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docs:modular_manual [2019/02/27 23:21]
Melissa Glidewell camera tilt adjustment 		docs:modular_manual [2022/10/25 22:47] (current)
Jon Daniels [Fine alignment]
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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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 ===== 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>.

Trace: