TITLE: Imaging cleared mouse brains on SmartSPIM
AUTHORS: John Rohde

[DESCRIPTION]
The SmartSPIM is an axially swept lightsheet microscope that produces uniform axial resolution across the entire imaging volume of large biological samples, such as whole mouse brains. Acquiring these datasets requires well prepared samples, carefully aligned hardware, and a broad knowledge of the acquisition software.

[STEPS]
SECTION: Mounting Samples:
1. Mounting agarose-embedded and unembedded brain samples. A small drop of super glue is sufficient to hold a brain for >24 hrs when submerged in the imaging bath (see figure (a) below). Placing the glue along one edge makes it easier to remove the brain after imaging.

  
SECTION: Configuring the microscope to acquire sample data:
4. Tiling parameters (Procedure Step #2 in the SmartSPIM acquisition software):
Specify the six faces of a rectangular solid surrounding the brain. Center and shift the tiling to align with the midline of the brain. Note that in order to perform these steps, one or more lasers will be ON and potentially bleaching the sample. Turn OFF the laser in between steps to reduce photo bleaching.
SECTION: Configuring the microscope to acquire sample data:
8. Multi-brain acquisitions:
Save configurations and parameters for the first brain before starting a second brain, using the Sequencer option.
a) Once Procedure steps 1)-to-5) are complete, the sequencer allows you to save all of the configurations for the previous sample. 
b) Click [Save Configuration] at the top. A pop-up window appears. 
c) Name the file, double-check the laser and filter selections that you will be aquiring in. Finally, click [Ok] to acquire a preview single-plane image at the mid-line (along the z-direction) that’s stitched. 
 
SECTION: Mounting Samples:
2. Mounting multiple samples: up to three brains can be loaded onto the sample arm at a time. The brains are embedded in 10mm x 15mm x 20mm agarose blocks. Align the blocks so the sides are perpendicular to the incident light sheets and the top is perpendicular to the detection path which is orientated vertically upward.
  

SECTION: Configuring the microscope to acquire sample data:
8.1. Save configurations for the first brain:
After clicking [Ok] in the previous step, the sample arm will translate in z to reach the midline (in z), and then translate in x- and y-directions to capture an image for every tile. The position of the rectangular solid around that brain that you set in “Procedure 2)” is now shown in an xy-plane corresponding to the imaging chamber.
 
Ensure the microscope’s box is closed-up (reattach the removeable panels):
  

SECTION: Configuring the microscope to acquire sample data:
4.1. Aligning the Right(xy) and Left(xy) edges or faces of the sample:

RIGHT(xy): Set the excitation side to "Right," use an excitation laser that is appropriate for the sample (often longer wavelengths like 639 nm are best), and click the "Preview" button to start a live preview. Use the XY joystick and the left control knob to locate the right side of the brain and position it near the right side of the live preview window. Rotate the control knob to move the sample up/down and ensure that the widest part of the brain is bounded by the right side of the preview.
  
LEFT(xy): Set the excitation side to "Left," and click the "Preview" button to start a live preview. Use the joystick and the left control knob to locate the left side of the brain and position it near the left side of the live preview window. Rotate the control knob to move the sample up/down and ensure that the widest part of the brain is bounded by the left side of the preview.
  
SECTION: Configuring the microscope to acquire sample data:
4.2. Aligning the Top(xy) and Bottom(xy) edges or faces: 

TOP(xy): Set the excitation side to "Left" or "Right" and click the "Preview" button to start a live preview. Use the joystick and the left control knob to locate the top(xy) surface of the brain and position it near the top(xy) side of the live preview window. Rotate the control knob to move the sample up/down and ensure that the edge of the brain is bounded by the top(xy) edge of the preview window.
  
BOTTOM(xy): Set the excitation side to either "Left" or "Right" and click the "Preview" button to start a live preview. Use the joystick and the left control knob to locate the bottom(xy) surface of the brain and position it near the bottom(xy) edge of the live preview window. Rotate the control knob to move the sample up/down and ensure that the edge of the brain is bounded by the bottom(xy) edge of the preview window.
 
SECTION: Configuring the microscope to acquire sample data:
4.3. Aligning the Top(z) and Bottom(z) edges or faces:

TOP(z): Set the excitation side to either "Left" or "Right" and click the "Preview" button to start a live preview. Use the joystick and the left control knob to locate the top(z) surface of the brain anywhere within the live preview window. Rotate the control knob to move the sample down to ensure that the edge of the brain is bounded and fully contained by the Set Top position.
  
BOTTOM(z): Set the excitation side to either "Left" or "Right" and click the "Preview" button to start a live preview. Use the joystick and the left control knob to locate the bottom(z) surface of the brain anywhere within the live preview window. Rotate the control knob to move the sample up to ensure that the edge of the brain is bounded and fully contained by the Set Bottom position.
 
SECTION: Configuring the microscope to acquire sample data:
4.4. Centering the tiling:
Keyboard shortcut: [C] + [Left-click]
With the live preview active, center the midline of the brain within the preview window (a). Use the keyboard/mouse shortcut [C] + [Left-click] to set this location as the tile grid center. You should see the white dashed line move to the center of the white box (c).

  
SECTION: Configuring the microscope to acquire sample data:
4.5. Shifting the tiling:
Keyboard shortcut: [S] + [Left-click]
 
Symmetrically dividing the tiling between the illumination paths: 
Finally, hit the blue [Calculate Tile] button (a), to (b) update the Left-light sheet (Green tiling) and Right-light sheet (red tiling) positions to now coincide with the centerline of the brain (c).
   

SECTION: Configuring the microscope to acquire sample data:
8.2. Saving configurations for additional brains:
Work through the Procedure 2)-to-5). Now, when you click [Save Configuration] the 2nd brain, its location will be shown, and L/R alignment (Procedure Step 3)) and focus set-up (Procedure Step 5)) will also update for the 2nd brain. These updated paramters aren’t visible in the UI, but the xy-plane is a visual indication that everything about the 2nd brain is now saved. 
  
SECTION: Starting an Acquisition:
9. Single vs. Multi-brain acquisitions:
SECTION: Set-up and align the instrument:
3. To set-up and align the instrument in preparation for a sample, follow the "SmartSPIM setup and alignment" protocol.

SECTION: Configuring the microscope to acquire sample data:
5. L/R Alignment of the two illumination paths (Procedure Step #3 in the SmartSPIM acquisition software):
Procedure step 3) opens another pop-up window. There are 4 steps to complete here, listed below, corresponding to the four buttons in the center of the window (three along the middle, and the fourth in the lower right). 
First, hit the “[Go to midline]” so both light sheets equally illuminate the region of interest. Be sure to select a location with bright and high-contrast features, and with features that change rapidly in the z-direction. Select a channel where there’s fluorescence to ensure high-contrast and low noise for this important set of steps. 
 
SECTION: Configuring the microscope to acquire sample data:
6. L/R Power adjustment (Procedure Step #4 in the SmartSPIM acquisition software):
The last step leaves the FOV zoomed-in. Zoom out before starting these steps:
  
SECTION: Configuring the microscope to acquire sample data:
7. Focus setup in "z" (Procedure Step #5 in the SmartSPIM acquisition software):
Make fine adjustments to the position of the detection objective to adjust the focus of microscope throughout the depth of the brain volume.
SECTION: Configuring the microscope to acquire sample data:
7.1. "Clear All" channels before beginning each brain (previous parameters are saved):
First, select which channels are required for imaging (a).
Second, uncheck "Focus Active" (b).
Third, use "Clear All" if another brain was previously acquired.
  
SECTION: Configuring the microscope to acquire sample data:
7.2. Adjusting laser power to prevent photodamage:
Especially for brains with endogenous fluorophores, LOWER the laser power during this step to lower the risk of photodamaging the sample. Alternatively, in samples with low SNR (signal-to-noise ratio), you may need to instead boost laser power here to have enough signal to ensure proper focus.
  

SECTION: Configuring the microscope to acquire sample data:
7.3. Selecting a vertical path through "z":
Recall that the right side of the brain is the “Reference” side of the instrument. Select a path through z that’s near the center of the brain, biased towards the refence side. If the brain is not fully cleared (b), or at short wavelengths where the light is more scattered and the interior of the brain is dim, it may be necessary to follow the edge of the brain for clear enough sample to focus on. Deep regions in z may require you to move to the olfactory bulbs in order to access the sharpness/focus of the image.
  

SECTION: Configuring the microscope to acquire sample data:
7.4. Readjust laser power:
Now that the manual alignment steps are complete, restore the laser power to a higher value that will be used for imaging. After these steps where the laser must be on for a long period of time (Procedure 3)-5)), be sure to adjust the power to the level you will image at. Typically, lower the power in the channels with fluorescence while doing these alignment steps to avoid bleaching, then increase the power to image, in order to maximize the number of photons reaching the camera.
  
SECTION: Configuring the microscope to acquire sample data:
5.2. Acquiring reference images and aligning to the reference:
Goal: capture a position for the "reference" = RIGHT lightsheet, and aligning the non-reference (LEFT lightsheet) to it.
First, turn on the laser and adjust the focus (right wheel) to achieve a tack-sharp image. 
Second, click on [Grab Reference]. A static grayscale image appears. Note that for this step the software automatically turns of the laser.
Third, click on [Adjust Non Ref] with the right wheel on the joystick controller. 
During this process the reference image (Left lightsheet) is overlayed with the live view of the Right lightsheet. The two images are well-align when green + magenta = white. Look for sharp features, such as small fluorescent cells, to align.
 
SECTION: Configuring the microscope to acquire sample data:
5.3. Additional example - Reference images and aligning the non-reference (LEFT light sheet) to it:
  
SECTION: Configuring the microscope to acquire sample data:
5.4. Troubleshooting misaligned L/R illumination paths:
Repeat previous alignment steps. a) Note that the left hemisphere is out-of-focus. When this occurs, repeat the L/R alignment before acquiring a dataset of the whole brain. 
Note that there are several possible locations to perform this alignment along the L/R midline (b). The top of the brain will be the sharpest. Be sure to explore front-to-back as well. Search for high-contrast, sharp features to focus on.
  

SECTION: Configuring the microscope to acquire sample data:
5.1. Preventing photobleaching:
Note that during imaging, the exposure time is just 2 ms slit width per plane. Work quickly and carefully, adjust laser power down if you have plenty of signal, and [STOP] the laser as soon as you’ve completed each step. Completing some steps does turn off the laser automatically, but others do not, and it’s easy to bleach the sample during these times. Some fluorophores and dyes will significantly bleach in just a few seconds.
Be careful to not bleach the sample. Some photodamage is inevitable, because you’re working in a channel with fluorescence for high-contrast, low-SNR images to align with. Lower the laser power to reduce the light on the sample, but keep a high enough power to ensure good SNR (signal-to-noise ratio) and image quality.
SECTION: Configuring the microscope to acquire sample data:
6.1. Adjusting laser power based on the L/R Power histogram:
Note that the relative Left and Right laser powers can be adjusted. By un-checking the “Link” box, each illumination path can be adjusted separately (a). When imaging along the center-line of the brain, overlap the two histograms (b). Depending on the instrument, this step rarely needs to be performed, because the L/R power is already closely matched.
 
SECTION: Starting an Acquisition:
9.1. Single brain acquisitions:
a) When not using the sequencer, click [Start Acquisition]. Name the file with the LabTracks ID, and confirm the channels you wish to acquire in are selected. Remember to adjust the laser power of each channel to a level you wish to image with. 
b) For the Sequencer (acquiring multiple brains in sequence, up to 4, all mounted on a single sample arm), select [Ok] (blue outline, here it’s accidentally obscured by the 5th and 6thchannel information). c) During this step, a pop-up window will appear and flash red-and-white. A time estimate will appear along the bottom of the software.
  
SECTION: Starting an Acquisition:
9.2. Viewing and adjusting a single plane of the brain. Composite images are stitched in the LifeCanvas software using TerraStitcher.
During this step the software creates and applies a flat to pre-correct the images as they’re being saved. The incident laser line that forms a sheet after passing through a cylindrical lens has a Gaussian profile, and the flat corrects for and "flattens" the intensity of the field of view. Warning: this step is not easily reversible, because the flat is applied to the saved data.
The SmartSPIM software, using TerraStitcher, generates a single, 2D, stitched plane image of the whole brain (typically 20-28 tiles). There are six sliders to adjust the brightness (Display Range), the amplitude and center of the tile-overlap adjustments (L amp, L center, R amp, R center), and the L/R brightness (Left/Right Balance). Adjust both the left and right hemisphere to create a clean “corrected” image. The “raw” or “original” image is pictured on the left, and the corrected image is on the right.
  
SECTION: Starting an Acquisition:
9.3. Additional examples of Creating and Applying flats within the acquisition software:
  
SECTION: Starting an Acquisition:
9.4. Starting a multi-brain (Sequencer) acquisition:
When imaging multiple brains, you no longer use the “Start Acquisition” button. Note that the Sequencer menu blinks red-and-white during the acquisition.