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docs:modular_manual [2019/02/05 21:08]
Melissa Glidewell [Phase 1] 		docs:modular_manual [2022/10/25 22:47] (current)
Jon Daniels [Fine alignment]
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 ===== Overview ===== ===== Overview =====
-Two objectives are placed at right angles to one another, each 45° from vertical, above a horizontally-mounted sample. A light sheet is created from one objective and then imaged through the other objective. A stack of images is collected by moving the light sheet through the sample.  +Two objectives are placed at right angles to one another, each 45° from vertical, above a horizontally-mounted sample. A light sheet is created from one objective and then imaged through the other objective. A stack of images is collected by moving the light sheet through the sample or the sample through the light sheet. The advantage of being dual-sided is that the role of the two objectives can be reversed to collect a second stack from a perpendicular direction. The two optical paths are usually symmetric, and are designated Path A and Path B. (The two paths are described in detail in the [[docs:mm_dispim_plugin_user_guide#diSPIM Plugin User Guide]].) The two datasets can be computationally merged for improved resolution isotropy.
-The core assembly, the SPIM head, can be mounted on various inverted microscopes. Thus far, microscope-specific adapter brackets((Attaching the SPIM precludes the use of the transmitted light optics because the brackets utilize the transmitted light pillar mounting point.)) exist for the following microscopes: +
-  * ASI RAMM frame (<imgref SPIM_Figure>) +
-  * Leica DMI-6000 +
-  * Nikon TE-300, Ti, TE-2000, Ti2 +
-  * Olympus IX-71/81, IX-73/83 +
-  * Zeiss Axio-Observer+
  
 ===== Mount the SPIM Head ===== ===== Mount the SPIM Head =====
 {{ youtube>TAgbr6IrTqw?start=215&end=377&rel=0&small}}  {{ youtube>TAgbr6IrTqw?start=215&end=377&rel=0&small}} 
-**Tools**: M6 hex driver\\ +**Tools**: 6 mm hex driver\\ 
 The video is cued up to show the process for mounting a diSPIM onto an ASI RAMM frame, but the principles apply to other scopes as well.\\  The video is cued up to show the process for mounting a diSPIM onto an ASI RAMM frame, but the principles apply to other scopes as well.\\ 
  
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 ===== Red Bracket ===== ===== Red Bracket =====
-**Tools**: M5 and M6 hex drivers\\ +**Tools**: 5 mm and 6 mm hex drivers\\ 
  
 RAMM frames are shipped with a red bracket supporting the inverted scope; it must be removed. For detailed instructions with pictures, please visit [[http://asiimaging.com/docs/removing_red_shipping_bracket|ASI's documentation site]]. RAMM frames are shipped with a red bracket supporting the inverted scope; it must be removed. For detailed instructions with pictures, please visit [[http://asiimaging.com/docs/removing_red_shipping_bracket|ASI's documentation site]].
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 ===== Scanners ===== ===== Scanners =====
 {{ youtube>TAgbr6IrTqw?start=480&end=697&rel=0?small}} {{ youtube>TAgbr6IrTqw?start=480&end=697&rel=0?small}}
-**Tools**: M2 hex driver\\ +**Tools**: 2 mm hex driver\\ 
 The video is cued up to show the process for mounting the scanners.\\  The video is cued up to show the process for mounting the scanners.\\ 
  
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 ===== Orientation ===== ===== Orientation =====
 {{ youtube>TAgbr6IrTqw?start=695&end=878&rel=0?small}}  {{ youtube>TAgbr6IrTqw?start=695&end=878&rel=0?small}} 
-**Tools**: M2 hex driver\\ +**Tools**: 2 mm hex driver\\ 
 The video is cued up to show the process for mounting and orienting the cameras.\\  The video is cued up to show the process for mounting and orienting the cameras.\\ 
 +
 +Depending on the accessibility of your system, it may be advantageous to connect all of the camera's cables (power, data, trigger) before mounting them to their tube lenses.
 +As with the scanners, a C-mount-to-tube-lens adapter is used. Screw the adapter's C-mount onto the camera before orienting and mounting them.
  
 For thin samples, having the beam enter the camera's field of view (FOV) from the top or bottom may be advantageous because the light sheet's intersection with the coverslip is a horizontal line and can be read out with fewer rows. However, the cameras are usually oriented so the beam enters the FOV from the side so the cameras’ light sheet mode, or rolling shutter, can be utilized. For thin samples, having the beam enter the camera's field of view (FOV) from the top or bottom may be advantageous because the light sheet's intersection with the coverslip is a horizontal line and can be read out with fewer rows. However, the cameras are usually oriented so the beam enters the FOV from the side so the cameras’ light sheet mode, or rolling shutter, can be utilized.
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 The convention the Shroff lab uses in their Nature protocols paper has the cameras rotated so that the beam enters from the same side of the field as the camera is on. On Path A, the beam from the left scanner appears to the right camera as entering from the right. The convention ASI uses has the beam cross the field of view in the same direction that it travels across (the front-view of) the microscope. On Path A, the beam from the left scanner appears to the right camera as entering from the left. The convention the Shroff lab uses in their Nature protocols paper has the cameras rotated so that the beam enters from the same side of the field as the camera is on. On Path A, the beam from the left scanner appears to the right camera as entering from the right. The convention ASI uses has the beam cross the field of view in the same direction that it travels across (the front-view of) the microscope. On Path A, the beam from the left scanner appears to the right camera as entering from the left.
 +
 +(After deciding how to orient the cameras, label the corner that goes "up" for future reference in case you have to remove and replace the camera.)
  
 ===== Camera Setup ===== ===== Camera Setup =====
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 ===== Filters and Mirrors ===== ===== Filters and Mirrors =====
 {{ youtube>TAgbr6IrTqw?start=1251&end=1721&rel=0?small}} {{ youtube>TAgbr6IrTqw?start=1251&end=1721&rel=0?small}}
-**Tools**: ASI ring tool, small Phillips head screwdriver, M2 hex driver\\ +**Tools**: ASI ring tool, small Phillips head screwdriver, 2 mm hex driver\\ 
 The video is cued up to show the process for installing an excitation filter, emission filter, and dichroic filter.\\  The video is cued up to show the process for installing an excitation filter, emission filter, and dichroic filter.\\ 
  
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 <WRAP left round info 50%> <WRAP left round info 50%>
-Notice: It is very easy to warp dichroic mirrors; only lightly tighten the retaining clip of the D-CUBE.  +Notice: It is easy to warp dichroic mirrors enough to induce aberrations; only lightly tighten the retaining clip of the D-CUBE.  
 </WRAP> </WRAP>
 \\ \\ \\ \\ \\  \\ \\ \\ \\ \\ 
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 ===== Stage limits ===== ===== Stage limits =====
-**Tools**: M1.5 hex driver\\ +**Tools**: 1.5 mm hex driver\\ 
  
 Setting the limit magnets on the motorized stages prevents crashes that can break coverslips or otherwise damage equipment; hardware limits are the most foolproof way to prevent crashes.((Limits may also be defined in software. Software-defined limits persist when the controller is powered down, but are lost when the firmware is updated or reset.)) The limit magnets move with the stage plates; when a magnet approaches a Hall effect sensor affixed in the stage body, the firmware detects a limit condition and stops the stage. The polarity of limit magnets relative to the stage is essential to their function.  Setting the limit magnets on the motorized stages prevents crashes that can break coverslips or otherwise damage equipment; hardware limits are the most foolproof way to prevent crashes.((Limits may also be defined in software. Software-defined limits persist when the controller is powered down, but are lost when the firmware is updated or reset.)) The limit magnets move with the stage plates; when a magnet approaches a Hall effect sensor affixed in the stage body, the firmware detects a limit condition and stops the stage. The polarity of limit magnets relative to the stage is essential to their function. 
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 ===== Single axis =====  ===== Single axis ===== 
-{{ youtube>qnOrg30NNuE?start=952&end=1217&rel=0?small}}**Tools**: M1.5 hex driver\\ +{{ youtube>qnOrg30NNuE?start=952&end=1217&rel=0?small}}**Tools**: 1.5 mm hex driver\\ 
 The video is cued up to demonstrate setting the F-axis limit magnet.\\  The video is cued up to demonstrate setting the F-axis limit magnet.\\ 
  
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 ===== XY-axes ===== ===== XY-axes =====
 {{ youtube>qnOrg30NNuE?start=952&end=1217&rel=0?small}} {{ youtube>qnOrg30NNuE?start=952&end=1217&rel=0?small}}
-**Tools**: M1.5 hex driver\\ +**Tools**: 1.5 mm hex driver\\ 
 The video is cued up to demonstrate setting the XY-axes limit magnets; note that we now recommend setting the X-axis magnets first.\\ The video is cued up to demonstrate setting the XY-axes limit magnets; note that we now recommend setting the X-axis magnets first.\\
  
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 ===== Beam position ===== ===== Beam position =====
-We are concerned with getting both the beam position and angle correct. Fortunately, the adjustments to do so are mostly independent of each other. <tabref what_affects_what> can be derived by understanding the light path.\\ +We are concerned with getting both the beam position and angle correct, along with getting both objectives "looking" at the same place. Fortunately, the adjustments to do so are mostly independent of each other. <tabref what_affects_what> can be derived by understanding the light path.\\ 
  
 <tabcaption what_affects_what| How alignment adjustments affect the beam position and angle.> <tabcaption what_affects_what| How alignment adjustments affect the beam position and angle.>
 ^ Adjustment                            ^ Position?        ^ Angle?          ^ Comments                                                                                                                                                                                                                                                                                      ^ ^ Adjustment                            ^ Position?        ^ Angle?          ^ Comments                                                                                                                                                                                                                                                                                      ^
-| Tilt of dichroic mirror               | Some             | Yes             | Position effect varies ((The dichroic tilt affects the angle at which the beam enters the back focal plane as well as the beam position at the back focal plane. It is possible to compensate by adjusting the scan center but we usually adjust the camera mirrors to compensate instead.))  | +| Tilt of dichroic mirror (Cube-III)              | Some             | Yes             | Position effect usually small ((The dichroic tilt affects the angle at which the beam enters the back focal plane as well as the beam position at the back focal plane. It is possible to compensate by adjusting the scan center but we usually adjust the camera mirrors to compensate instead.))  | 
-| Tilt of camera mirror                 | Yes              | No              | Set during scanner alignment and not after                                                                                                                                                                                                                                                    | +| Tilt of camera mirror (Cube-III)                | Yes              | No              | Set during scanner alignment and not after                                                                                                                                                                                                                                                    | 
-| Position of upper Z stage             | No               | No              | Angle varies if scanners are not connected to dichroic tubes                                                                                                                                                                                                                                  |+| Z position of SPIM head             | No               | No              |                                                                                                                                                                                                                                   |
 | Objective bushing (focus)             | Maybe minor      | Maybe minor     | If the bushing isn't perfectly concentric then slight movements can occur but this isn't usually a problem                                                                                                                                                                                    | | Objective bushing (focus)             | Maybe minor      | Maybe minor     | If the bushing isn't perfectly concentric then slight movements can occur but this isn't usually a problem                                                                                                                                                                                    |
 | Linear objective adjustment           | Yes              | Maybe minor     |                                                                                                                                                                                                                                                                                               | | Linear objective adjustment           | Yes              | Maybe minor     |                                                                                                                                                                                                                                                                                               |
-| <del>Tilt of adjustable mirror</del>  | <del>Some</del>  | <del>Yes</del>  | <del>Set during scanner alignment and not after</del>                                                                                                                                                                                                                                         | +| <del>Tilt of adjustable mirror</del>  | <del>Some</del>  | <del>Yes</del>  | <del>Set during scanner alignment, not user-adjustable</del>                                                                                                                                                                                                                                         | 
-| <del>Tilt of main micro-mirror</del>  | <del>Yes</del>   | <del>No</del>   | <del>Set during scanner alignment and not after</del>                                                                                                                                                                                                                                         |+| <del>Tilt of main micro-mirror</del>  | <del>Yes</del>   | <del>No</del>   | <del>Set during scanner alignment, not user-adjustable</del>                                                                                                                                                                                                                                         |
 </tabcaption> </tabcaption>
  
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 **Outline**\\  **Outline**\\ 
 +  * Phase 0 (coarse alignment by eye as described above)
   * Phase 1 (iterative)   * Phase 1 (iterative)
-    * 1. Co-align SPIM objectives to focus and center beams. +    * 1.1 Co-align SPIM objectives to focus and center beams. 
-    * 2. Tilt dichroic mirrors for horizontal beams __uniformly__ in/out of focus.  +    * 1.2 Tilt dichroic mirrors for __horizontal__ beams __uniformly__ in/out of focus.  
-    * 3. Tilt imaging mirrors to center epi spots.+    * 1.3 Tilt imaging mirrors to center epi spots. 
 +    * 1.4 Adjust lateral position of objective. 
 +    * 1.5 Go back to 1.1 and repeat until no adjustments needed
   * Phase 2 (may have to be repeated for initial alignment, and again when scanners or cameras are changed or disturbed)   * Phase 2 (may have to be repeated for initial alignment, and again when scanners or cameras are changed or disturbed)
-    * 4. Focus collimators to center beam waists in FOV. +    * 2.1 Focus collimators to center beam waists in FOV. 
-    * 5. Rotate scanners for __uniform__ focus of the sheets. +    * 2.2 Rotate scanners for __uniform__ focus across the sheets. 
-    * 6. Rotate cameras for vertical sheets from epi view. +    * 2.3 Rotate cameras for vertical sheets from epi view. 
-  * Phase 3 (performed just once for initial alignment and again for objective or sample changes) +    * Go back to 1.1 
-    * 7. Establish the coverslip location. +  * Phase 3 (performed just once for initial alignment and again for objective change) 
-    * 8. Align the inverted scope with the SPIM objectives. +    * 3.1 Establish the coverslip location. 
-    * 9. Cross-calibrate the piezo and light sheet movement.+    * 3.2 Align the inverted scope with the SPIM objectives. 
 +    * 3.3 Cross-calibrate the piezo and light sheet movement. 
 +  * Phase 4 (performed for any new sample)  
 +    * 4.1 Check that light sheet and detection plane are coincident; adjust offset in plugin if needed.
  
 ==== Phase 1 ==== ==== Phase 1 ====
-Repeat the steps of Phase 1 steps multiple times (after the first iteration, in no particular order) as you spiral in on alignment.+Repeat the steps of Phase 1 steps multiple times as you spiral in on alignment.
  
 === Cofocus SPIM objectives === === Cofocus SPIM objectives ===
 {{ youtube>qnOrg30NNuE?start=1694&end=2379&rel=0?small}} {{ youtube>qnOrg30NNuE?start=1694&end=2379&rel=0?small}}
-**Adjust**: OBLPA (<imgref piezo_with_bushing>) for steering the beams up and down in the Live mode window((i.e. aligning the beams in the Y-axis)) and threaded objective bushings for focusing\\  +**Configure** the [[docs:mm_dispim_plugin_user_guide#setup_path_tabs|Setup Path tabs]]:\\ 
-**End goal**: Both beams in focus with the epi-spot sitting on the beam waist (roughly at first, as in <imgref BeginAlign>, but better and better in subsequent iterations of Phase 1, as in <imgref ImprovingAlign>)\\  +select the checkboxes for 1) the "excitation beam" and 2) "Change settings on tab activate", and\\  
-The video is cued up to demonstrate cofocusing the SPIM objectives using Micro-Manager.\\+use the "On tab activate" drop down menu to select the Imaging camera.
  
-In the [[docs:mm_dispim_plugin_user_guide#navigation_tab|Navigation tab]], select the checkboxes for both Path A beam and Path B beam, and click on the **Multi** (for Multi camera overlay) and **Live** buttons.+**Adjust**: 
 +  * both objective bushings, and  
 +  * the knob on the OBLPA (<imgref piezo_with_bushing>) to center the beams in the Live window ((This aligns the beams in the Y-axis.))\\  
 +**The goal** is to have both beams in focus (roughly at first, as in <imgref BeginAlign>, but better and better in subsequent iterations of Phase 1, as in <imgref ImprovingAlign>).\\  
 +The video is cued up to demonstrate cofocusing the SPIM objectives using Micro-Manager.\\
  
 This is best done by alternating between steering and focus adjustments, alternating sides, __moving halfway to focus at a time__  until both beams are centered and focused. (See the [[hardware:troubleshooting#lack_of_bushing_travel|troubleshooting]] page if you cannot focus for lack of bushing travel.) Keep in mind that the nominal spacing between the objective bushing and piezo is 1 mm as pictured in <imgref piezo_with_bushing>. This is best done by alternating between steering and focus adjustments, alternating sides, __moving halfway to focus at a time__  until both beams are centered and focused. (See the [[hardware:troubleshooting#lack_of_bushing_travel|troubleshooting]] page if you cannot focus for lack of bushing travel.) Keep in mind that the nominal spacing between the objective bushing and piezo is 1 mm as pictured in <imgref piezo_with_bushing>.
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 The video is cued up to demonstrate adjusting the tilt of dichroic.\\ The video is cued up to demonstrate adjusting the tilt of dichroic.\\
  
-In the [[docs:mm_dispim_plugin_user_guide#navigation_tab|Navigation tab]], verify that the the piezos and scanners are in their neutral positions (at zero); also turn off Multi camera mode and use only the imaging camera, working on one beam at a time.  +In the [[docs:mm_dispim_plugin_user_guide#navigation_tab|Navigation tab]], verify that the the piezos and scanners are in their neutral positions (at zero). Use only the imaging camera, working on one beam at a time.  
  
-Use CUBE-III kinematic adjuster A alone, or B and C in tandem, to get the beams uniformly in/out of focus; an easy way to check it is by adjusting the imaging piezo (e.g. using the joystick knob) to see if the beam comes in and out of focus uniformly rather than with a horizontally rolling focus. (It may be helpful to use a false-color heat map on a single-camera view to aid in judging uniformity.) If the iris is fully opened to create a pronounced beam waist, the waist position will remain unchanged as you focus and defocus via the imaging piezo. +Check the //uniformity// of focus across the beam by adjusting the imaging piezo (e.g. using the joystick knob) or the slice position to see if the beam comes in and out of focus uniformly or with a horizontally rolling focus. Use CUBE-III kinematic adjuster A alone, or B and C in tandem, to get the beams properly positioned. It may be helpful to use a LUT (false-color heat map) on a single-camera view to aid in judging uniformity. 
  
 Next, adjust kinematics B or C (alone or in opposition) to get the beam horizontal. Confirm that this did not affect the uniformity of focus. The grid overlay in Micro-Manager (**Plugins** > **Acquisition Tools** > **Pattern Overlay**) can be helpful by providing reference lines. Next, adjust kinematics B or C (alone or in opposition) to get the beam horizontal. Confirm that this did not affect the uniformity of focus. The grid overlay in Micro-Manager (**Plugins** > **Acquisition Tools** > **Pattern Overlay**) can be helpful by providing reference lines.
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 ==== Phase 2 ==== ==== Phase 2 ====
-Phase 2 should only have to repeated once or twice for the initial alignment, but should be revisited anytime the collimators, scanners, or cameras are changed or disturbed. It is callous to sample changes.+Use a solution or 2D sample of fluorescent beads((Certain highlighters, e.g. Zebra Mildliner and Zebra Eco Zebrite, create bead-like fluorescent droplets when briefly dipped in water)) for Phase 2. Phase 2 should only have to repeated once or twice for the initial alignment and revisited anytime the collimators, scanners, or cameras are changed or disturbed; it is callous to sample changes.
  
 === Collimator === === Collimator ===
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 **Adjust**: Rotation of the camera (and camera's tube lens)\\  **Adjust**: Rotation of the camera (and camera's tube lens)\\ 
 **End goal**: Epi-view of the sheets produces a vertical line in the camera image\\  **End goal**: Epi-view of the sheets produces a vertical line in the camera image\\ 
-**Tools**: M5 hex driver+**Tools**: 2 mm hex driver
 The video is cued up to demonstrate adjustment of the camera's rotation.\\  The video is cued up to demonstrate adjustment of the camera's rotation.\\ 
  
-If the epi-view of the sheet is not vertical, adjust the camera via its tube lens (loosen the two bolts in the wide split ring holding the camera tube lens until the tube lens will rotate). Because we already verified the scanner orientation, any remaining tilt must be in the camera. Rotate as needed and tighten the bolts. Check both cameras. +If the epi-view of the sheet is not vertical, slightly loosen the set screws holding the camera's C-mount adapter to the tube lens, rotate the camera/adapter as needed, and retighten the screws. Because we already verified the scanner orientation, any remaining tilt must be in the camera. Check both cameras. 
  
 If you make any changes in Phase 2, return to the start of Phase 1. If you make any changes in Phase 2, return to the start of Phase 1.
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 <imgcaption BottomCamera|  Inverted microscope's view of the light sheets shown in <imgref ConvergingSheets>>{{:docs:manual/BottomCamera.jpg?300| Bottom camera aligned to the light sheets}}</imgcaption> <imgcaption BottomCamera|  Inverted microscope's view of the light sheets shown in <imgref ConvergingSheets>>{{:docs:manual/BottomCamera.jpg?300| Bottom camera aligned to the light sheets}}</imgcaption>
  
-=== Cross calibrate piezo and scanner movement[sub:Cross-calibrate-piezo] ===+=== Cross calibrate piezo and scanner movement ===
 {{ youtube>qnOrg30NNuE?start=4269&end=4571&rel=0?small}} {{ youtube>qnOrg30NNuE?start=4269&end=4571&rel=0?small}}
 **Adjust**: Values in the Piezo/Slice Calibration section of the [[docs:mm_dispim_plugin_user_guide#setup_path_tabs|Setup Path tabs]] via the **2-point** button\\  **Adjust**: Values in the Piezo/Slice Calibration section of the [[docs:mm_dispim_plugin_user_guide#setup_path_tabs|Setup Path tabs]] via the **2-point** button\\ 

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