SLICE alignment workflow

Purpose

Use the SLICE alignment workflow to adjust the position of the light sheets so that they intersect with the focal plane of the detection objective. Proper alignment is necessary to achieve sharp in focus images.

Alignment enables the ability to get crisp focus at a single Z plane. Proper alignment in conjunction with a correctly adjusted offset setting enables crisp focus at different Z planes. You will need to adjust the offset value as you navigate around a tissue sample. Refractive Index (RI) compensation mapping enables you to record the proper offset position at different locations of the tissue sample and an RI compensation map is required for acquiring a Tissue Scan. However, if you are just navigating around the tissue live, you can manually adjust the offset in the SLICE Light sheet panel as you go.

Procedure

Before you start

  • Remove the oil reservoir drip tray from the microscope.

  • The alignment pattern is a series of lines etched into the alignment prism mirror in random orientations. This may make the mirror appear filmy or dirty upon visual inspection before being attached to the microscope stage.

  • When using the joystick, adjust the slider to lower the step size for finer stage movements. This will enable more exact positioning of the mirror alignment patterns and light sheets in the field of view as needed.

  • As you move through the workflow, the light sheet controls will automatically adjust as needed for each step.

Click SLICE Alignment on the Advanced ribbon to get started.

If the Neutral density filter was not automatically selected upon launching the workflow, manually select it in the SLICE Light Sheet panel, Emission Filter dropdown menu.
Follow this procedure to configure the Neutral density filter to be automatically selected when the SLICE alignment workflow starts.

SLICE alignment workflow steps

  1. Attach Mirror

    Attach the alignment prism mirror to the microscope stage. The stage will automatically move to the load position to attach the mirror.

    The prism mirror is designed to only attach in the correct orientation.

  2. Start left sheet alignment

    Turn on the left light sheet and bring the alignment pattern on the mirror into the field of view.

    It is ok if the alignment pattern is blurry for this step.

    1. Click Turn on light sheet to enable the left light sheet.

    2. Use the up arrows of the XY joystick to lower the stage and submerge the mirror into the oil until the mirror is roughly centered in the observation window.

    3. Use the right/left arrows of the XY joystick to move the stage in X until the a portion of the alignment pattern in the field of view.

      • In order to see the alignment pattern, you may need to adjust the white point or optimize the camera histogram.

      • If alignment has been performed previously, it may be easier to click Move stage to saved alignment position to get the alignment pattern into or close to the field of view.

      • It is ok if the alignment pattern is blurry.

    4. (Optional) If there is a rolling line pattern in the field of view, you can adjust the camera exposure time to minimize the rolling effect:

      • If there is a bright rolling pattern, decrease the exposure.

      • If there is a dark rolling pattern, increase the exposure.

      Adjusting the rolling pattern in not necessary. It will only be seen during alignment and will not affect the alignment in any way.

  3. Find center in X

    Find the center of the alignment pattern on the alignment prism mirror.

    It is ok if the alignment pattern is blurry for this step.

    1. Locate the center mark (small red circle with a plus sign inside of it) in the field of view.

    2. Use the left arrows of the XY joystick to move the stage in X until the left edge of the alignment pattern is lined up with the center mark.

      The edge of the alignment pattern is where the pattern (with light you can see) butts up against solid black.

    3. Click Left edge

    4. Use the right arrows of the XY joystick to move the stage in X until the right edge of the alignment pattern in lined up with the center mark.

    5. Click Right edge

    Once both edges have been set, the center point between the edges is calculated and the stage will automatically move to line up the center of the alignment pattern with the center mark.

  4. Translate Detection Objective

    Physically adjust the position of the detection objective so that its focal plane intersects with the left light sheet.

    This step is made up of 2 sub-steps. The light sheet settings will automatically adjust between the two sub-steps to give proper illumination for performing each step.

    • Sub-step A

      • Adjust the detection objective knob located below and directly in the center of the SLICE unit until the pattern over the center mark is in crisp focus. The knob will turn left and right.

        The focus will become blurry to the right and left of the field of view. There isn’t an exact line of perfect focus. You want the image to be balanced in the field of view, with the most crisp focus near center mark.

    • Sub-step B

      1. Move the stage in X with the joystick until you find the light sheet, which will look like one non-continuous vertical bright line. The light sheet may initially be out of the field of view.

      2. Adjust the stage in X with the joystick until the light sheet is positioned over the center mark. If the light sheet line is too broken to see well, move the stage in Y until you find a more continuous line (where there are fewer etched lines on the mirror).

    Sub-steps A and B are iterative. Keep switching between A (center the most in focus part of the alignment pattern by adjusting the detection objective knob) and B (center the light sheet by moving in X with the joystick) until the light sheet and the most in focus part of the alignment pattern are over the center mark at the same time.

  5. Adjust left sheet Y center

    Adjust the Center setting in the Left Sheet Controls section of SLICE Light sheet panel to center the light sheet vertically in the field of view. When entering this step, the height of the light sheet will automatically adjust to the lowest setting = 1.

  6. Translate Left Illumination Objective

    Focus the left light sheet by adjusting the position of the left illumination objective. Physically turn the knob below the left illumination objective underneath the left side of the SLICE unit until the light sheet is as thin as possible and in sharp focus. The knob will turn forward and backward.

  7. Balance illumination left

    Balance the left sheet illumination in the field of view.

    Adjust the light sheet so that it is in focus in the center of the field of view and is equally blurry at the edges of the field of view.

    1. Move the stage in X with the joystick until the light sheet is close to, but not on, the left edge of the field of view. Leave a little space between the edge and the light sheet.

    2. Click Light sheet is near left edge

    3. Take note of how blurry the alignment pattern is.

    4. Click Go to left edge and then Go to right edge to see if the blurring is equal. If one side is more blurry than the other, click the button to go to that side and turn the left illumination objective knob to make it a little more in focus.

    5. Keep going back and forth, comparing and adjusting as needed, until the light sheet is in focus in the center of the field of view and is equally blurry at the edges of the field of view.

    6. Click Go to center to verify that it is in focus in the center.

  8. Start right sheet alignment

    Adjust the right light sheet so that it intersects with the focal plane of the detection objective.

    This step is made up of 2 sub-steps. The light sheet settings will automatically adjust between the two sub-steps to give proper illumination for performing each step.

    • Sub-step A

      1. Move the stage in X with the joystick until the light sheet comes into the field of view. The light sheet may initially be out of the field of view.

      2. Adjust the X position with the joystick until the most in focus part of the alignment pattern is positioned over the center mark.

    • Sub-step B

      • Adjust the Offset setting in Right Sheet Controls section of SLICE Light sheet panel to position the light sheet over the center mark.

    Sub-steps A and B are iterative. Keep switching between A (center the most in focus part of the alignment pattern by moving in X with the joystick) and B (center the light sheet by adjusting the right sheet offset) until the light sheet and the most in focus part of the alignment pattern are over the center mark at the same time.

  9. Adjust right sheet Y center

    Adjust the Center setting in the Right Sheet Controls section of SLICE Light sheet panel to center the light sheet vertically in the field of view. When entering this step, the height of the light sheet will automatically adjust to the lowest setting = 1.

  10. Translate Right Illumination Objective

    Focus the right light sheet by adjusting the position of the right illumination objective. Physically turn the knob below the right illumination objective underneath the right side of the SLICE unit until the light sheet is as thin as possible and in sharp focus. The knob will turn forward and backward.

  11. Balance illumination right

    Balance the right sheet illumination in the field of view.

    Adjust the light sheet so that it is in focus in the center of the field of view and is equally blurry at the edges of the field of view.

    1. Move the stage in X until the light sheet is close to, but not on, the left edge of the field of view. Leave a little space between the edge and the light sheet.

    2. Click Light sheet is near left edge

    3. Take note of how blurry the alignment pattern is.

    4. Click Go to left edge and then Go to right edge to see if the blurring is equal. If one side is more blurry than the other, click the button to go to that side and turn the right illumination objective knob to make it a little more in focus.

    5. Keep going back and forth, comparing and adjusting as needed, until the light sheet is in focus in the center of the field of view and is equally blurry at the edges of the field of view.

    6. Click Go to center to verify that the light sheet is in focus in the center.

  12. Remove mirror

    The stage will automatically move to load position and you can remove the alignment prism mirror from the microscope stage.

    When finished, click Close SLICE align workflow