====== CT-dSPIM Manual ======

The CT-dSPIM is a flexible and easy-to-use implementation of Selective Plane Illumination Microscopy (SPIM) that allows for dual views (d) of large samples such as cleared tissue (CT). Standardly, this SPIM configuration forgoes any standard microscope frame instead utilizing a Focusing Translation Platform (FTP)- two linear positioners mounted to an optical table as "stilts" for an XY stage. The SPIM head is installed over the stage and supported by solid stainless steel bars. This concept creates an extremely stable, versatile architecture that makes loading and viewing large samples easy.

Despite numerous differences, it shares many similarities with its older brother diSPIM and therefore this document will often reference the diSPIM and indicate similarities/differences as needed. Most users will benefit from controlling the CT-dSPIM with the free, open-source Micro-Manager diSPIM plugin for both [[ct-dspim#alignment|alignment]] and [[mm_dispim_plugin_user_guide#acquisition_tab|acquisitions]]. 

This document concentrates on the assembly, setup, and alignment of the CT-dSPIM. 

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

<imgcaption SPIM_Figure|Typical diSPIM system on RAMM frame showing many major components in common with the CT-dSPIM: 3) SPIM arm mount (RAO-0046), 4) objective mounting assembly, 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) 50 mm 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>

<imgcaption ctdSPIM|Typical ct-dSPIM system has the SPIM head supported by stainless steel posts over a FTP (an XY stage mounted to Z-axis stilts).>{{:docs:ct-dspim.jpg?400|Typical ct-dSPIM}}</imgcaption>

====== Assembly ====== 
The relevant sections of the diSPIM [[https://youtu.be/TAgbr6IrTqw|assembly]] video are embedded. (For now, there is not a CT-dSPIM specific assembly video.) 

===== FTP (XYZ Stage) =====
**Tools**: 4 mm and 5 mm hex drivers\\

Individually, the stilts of the FTP are linear, or single axis, stages. In the firmware, one of them is designated the master axis and the other the slave.((Usually it is master-Z-axis and slave-F-axis, but it can vary depending on the system.)) Any time [they are both plugged in and] the master is commanded to move, the slave will move as if given the same command.((The only reason to command the slave axis directly is for alignment purposes.))

Together, the stilts of the FTP produce movement in the Z-axis, raising and lowering the entire stage, insert/chamber, and sample.

Mount the stilts to the breadboard, align them if necessary, and mount the XY stage to the stilts. ASI has a detailed [[http://www.asiimaging.com/docs/ftp_setup|FTP setup guide]] on their documentation site.

===== Post Mounts =====

ASI has a detailed [[http://asiimaging.com/docs/post_mount_setup|Post Mount Setup guide]] on their documentation site.
Mount the post mount rods, ~55 cm center to center, to the optic table with four M6 x 25 mm bolts.
The retaining collars are already installed on your posts at the same height; they can be adjusted later.  
Place the black rod-to-rod 90° connector on top of the retaining collars on both posts as in Figure 1.  Do not tighten them yet. 
Insert the shorter horizontal rod (attached to a black S-shaped arm with a split ring on the end) all the way into the black connector.  
Tighten the clamp so that the split rings are symmetrical: up ~45°, towards the center, and almost exactly 20 cm center to center (the distance between CUBE-IIIs, center to center). 
Leave the connection around the vertical post loose.

===== SPIM Head =====
**Tools**: 6 hex driver
from ct-spim document in Google Drive
Post Mount Rods

SPIM Head
Attach the camera and scanner tube lenses using a 1.5 mm hex driver, see Figure 3.

Figure 2: The camera and scanner tube lenses mount via three set screws in the CUBE-IIIs.

Lift the SPIM head and insert the tube lenses all the way, until the cubes are up against the split rings.  You may need to loosen the clamps around the horizontal bars. Make sure you support the SPIM head any time the horizontal bars are not tightly clamped.
Level the SPIM head; support its weight and loosen all 4 clamps. After it is level, engage the clamps on all 4 rod connection points. The two sides should be symmetric.
Tighten the bolts on the split ring, alternately tightening one bolt a bit (less one turn) and then the other, otherwise the asymmetric tension can cause the split ring to bind up.
If you need to move the SPIM head up or down, support the SPIM head, loosen the retaining collars on the vertical rods and move them along the posts, and finally re-lock onto the vertical rods.


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

<imgcaption Scanner_assembly|Scanner attached to C-mount adapter (left); 160 mm focal length tube lens and 10 mm spacer (right) ready to be mounted>{{:docs:manual/Scanner_assembly.jpg?700|Scanner attached to C-mount adapter (left); tube lens and spacer (right)}} </imgcaption> 
<imgcaption Scanner_tube_install|160 mm F.L. tube lenses and spacers mount to the lower, or inner, CUBE-IIIs before the scannner / C-mount adapter are installed>{{:docs:manual/Scanner_tube_install.jpg?300|Tube lens assembly attaches 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}}

==== 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**: 1.5 mm hex driver\\ 
The video is cued up to show the process for mounting the objectives (on a diSPIM) with a discussion of the two optical paths, Path A and Path B at the end.\\ \\ 

The objective mounting assembly includes an __ob__jective __l__ateral __p__ositioner __a__ssembly (OBLPA) for one side and a replacement spacer for the other, each with a bushing that the objective screws into. The assembly mounts to the right angle bracket via dovetail joint and is secured into position via set screw. Due to the long working distance of the objectives used by the ct-dSPIM, it is not necessary to remove the objective mounting assemblies in order to install the objectives; if you choose to do so, 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 assembly by the front and back, and pull towards you (while pushing on the right angle bracket). When you re-insert the assembly, it will “click” into position, and then the set screw must be re-tightened.

After the objectives are screwed into the bushings, 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 objective mounting assembly from the SPIM arm}} </imgcaption>
<imgcaption two_objectives_closeup| Special Optics ~17x, 0.4 NA, 12 mm WD objectives co-focused with ~12 mm gap>{{:docs:clearedtissueobjectives.jpg?400|Nominal spacing between these objectives is ~12 mm}}</imgcaption>
<imgcaption ct_objectives| Cleared tissue objectives in dipping media; the silver knob near the right objective is part of the OBLPA which produces lateral movement for the in the Y-axis to facilitate co-focusing the objectives>{{:docs:ct-dspimobjectivesmounted.jpg?400|Cleared tissue objectives mounted to SPIM head}}</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:
  * epi spot centered on epi camera
  * beam waist centered on imaging camera
  * beam/sheet plane coincident with imaging focus plane near the center of the piezo travel
  * beam perpendicular to imaging camera field of view
  * sheet centered in imaging camera field of view
  * sheets centered in bottom camera’s field of view (i.e. co-alignment of all three objectives)

==== 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}}