the feature illustrated at the right. By turning these channels on and off, very different images and features can be observed. When multiple dyes were selected, the Operator can only have two activated at one time during the image observation. As the operator switches between lasers, an image is generated that combines all the different channels into a single image. As the Operator moves around the map image, the image will change (ensure that “Repeat” is ON). Each square represents a different wavelength being recorded. By adding and removing channels, the individual squares that are associated with those channels will alter the combined image. By clicking and dragging within these squares the Operator can move the focus of the laser. The bottom right square combines all other images into one. This will not affect the final image capture. This allows for precise feature alignment. This section of the screen affects the depth of focus By clicking on the “10X” button, the Operator will
Clicking “Save” and Save As” will save only the image. Anything added to it will NOT BE SAVED. Clicking on either of the “Export” buttons will allow the operator to save the image AS IT HAS BEEN EDITED in various formats. Clicking “Save Display” will capture whatever is on the image screen at that time. Image editing in 3D (All the previous editing techniques can also be applied to 3D images). When the Operator opens a 3D image, a slightly different window appears. The two boxes in the upper right corner will be highlighted and explained in the following two steps. The box to the left opens “3D Animation.” This takes the entire stack of images and creates a slideshow that makes them appear as on rotating image. The box to the right combines all the images into a 3D rendering for further editing. After clicking the right box, the windo will look like the one to the right.In this view, the Operator can click and drag around the image to rotate it 360 degrees in any direction. In this view the Operator also has access to the 3D control panel and the 3D image settings and can access these panels the same way as in the 2D view. In the “3D Control Panel” switch between the two rendering modes; both will give a unique image. In the “3D Image Settings” window, go to the