====== diSPIM Manual ======
The diSPIM is a flexible and easy-to-use implementation of Selective Plane Illumination Microscopy (SPIM) that allows for dual views (d) of a sample mounted on an inverted (i) microscope. This document concentrates on the assembly, setup, and alignment of the diSPIM. The vast majority of these instructions apply regardless of the inverted microscope frame on which the SPIM head is mounted, but configuration specific manuals are in the works. 

====== Overview ====== 
{{section>modular_manual#overview&noheader}}

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

<imgcaption SPIM_Figure|Typical complete diSPIM system on RAMM frame showing major components. 1) SPIM-MOUNT , 2) SPIM-RAMM , 3) SPIM arm mount (RAO-0046 ), 4) piezo objective mover APZOBJ-200 or similar, 5) MIM inverted microscope with LS-50M stage, 6) CDZ-1000 centering stage or CDZ-R block, 7) LS-50M stage, 8) MIM-CUBE-II w/ mirror, 9) MIM-CUBE-II w/ fluorescence filters, 10) C60-TUBE_B imaging tube lens, 11) C60-TUBE_160 scanner tube lens, 12) 50mm extension tube, 13) MM-SCAN_1M light sheet scanner, 14) MS-2500 XY stage.>{{:docs:manual:dispim_figure.jpg?600| Typical complete diSPIM system on RAMM frame showing major components.}}</imgcaption>

====== Assembly ====== 
The relevant sections of the diSPIM [[https://youtu.be/TAgbr6IrTqw|assembly]] video are embedded. 

===== SPIM Head =====
{{section>modular_manual#mount_the_spim_head&noheader}}
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<imgcaption SPIM_mount_to_RAMM| SPIM head mounts via 2 dowel pins and a bolt at the indicated location regardless of the microscope frame. (RAMM frame pictured)>{{:docs:manual/SPIM_mount_to_RAMM.png| The SPIM head mounted to the microscope frame via 2 dowel pins and a bolt.}}</imgcaption>

===== Red Shipping Bracket ===== 
{{section>modular_manual#red_bracket&noheader}}

===== Scanners ===== 
{{section>modular_manual#scanners&noheader}}

<imgcaption Scanner_assembly|Scanner attached to C-mount adapter; tube lens ready to be installed on the CUBE-III>{{:docs:manual/Scanner_assembly.jpg?700|Scanner attached to C-mount adapter with tube lens assembly ready to be installed.}} </imgcaption> 
<imgcaption Scanner_tube_install|Scanner tube lens assembly mounted before the scannner with C-mount adapter is installed>{{:docs:manual/Scanner_tube_install.jpg?300|Tube lens assembly is attached to lower cubes on the microscope.}}</imgcaption>
<imgcaption NotWellAligned|Path A scanner with poor alignment>{{:docs:manual/NotWellAligned.jpg?300|Scanner A poorly aligned}}</imgcaption> 
<imgcaption BetterAligned|Path A scanner with good alignment>{{:docs:manual/BetterAligned.jpg?300|Scanner A well aligned}}</imgcaption>

===== Cameras =====
==== Compatibility ====
{{section>modular_manual#camera_setup&noheader}}

==== Mount Camera Tube Lenses ====
Tools: M5 hex driver\\ 
In the standard configuration for diSPIM on a RAMM frame, as in <imgref SPIM_Figure>, each camera tube lens is fastened to the SPIM-MOUNT((Other common configurations use a post mount system to minimize vibration from the camera's fans, or to accommodate an FTP system in lieu of a RAMM frame.)) with a ring clamp. The end of the tubes should come within approximately 1 mm from the upper (mirror) CUBE-IIIs. When the objectives are lowered into position for viewing a sample, adjust the bolts holding the tube support arms so the camera tube lenses are centered on the CUBE-IIIs. \\ 

==== Orient & Connect ====
{{section>modular_manual#orientation&noheader}}

<imgcaption cameraTable|Currently supported sCMOS cameras oriented in Shroff Lab and ASI conventions as seen from the back of the microscope; power switches highlighted in red>{{:docs:cameraorientation.png?600|Camera orientation table}}</imgcaption>

<imgcaption Scanners_Zylas| Andor Zyla cameras oriented according to ASI convention.>{{:docs:manual/Scanners_Zylas.jpg?600| Andor Zyla cameras according to ASI convention}}</imgcaption>

<imgcaption Scanners_Edges| PCO Edge cameras oriented according to Shroff lab convention.>{{:docs:manual/Scanners_Edges.jpg?600| PCO Edge cameras according to Shroff lab convention}}</imgcaption>

===== Objectives =====
{{ youtube>TAgbr6IrTqw?start=889&end=1246&rel=0?small}} 
**Tools**: M1.5 hex driver\\ 
The video is cued up to show the process for mounting the objectives with a discussion of the two optical paths, Path A and Path B at the end.\\ \\ 

If your system was delivered before 2015, refer to the document on [[hardware:dated#piezos|pre-2015 piezos]]. 

The objective mounting assembly includes a __ob__jective __l__ateral __p__ositioner __a__ssembly (OBLPA) for one side of the SPIM head and a replacement spacer for the other, a piezo objective mover (POM), and the bushing that the objective screws into; see <imgref piezo_with_bushing>. The assembly mounts to the right angle bracket via dovetail joint and is secured into position via set screw.\\ 

<imgcaption piezo_with_bushing| Objective mounting assembly top to bottom: objective lateral positioner assembly (OBLPA), piezo objective mover (POM), bushing, objective>{{:docs:manual/piezo_with_bushing.jpg?300| Objective mounting assembly}}</imgcaption>

Remove either one of the objective mounting assemblies; use a 1.5 mm hex driver to loosen the center set screw on the end of the right angle bracket, as in <imgref piezo_dovetail>. Grip the OBLPA or replacement spacer by the front and back (avoiding unnecessary stress on the piezo), and pull towards you (while pushing on the right angle bracket). Screw an objective into the bushing that is still mounted; add the other objective to the assembly you just removed. Re-insert the assembly (it will “click” into position) and tighten the set screw.

<WRAP left round info 50%>
Notice: If your system uses POMs, take care that you do not unnecessarily apply mechanical stress to the piezos when screwing the objectives into their bushing; they can be permanently damaged.  
</WRAP>\\ \\ \\ \\ \\ 

Use the OBLPA adjustment screw and the two objective bushings to move the objectives until they are visually symmetric from the front, as in <imgref two_objectives_closeup>, and visually aligned when viewed from the side of the microscope.

<imgcaption piezo_dovetail| Side view of SPIM head: set screw for securing the piezo assembly to the SPIM arm and on either side is a screw with ball-nose spring plunger that allows causes the assembly to "click" into place>{{:docs:manual/piezo_dovetail.png?300| Set screw for loosening the piezo assembly from the SPIM arm}} </imgcaption>
<imgcaption two_objectives_closeup| Nikon 40x, 0.8 NA, 3.5 mm WD objectives co-focused with ~0.5 mm gap>{{:docs:manual/two_objectives_closeup.jpg?463|Co-focused objectives with ~0.5 mm nominal gap}}</imgcaption>

===== Cables ===== 
==== Tiger controller ==== 
The electrical components of the diSPIM are controlled by ASI’s modular TG-1000 which comes in an 8- or 16- variety. Set up the controller in a location where the indicator lights are visible. See the [[hardware:controller|controller page]] for details on Tiger firmware; full documentation of the controller is on  [[http://asiimaging.com/docs/products/tiger|ASI's website]].
The Tiger controller’s power switch should always be switched to “off” when connecting or disconnecting cables.
The Tiger controller uses a universal power cable (as for a computer) that plugs into the back of the unit. 

<imgcaption TG8| Tiger controller with 8 slots>{{:docs:tg8.jpg?350|TG-8}}</imgcaption>
<imgcaption TG16| Tiger controller with 16 slots>{{:docs:tg16.jpg?600|TG-16}}</imgcaption>

**Connect each card’s cables:**\\ 
(0) TIG/COM
  * USB 2.0 Type B to host computer USB 2.0 Type A
  * DE-15 to four-axis Joypod (joystick/knob) DE-15
1: X/Y
  * DB-25 to X-axis then Y-axis
  * Hirose 4 pin to dimmable LED illuminator SubMiniature v.B
2: Z/F
  * DB-25 to inverted microscope focus Z-axis then SPIM focus F-axis
  * Hirose 4 pin to dimmable LED illuminator SubMiniature v.B
3: MICROMIRROR
  * DE-09 (like VGA) to either Path A scanner (axes A/B((Before March 2014 other cables and scanner orientations were used. Contact ASI for new cables.))) then Path B scanner (axes C/D), or scanner with anti-striping micromirrors
4: P
  * DE-09 to P-axis (right) POM
5: Q
  * DE-09 to Q-axis (left) POM
  * Check serial numbers on the POMs and controller faceplates to be sure to connect the cables to the correct controller module.
6: PLC((If your system was delivered before 2015, refer to the document on the [[Hardware:dated|older style TTL Card]].))
  - BNC to Light Path A camera trigger
  - BNC to Light Path B camera trigger
  - N/A standardly; can be configured as global shutter or ongoing acquisition signal
  - BNC to Laser side select
  - BNC to Laser #1 on/off
  - BNC to Laser #2 on/off
  - BNC to Laser #3 on/off
  - BNC to Laser #4 on/off

==== Other cables ====
Connect the power and/or data cables to the two cameras and two scanners if you have not already done so. 

Specialty cables from ASI are labeled, but the two cameras and scanners are identical, so take special care to keep straight which cables go where so that the control software can work as intended. The POMs look identical but are labeled "P" and "Q"; take care to install them correctly as well.

===== Laser and Fiber Optics =====
{{section>modular_manual#laser_and_fiber&noheader}}

===== Filters and Mirrors =====
{{section>modular_manual#filters_and_mirrors&noheader}}

<imgcaption D-Cube|Disassembled dichroic/filter cube, D-CUBE; installed into CUBE-III>{{:docs:manual/D-Cube.jpg?300|Internal dichroic/filter cube (D-CUBE)}}</imgcaption>

<imgcaption Dove-II|D-CUBE installed on the dovetail mount (CUBE-II pictured), left, with a bottom view of the filter cube, right>{{:docs:manual/Dove-II.jpg?300|Cube installed on the C60-DOVE-II adjustable cube dovetail mount. There are two set screws on the dovetail mount. One has a small cam end that engages a slot in the internal cube. Align the filter cube slot with this screw and tighten until the cam engages the slot. The cam will allow the cube to be moved slightly up and down. Place in the neutral position and tighten the nylon-tipped set screw to hold the cube in place.}}</imgcaption>

<imgcaption RA_Mirror| Right angle mirror installed on the dovetail mount>{{:docs:manual/RA_Mirror.jpg?300| Right angle mirror on dovetail mount}}</imgcaption>


====== Alignment ======
The relevant sections of the diSPIM [[https://youtu.be/qnOrg30NNuE|alignment]] video are embedded. 

===== Getting started with Micro-Manager =====
{{section>modular_manual#getting_started_with_micro-manager&noheader}}

=== Hardware Configuration ===
{{section>modular_manual#hardware_configuration&noheader}}

=== Plugin ===
{{section>modular_manual#plugin&noheader}}

===== Stage limits ===== 
{{section>modular_manual#stage_limits&noheader}}

==== Single axis stages ==== 
{{section>modular_manual#single_axis&noheader}}

==== XY-axes ====
{{section>modular_manual#XY-axes&noheader}}

Figure 18: The limit magnets are in housings that can be positioned along a track, secured by a set screw.

Figure 19: Small sample chambers may require the limit magnet housings be rotated 180° in order to get the magnets close enough together while still being able to secure the housings via their set screw.
 
The relevant sections of the diSPIM [[https://youtu.be/qnOrg30NNuE|alignment videos]] are embedded. 

===== Optical alignment =====

**Goals:**
The qualities of final alignment (for both SPIM optical paths), include all of the following:
  * beam/sheet plane coincident with imaging focus plane, near the center of the piezo and/or slice travel
  * sheet centered in imaging camera field of view
  * beam perpendicular to imaging camera field of view
  * beam waist centered on imaging camera
  * for dual view, epi spot centered on epi camera
  * when mounted on an inverted microscope, sheets centered in bottom camera’s field of view (i.e. co-alignment of all three objectives)

For single-view systems there are more adjustments than required to achieve good alignment of the single light sheet plane and detection focal plane and does not have an epi view. This makes it easier to align, but also makes it possible to adjust things so that the illumination is far from the center of the illumination objective so this should be checked by eye.

==== Overview ==== 
{{section>modular_manual#alignment_overview&noheader}}
{{section>modular_manual#beam_position&noheader}}

==== Coarse alignment ==== 
{{section>modular_manual#coarse_alignment&noheader}}

==== Fine alignment ==== 
{{section>modular_manual#fine_alignment&noheader}}