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User Guide to the IBIF Leica TCS SP8 MP Confocal Microscope
This version: 7.24.14.
Introduction
The IBIF confocal microscope is made available on a fee-for-use-hour basis to all users who have been
trained. Unlike our previous instrument, it has a number of hardware features, specifically the
Objectives, the stage, and the condensers, that can be added or removed by the user, with the
permission of the Imaging Specialist. To minimize inconvenience to other users, and to reduce the risk
of inadvertent damage to the equipment, we ask that users obey two rules:
1) Leave the confocal as you found it. Replace and/or reset everything that you modified.
2) Be aware that other users may have failed to obey rule 1. Be alert, especially in regard to
mechanicals such as stage translation, condenser height, and objective rotation.
Thank you, and we wish you every success in your imaging. We are here to help.
The Confocal Microscope and Accessories
The items on the desk (along with the monitor,
keyboard, and mouse) consist of a confocal
controller, a microscope controller, a metal-
halide power source (which performs the
function of the Hg bulb in the previous system),
and a controller for the Dodt contrast
enhancement system (which replaces the
Nomarski contrast enhancement method of the
previous system).
The unlabeled switch is a shutter for the
Mai Tai laser and should only be needed in
emergencies in which the beam is exposed.
The instrument is a Leica TCS SP8 MP confocal
microscope, equipped for multi-photon confocal
microscopy. The components are the microscope
body, two automated stages, the scan head, the
electro-optical modulator (EOM), the monitor
and computer, and the Spectra-Physics Mai-Tai
tunable near-IR laser.
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Summary of Contents for Leica TCS SP8 MP
- Page 1 User Guide to the IBIF Leica TCS SP8 MP Confocal Microscope This version: 7.24.14. Introduction The IBIF confocal microscope is made available on a fee-for-use-hour basis to all users who have been trained. Unlike our previous instrument, it has a number of hardware features, specifically the objectives, the stage, and the condensers, that can be added or removed by the user, with the permission of the Imaging Specialist.
- Page 2 Two photomultiplier tube (PMT) detectors and two hybrid-PMT (HyD) detectors are available for conventional (single-photon) confocal microscopy. Some cell-phone carriers use frequencies that interfere with the HyD detectors. Switch your phone off or leave it at the front of the room. Two high-sensitivity Super HyD detectors are installed for non-descanned (multi-photon) confocal microscopy.
- Page 3 The microscope has no manual focusing adjustment. Instead, it has two stages and two computer-controlled focusing mechanisms. The large X-Y stage performs lateral translation in all modes. The nosepiece has its own focusing drive, controlled from the microscope controller, or the confocal controller, or from the software.
- Page 4 System Specification Lasers The system has four visible-light lasers: 405 nm, 488 nm, 514 nm, 552 nm, and 633 nm. The 405 nm should be used to excite DAPI or similar blue-emitting dyes, therefore use of the multi-photon laser will not be necessary.
- Page 5 Condenser A transmitted light condenser is available if high-quality visualization of unstained tissue or cells is required. However, there is enough unfocussed light available in transmitted light mode to enable simple localization of specimens without a condenser. The Imaging Specialist will demonstrate the user of the condenser if requested.
- Page 6 The Microscope Controller Control of Stage Movement Manual x, y and z stage control is via the knobs attached to the microscope controller, or by the manual X-Y stage control on the stage itself. The microscope controller moves the large X-Y stage only.
- Page 7 Screen 4: Nosepiece (i.e., objective) Z-control or stage X-Y control. Z-mode: The Home position is the highest point to which the nosepiece can currently be raised. It is usually set at the upper limit of travel of the nosepiece. The Focus Stop position is the approximate focus position (actually, a little above) for the 10x objective.
- Page 8 Setting and Restoring Home and Focus Positions (Screen 5). In some situations, for example when using the large X-Y stage in a lowered position, it may be desirable to adjust the Home and Focus positions. Here’s how to do it. First, select Screen 3 on the microscope controller display and select the 10x objective.
- Page 9 Setting the Focus Stop position Raise the nosepiece and set a glass slide specimen on the stage. If you do not have a suitable slide, there are some H & E stained slides in the top drawer of the desk. The slides are autofluorescent to GFP or YFP excitation, so you may use fluorescence as an alternative to transmitted light.
- Page 10 Positioning a Slide Raise the objectives using the microscope controller. Displace the stage to the left manually or using the stage X-Y manual control. For extra room, rotate the objectives manually so that the 10x is over the stage. Be sure that the clips are positioned at the edges of the slide recess.
- Page 11 Confocal Operation without Multi-photon Users will not need multi-photon microscopy for DAPI excitation because of the availability of the 405 nm laser. Metal-Halide Power Supply Be sure that the metal halide power supply shutter control button is set to remote (out). Application Start Turn on the computer, monitor, confocal power strip, and metal-halide power supply, in any order.
- Page 12 LAS AF Application The application screen will start up in the Acquire pane and will look like this: Buttons and Sliders Most functions are controlled by sliders. Click on the slider button to change the slider value. Drag the button or, for more precise adjustment, use the mouse wheel.
- Page 13 Program Panes The program has four panes – Configuration, Acquire, Process, and Quantify, selected by the tabs at the top left of the screen. Many Configuration pane options can be selected from the Acquisition pane, which reduces the need to use the configuration window. Configuration Pane The Configuration Pane allows setting of multiple hardware parameters.
- Page 14 Acquisition Pane Selection of Configuration Sixteen standard configurations are available to all users from the pull-down menu above. Please don’t modify them! You can also create your own configurations and store them. Acquisition Mode The acquisition mode defaults to xyz, with z = 1, that is, a single frame.
- Page 15 Acquisition Rate The acquisition rate window selects the acquisition scan parameters, including the number of pixels in the image and the scan speed, via pull-down menus. We recommend 400 Hz as an initial rate. The image scan may be zoomed down to 0.7 and up to Averaging may be by line or by frame, using pull-down menus –...
- Page 16 HyD detectors have a gain selected by clicking on its slider button. PMT detectors have both gain and offset functions, controlled by clickable sliders. Leica recommends a starting offset of -0.3%. Modifiying the Detector Bandwidth The detector bandwidth may be modified in either direction by simply clicking on and dragging its edges.
- Page 17 At right, the Capture Image button is used to acquire a single image. The Start button is used to initiate acquisition of a stack or a sequence. Display The displayed image is rotated 90° anti-clockwise with respect to the image seen in the objectives. Thus x-axis stage translation moves the image vertically on the screen, and y-axis stage translation moves the image horizontally.
- Page 18 Programming a Stack Programming a stack works similarly to the Mark First/Last mechanism used in Zeiss LSM. It requires the following steps: 1. With Live acquisition going, adjust the focus to the top of the stack. Use the Confocal Controller, or the microscope controller fine focusing knob.
- Page 19 Click on the ROI button to set the foreground conditions. Acquisition conditions within the ROI will be as specified in the Detectors panel. Draw the ROI using the tools above the image window. In this example, a square ROI has been selected.
- Page 20 Alternate Acquisition Modes – Sequential Click on the right-most icon in the Acquisition Mode menu. The Sequential Mode menu appears at the end of the acquisition parameters window. Click ‘+’ to add sequential events, ‘-’ to remove them. Up to six sequential acquisition events are possible. Events may be any of the available modes, e.g., xy, xyz, xyt, xyλ.
- Page 21 Alternate Acquisition Modes - Line Scan...
- Page 22 Alternate Acquisition Modes - Spectral Select xyλ or similar acquisition mode. The laser and detector section of the acquisition pane will appear as shown here. The number of bands and the bandwidth is set in the lambda- Scan Range Properties window. The maximum spectral range is 380 nm to 785 nm.
- Page 23 Alternate Acquisition Modes - Time Series Select xyt or similar acquisition mode.
- Page 24 Alternate Acquisition Modes - Tile Scan Select Tile Scan in the Acquisition Mode menu. The Tile Scan window will appear below in the Acquisition window. Note: Tile Scan should not be combined with image rotation or erroneous results will be obtained. Tile Scan Window The Tile Scan window features an X-Y map showing the current position of the stage.
- Page 25 Setting up a Tile Scan The approach to setting up a tile scan is different from that in Zeiss LSM. Step 1: Click on the Select Position button to select the current position as Position 1. Step 2: Move the stage to a position at one extreme of the area to be tiled. Use the microscope controller X-Y controls to move to the new position, while observing through the eyepieces or observing the display in Live mode.
- Page 26 Alternate Acquisition Modes – Mark and Find Mark and Find enables you to store multiple locations on your specimen so that you can return to them individually. It functions much as the way you store locations in the X-Y plane using Screen 4 of the Microscope Controller.
- Page 27 Storing Experiments Click on the Experiments tab to show a list of completed experiments. Double-click on an experiment to inspect the result or analyze it. Right-click on an experiment or sub-experiment to change its name. Use the Save button below in the Experiments window to save experiments.
- Page 28 Using the Experiment Metadata If you desire to repeat the acquisition parameters of a previous sub-experiment, you can do so by two different methods. If you are confident that the sub-experiment represents the correct parameters Load the experiment if it is not already loaded. Click on the sub-experiment.
- Page 29 Note - Not all parameters will be applied. These parameters are applied from an image file: Laser status, including multiphoton (assuming multiphoton laser is on) Multiphoton gain and offset All detector settings (including acquisition type for HyDs - Standard vs Photon-Counting) ...
- Page 30 Process Pane...
- Page 31 Quantify Plane...
- Page 32 Confocal Operation with Multi-photon Detector Filters and Barrier Filter The Super HyD detectors have a 620 nm barrier filter in front of them to protect against reflected IR light. For bandwidth selection, you will need a suitable filter in front of the detector or detectors. Ask us how to set this up.
- Page 33 Pinhole The pinhole is not in the emission path for the non-descanned detectors, so it can be ignored. Operation of the confocal with multi-photon excitation and descanned detection is ineffective and should be avoided. Enabling the MP Laser Unlike the semiconductor lasers, the MP laser must be turned on in the Configuration pane.
- Page 34 Dispersion Compensation Dispersion compensation of the near-IR laser is set in the GDD pull-down panel. Select the appropriate objective form the table. The default (zero GDD) is adequate for multi-photon operation. The fine tuning slider can be adjusted for special circumstances (e.g., operation in deep tissue).
- Page 35 On the left of the window are two controls that are misleadingly labeled. They are, effectively, coarse (left) and fine (right) control of laser intensity at the specimen. The number at the base of the left slider is the excitation wavelength, in nm. Best practice is initially to set the coarse slider to zero and the fine slider to a low value.
- Page 36 Shut Down The order is not important. Switch off the EOM controller and the Super HyD power supply, if used. Close the application – you will be prompted to save the files that you want to save. Save to folders on drive D: Log off the computer.
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