SLICE Light Sheet panel

Purpose

Use the SLICE Light Sheet panel to enable the light sheets and adjust illumination settings.

The controls in the SLICE Light Sheet panel are used to optimize illumination of the sample by enabling a light sheet, choosing a color channel, and adjusting settings, such as the excitation intensity, and light-sheet offset. The offset is used to match the Z position of the light sheet to the current focal plane of the detection objective in order to achieve a sharp image. The offset may need to be adjusted frequently as you navigate to different focus positions in the specimen with the software joystick.

Best practices

• Determine the illumination settings in the SLICE Light Sheet panel that provide good illumination of the tissue with minimal photobleaching. To do this, you will primarily adjust the Excitation intensity slider.

• Use the Camera Histogram and Camera Settings to optimize visualization.

• To keep the live image in focus, you may need to frequently adjust the Offset slider for the active light sheet(s) in the SLICE Light Sheet panel as you move the to stage to different locations in the sample.

• You can illuminate the sample with one or both light sheets (left and right) throughout the tissue scanning/imaging process. The approaches have a trade-off between minimizing imaging artifacts and achieving maximum resolution. Illuminating with both light sheets together helps reduce striping or banding artifacts that represent shadows cast by opaque material in the sample; this is common in light-sheet microscopy of cleared tissue and leads to signal loss in the horizontal direction. The SLICE system can mitigate such permanent signal loss by providing complementary illumination from opposite directions and can correct striping-pattern artifacts using a destriping algorithm. While differences in scattering and attenuation between the two light sheets may slightly degrade resolution, in well-cleared samples, the reduction in striping artifacts generally outweighs this minimal change in resolution.

• Save your settings to the Multichannel Control panel: Once you have optimized all settings for a specific color channel save them to the Multichannel Control panel. Whenever you switch channels in the multichannel control window, BrightSLICE will automatically switch all settings to the most recently saved values for that color channel.

Light sheet panel functions

Emission Filter

Emission Filter: Shows the filter that is currently in position.

• The dropdown menu contains the filter for each color channel, and a neutral density filter.

• The emission filter will automatically switch to match the channel selected in the Multichannel Control window and to the neutral density filter when you start the SLICE alignment workflow.

• Alternatively, you can manually select a filter from the dropdown menu.

Light Sheets

Enable Left/Right When enabled, the indicator will be green. Enable the left or right light sheet as follows:

• Click Enable Left or use the keyboard shortcut Z to toggle the left light sheet on/off

• Click Enable Right or use the keyboard shortcut X to toggle the right light sheet on/off

Enable Split Enable automatic switching from the one light sheet to the other during an image acquisition.

Configure Split Open the Split Sheet Configuration window to set a split threshold position where the switch from one light sheet to the other will take place when Enable Split is active.

Advanced: Check the box to show additional settings for the Left sheet controls and Right sheet controls. The Advanced view also includes commands for applying light sheet offset values from current and previous SLICE alignment workflow results.

Excitation

Click Excitation next to Red, Green, or Blue to turn on the excitation for that color. When on, the indicator light will be white.

Use the sliders, or type in a value to change the excitation intensity percentage.

For all sliders, click the arrows to move 1 unit at a time, click on the slider bar to move 10 units at a time, or drag the slider to dynamically adjust the value.

Right matches left checkbox:

• When checked, any changes to the excitation intensity percentage will be applied to both light sheets.

• When unchecked, left and right excitation controls will be displayed in separate panes, with the Excitation Right settings below the Right Sheet Controls. Changing the excitation value for one light sheet will not be applied to the other.

All Off / Turn Back On Toggle to turn off the current excitation or turn on the most recent excitation.

Left sheet controls, Right sheet controls

Offset: Adjust the light sheet so it aligns with the current focal plane of the detection objective. The offset will need to be adjusted frequently as you navigate around the tissue specimen to achieve a sharp image.

Use any of the following methods to adjust the Left and Right Offset values:

• Place the cursor in the open box and scroll the mouse wheel.

  Hold the Shift key while scrolling to adjust the offset value by 10 instead of by 1

• Move the sliders with the mouse

• Type in offset values into the open boxes

Refractive Index (RI) compensation mapping enables you to record the proper light sheet offset position at different locations of the tissue sample. Creating an RI compensation map is recommended for acquiring images of an entire specimen using the Tissue scan workflow.