Biology & Biochemistry Imaging Core (BBIC)

Know your Leica controls, The Acquisition Panel, part II- the center

Setting up your detection windows in the SP8 software

The central portion of the acquisition panel controls your detector settings. Our SP8 has 3 photo-multiplier tubes (PMTs) and 2 Hybrid (HyD) detectors. This is a diagram of their arrangement in the hardware of the system:

leica-detector-hardware

The collected light is sent to PMT3 first. The position of the sliders (2) is adjusted from the acquisition panel. They allow light of the specified wavelength range into the detector, and reflect the blocked wavelengths to the next two detectors (HyD2 & 4). Those sliders will admit selected wavelengths and reflect the remainder to the last two detectors (PMT1 & 5). This is why if you are using only 1 fluorescent detector, and you do not required the extra-sensitivity of the HyD, you should use PMT3, because it can directly “see” all the wavelengths.

This is the central region of the Leica acquisition panel. The top portion is used to control the laser intensity. There are on switches for both the UV and visible light lasers, and slider bars for adjusting the power of each laser line. If a laser has not been turned on via the laser configuration panel, the bars will be greyed and cannot be adjusted. You can click and drag, use the scroll wheel on the mouse, or click on the number above a bar and type in the desired value.

panel-1.2

The lower region diagrams the visible spectrum at the top; controls for the 5 fluorescence detectors and a transmitted light PMT are at the bottom. In between are the emission windows or slider bars for the fluorescence detectors; these control the positioning of the sliders for each detector, which will determine which wavelengths of light are passed through to the detector, and which ones are blocked or reflected to another detector. When a detection window is active, a grey “ramp” (white arrow) will connect it with its corresponding slider bar (red arrow):

on-ramp.2

Inactive windows will have no ramp.

When a laser is active and ready to use, a corresponding line will appear on the spectrum diagram:

panel-2-lasers-on

The software also allows a user to display the emission and/or excitation curves for the fluors detected. They are not required to scan, but are very helpful for optimal setting of the slider bars. Click on the top drop down menu of a detection window and select your fluor:

panel-4-dye-spectra

If your dye is not listed, choose the closest match. You can do a lambda scan of any unlisted dye and add it to the drop down menu. When a dye is selected, an emission curve (solid line) will appear:

panel-7-em-curve

To see the excitation curve, right click on the spectra display to get this new window:

panel-8-r-click

Clicking on “Show Excitation Graph” adds a dotted line excitation curve:

panel-9-add-ex-curve

This is obviously quite helpful in verifying that you have picked an optimal laser (note that the 496 laser line is near the peak of the excitation curve in the example above). You can right click again to remove either curve if you wish.

The laser lines and emission curves are good guides for setting the spans of the emission windows. With 5 bars things can get confusing, so look for the grey ramps to keep track of which bar goes with which window. Note that the bars cannot be moved past each other (a reflection of the physical arrangement of the detectors and sliders), so you need to use the windows on the left/center for the shorter wavelengths, and the center/right windows for collecting longer wavelength light. You can click on a bar and move it from side to side to position it. Another option is to double left-click on a bar to bring up a wavelength setting window, and type in the numbers:

panel-10-set-range

The lower end of the range should never be less that 10nm higher than the wavelength of the laser line (in this example the “Begin” setting should be changed to 506 nm for the 496 laser). This will avoid any problems with unwanted reflection from the laser into the collected light.

Clicking the down triangle at the bottom of a detection window gives the user access to the gain and offset adjusters.

panel-11-settings1

Increasing the gain boosts the power, so your signal will get stronger, but so will your background. The offset is used to set the floor threshold, i.e., define the zero or totally black pixel (which will appear green in the LUT window Hi-Lo view). The proper adjustment of these settings is covered in the next section about the right side of the acquisition panel.

The PMT-Trans window has a drop-down menu that gives you 5 bright field scanning options:

pmt-trans

The Rest of the Center Panel

A few features in the center of the center panel not related to detection windows that users should know about:

The objective setting. A number of the microscope’s functions, such as defining the working distances for autofocus or calculating the optimal thickness of an optical slice, take the objective’s specifications into account. Therefore it is always a very good idea to make sure that the objective listed in the software actually matches the one that you are using. If they don’t match, the objective is in the wrong place on the turret and should be moved to the correct position. Only 6 objectives can be listed in the software/mounted on the turret at any given time, but if you need to use a different one, the Core manager can adjust the settings.

This is the objective drop down menu. It lists the objectives in the order of their turret positions (1 at the top to 6 at the bottom), with the objective currently in the home position (i.e., over the sample) highlighted in red.

obj-drop-down

Clicking on the objective icon to the right will open a window that provides detailed information about the objectives currently mounted:

obj-info

A second button of interest to most users, the USB control panel settings:

usb-1

The drop-down menus underneath each knob icon allow you to adjust the sensitivity of that knob to your personal preferences. You can change the functions controlled by a knob using the drop-down menu above its icon.

usb-2

Most people will use the Leica default settings for knob assignments (Smart Gain/ Smart Offset/ Scan Field Rotation/ Pinhole/ Zoom/ Z-Position). If you need a different combination for your imaging, you should save your personalized settings (bottom right of the panel) for future use.

The other buttons in the center of the center panel will likely never be needed by almost all users, but for the edification of the curious:

The Acoustical Beam Splitter panel button

panel-33-aobs

The Fluorifier Disc Setting window:

fluo-disc

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