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docs:ospim [2017/07/18 16:22]
Jon Daniels [oSpim] 		docs:ospim [2022/11/01 23:44] (current)
Steve Saltekoff [Micro-Manager plugin]
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 There is a Micro-Manager plugin for the oSPIM, based heavily on the diSPIM plugin but with a few changes.  In the future hopefully the oSPIM vs. diSPIM will hopefully be a setting changed by the user in the plugin, but for now there is a flag in the source code that needs to be changed and the code be rebuilt. There is a Micro-Manager plugin for the oSPIM, based heavily on the diSPIM plugin but with a few changes.  In the future hopefully the oSPIM vs. diSPIM will hopefully be a setting changed by the user in the plugin, but for now there is a flag in the source code that needs to be changed and the code be rebuilt.
  
-Start by installing a recent nightly build of Micro-Manager 1.4.x.  The latest official build is quite old at this point, so make sure to grab a nightly build.  For Windows the nightly builds are [[http://valelab.ucsf.edu/~MM/nightlyBuilds/1.4/Windows/|here]].+Start by installing a recent nightly build of Micro-Manager 1.4.x.  The latest official build is quite old at this point, so make sure to grab a nightly build.  For Windows the nightly builds are [[http://valelab.ucsf.edu/~MM/nightlyBuilds/1.4/Windows/|here]].  It can be helpful to install the build from the same timeframe as the JAR file was generated (see below).
  
-Download the latest copy of the plugin [[https://www.dropbox.com/sh/scifwh3toxejzes/AAD_o2yeAJwUAMjy8gIwiHgMa?dl=0 | here]] (last update 22-Jun-2017) or else one from the [[https://www.dropbox.com/sh/t5nm1uk6qr05xfe/AACKSzLkLZmQQDdyJaBK32vGa?dl=0|archives]].  Make sure Micro-Manager is not running and then copy the JAR file into C:\Program Files\Micro-Manager-1.4\mmplugins (it will take precedence over the ASIdiSPIM plugin in the Device_Control folder, or you can delete ASIdiSPIM.jar from that folder and replace it with this one).+Download the latest copy of the plugin [[https://www.dropbox.com/sh/scifwh3toxejzes/AAD_o2yeAJwUAMjy8gIwiHgMa?dl=0 | here]] (last update 08-Jan-2021) or else one from the [[https://www.dropbox.com/sh/t5nm1uk6qr05xfe/AACKSzLkLZmQQDdyJaBK32vGa?dl=0|archives]].  Make sure Micro-Manager is not running.  Then copy the JAR file into C:\Program Files\Micro-Manager-1.4\mmplugins\Device_Control.  **Finally delete ASIdiSPIM.jar from the same folder.**
  
 ===== Controller firmware ===== ===== Controller firmware =====
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 A good rule of thumb is to use the newest Tiger controller firmware that is older than the Micro-Manager plugin date. A good rule of thumb is to use the newest Tiger controller firmware that is older than the Micro-Manager plugin date.
  
-See the [[hardware:controller#firmware|controller page]] for general information about firmware including instructions to flash it.  Typically oSPIM systems have a scan-optimized XY stage so be sure to do the extra step to set the leadscrew pitch as described on the controller page.+See the [[hardware:controller#firmware|controller page]] for general information about firmware including instructions to flash it.  Typically oSPIM systems have a scan-optimized XY stage so be sure to do the extra step to set the leadscrew pitch as described on the controller page if the firmware is before version 3.20.
  
-Here is the download link for the [[https://www.dropbox.com/sh/sq2m4qpsrjl2hib/AAA61R_C5PrVsxE7TnKsaoCqa?dl=1|latest oSPIM firmware package from ASI]].+Here is the download link for the [[https://www.dropbox.com/sh/n93bzxtkrn31us5/AAA_moo_Y06RohB1bXbYjoiAa?dl=1|oSPIM firmware package from ASI]] (last updated 22-Mar-2018).  If you have newer firmware from ASI then use it (i.e. this package here is out of date).
  
 Standard firmwares for card addresses on oSPIM controllers are as follows Standard firmwares for card addresses on oSPIM controllers are as follows
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 | 5/35     | F/G stages    | STD_ZF%%__%%FG_Adr5.hex          |                                            | | 5/35     | F/G stages    | STD_ZF%%__%%FG_Adr5.hex          |                                            |
 | 6/36     | Progr. Logic  | PLOGIC_16%%__%%E_Adr6.hex        |                                            | | 6/36     | Progr. Logic  | PLOGIC_16%%__%%E_Adr6.hex        |                                            |
 +
 +===== Accessible range of illumination NA =====
 +
 +The light sheet thickness can be changed (within limits) by the iris on the "scanner" or [[hardware:scanner|light sheet generator]].  A more open iris means higher illumination NA which is a thinner waist but the waist is much shorter along the propagation axis (i.e. the confocal length or Rayleigh length is less).  A more closed iris means lower NA illumination which is a thicker sheet but uniform over longer distance.  The underlying physics is that for a 2x reduction in thickness you have a 4x reduction in the confocal length.
 + 
 +Usually you choose the illumination NA so that the confocal length matches the size of your sample and then the beam/sheet thickness simply is what it is.  E.g. for adherent cells 10um is a common target confocal length.  This corresponds to illumination NA of ~0.2 ([[docs:gaussian_beam|equations]]).
 +
 +The upper end of illumination NA can be limited by multiple factors:
 +  - the size of the MEMS mirror (larger MEMS mirrors can be used, but then the maximum speed goes down)
 +  - the "extra NA" of the objective beyond tilting the beam at 60°.
 + 
 +With a 1.2mm diameter MEMS mirror with (fixed) internal 22.5 degree mounting, the effective diameter is 1.1mm.  The standard light sheet scanner has a 75mm f.l. lens built in.  For the oSPIM the scanner tube lens is usually 200mm f.l. (different from diSPIM).  Assume you use the Olympus 60x/1.42 oil objective for illumination; this has EFL of 3mm.  Thus the maximum illumination NA considering these lenses and the MEMS is
 + 
 +1/2 * 1.1mm / 75mm * 200mm / 3mm = 0.49 
 + 
 +However, because the light sheet emerges tilted from the objective at 60 degrees, the actual "remaining NA" for this particular objective is
 +
 +1.42 * (1-sin(60°)) = 0.19
 +
 +so really the maximum illumination NA supported by the objective is twice that or 0.38 (clipped on one side by the objective back aperture).  Accessing a typical NA 0.2 shouldn't be a problem with this particular objective.
 ===== Assembly ===== ===== Assembly =====
  
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 Move the bottom objective until it's focused on the coverslip surface using transillumination (probably room lights are enough) as viewed with the camera on the inverted microscope.  A dirty coverslip helps, or put a dot of marker on the top (sample) surface of a coverslip and look for that. Move the bottom objective until it's focused on the coverslip surface using transillumination (probably room lights are enough) as viewed with the camera on the inverted microscope.  A dirty coverslip helps, or put a dot of marker on the top (sample) surface of a coverslip and look for that.
  
-Once the light sheet is aligned (see steps below) then you can adjust the tilt of the mirror between the dichroic and the camera tube lens to make sure the bottom camera is centered where the light sheet is being generated.  That way objects of interest can be identified with the inverted microscope and then immediately imaged with the light sheet.+Once the light sheet is nearly aligned (see steps below) then you can adjust the tilt of the mirror between the dichroic and the camera tube lens to make sure the bottom camera is centered where the light sheet is being generated.  That way objects of interest can be identified with the inverted microscope and then immediately imaged with the light sheet.
  
  
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 The hardest step is finding the pencil beam with the upper microscope.  There are 3 position adjustments: the SPIM head height, the SPIM head horizontal, and the manual linear positioner above the objective piezo for moving front to back as you view the microscope.  Use these to move the upper objective to the right neighborhood judging by eye.  The objective should be almost touching the coverslip.  You can judge front to back (adjusted with the manual linear positioner) pretty well by eye.  The hardest one for me is the horizontal adjustment. The hardest step is finding the pencil beam with the upper microscope.  There are 3 position adjustments: the SPIM head height, the SPIM head horizontal, and the manual linear positioner above the objective piezo for moving front to back as you view the microscope.  Use these to move the upper objective to the right neighborhood judging by eye.  The objective should be almost touching the coverslip.  You can judge front to back (adjusted with the manual linear positioner) pretty well by eye.  The hardest one for me is the horizontal adjustment.
  
-Once you think you are in the right neighborhood turn on the main (top) imaging camera.  Move around until you can see a line.  It will start out very blurry.  You can verify that you have found the beam by moving the manual linear positioner and see if the beam moves.  If the beam is vertical, rotate the camera to get it horizontal (you can get the SPIM imaging camera rotated very close to perfect by sighting off the edge of the cube in front of it; the camera should be rotated square with the cube).  Later you will fine tune the camera rotation.  Once you have found the beam, move the SPIM head height and horizontal to go down to where you can see the beam coming out of the coverslip.  You can move along the beam by adjusting the SPIM horizontal by a factor of -0.5 relative to the SPIM head height movement due to the 60 degree geometry (e.g. set the increment of the height to 20um and the increment of the horizontal to -10um in the Navigation panel of the plugin and then click one-to-one.)+Once you think you are in the right neighborhood turn on the main (top) imaging camera.  Move around until you can see a line.  It will start out very blurry.  You can verify that you have found the beam by moving the manual linear positioner and see if the beam moves.  If the beam is vertical, rotate the camera to get it horizontal (you can get the SPIM imaging camera rotated very close to perfect by sighting off the edge of the cube in front of it; the camera should be rotated square with the cube).  Later you will fine tune the camera rotation.  Once you have found the beam, move the SPIM head height and horizontal to go down to where you can see the beam coming out of the coverslip.  You can move along the beam by adjusting the SPIM horizontal by a factor of -0.5 relative to the SPIM head height movement due to the 60 degree geometry (e.g. set the increment of the height to 20um and the increment of the horizontal to -10um in the Navigation panel of the plugin and then click one-to-one; alternatively you can add the device "MultiStage" from the Utilities to the configuration and use System/Startup group/preset combination to define a synthetic stage with those relative motions).
  
 Use the mirror tilt of the top cube and/or the front to back knob just below to center the beam in the FOV (there is no good way of knowing which one is "right" and it doesn't matter much anyway in most cases). Use the mirror tilt of the top cube and/or the front to back knob just below to center the beam in the FOV (there is no good way of knowing which one is "right" and it doesn't matter much anyway in most cases).
  
-Now make sure that the beam is tilted correctly.  You can judge correct tilt by seeing if you move in and out of focus uniformly when you move the piezo, the SPIM head height, or the slice axis.  Understanding that the beam (and hence sheet) is diverging as it goes into solution.  Adjust the beam tilt with the dichroic of the inverted microscope.  It may help to stop the iris down so that there is less of a beam waist for this.  You can probably get it pretty close in dye, in the long term you also want to do this with beads in solution.+Now make sure that the beam is tilted correctly.  You can judge correct tilt by seeing if you move in and out of focus uniformly when you move the piezo, the SPIM head height, or the slice axis.  Understand that the beam (and hence sheet) is diverging as it goes into solution.  Adjust the beam tilt with the dichroic tilt of the inverted microscope.  It may help to stop the iris down so that there is less of a beam waist for this.  You can probably get it pretty close in dye, but ideally do it with beads in solution before finalizing the alignment.
    
 Next verify that the beam waist is right near the coverslip.  The way I suggest is first make sure the iris on the scanner is relatively wide open (wider beam on the edges, narrower waist).  Then make sure the bottom objective is focused on the top of the coverslip using the bottom camera with transillimunation (a flashlight or even room lights from above work well).  It is convenient to set this position of the lower Z axis to 0 so you can easily return.  Then switch to the SPIM camera view and on the navigation tab move the lower Z negative, so the objective is focused into the solution.  You should see the beam waist move into solution.  The beam waist should be very near the coverslip, if not the collimator of the scanner needs adjusting (see below). Next verify that the beam waist is right near the coverslip.  The way I suggest is first make sure the iris on the scanner is relatively wide open (wider beam on the edges, narrower waist).  Then make sure the bottom objective is focused on the top of the coverslip using the bottom camera with transillimunation (a flashlight or even room lights from above work well).  It is convenient to set this position of the lower Z axis to 0 so you can easily return.  Then switch to the SPIM camera view and on the navigation tab move the lower Z negative, so the objective is focused into the solution.  You should see the beam waist move into solution.  The beam waist should be very near the coverslip, if not the collimator of the scanner needs adjusting (see below).
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     * get the beam coming into the BFP at the correct position (essential) and angle (some wiggle room)     * get the beam coming into the BFP at the correct position (essential) and angle (some wiggle room)
  
-ASI is designing mounting hardware to overcome the first hurdle as of June 2017; it will leverage the same adapter bracketry as used for the diSPIM.  Overcoming the second hurdle will be up to the end user.+ASI has designed mounting hardware to overcome the first hurdle as of March 2018 which uses the same adapter bracketry as the diSPIM.  Overcoming the second hurdle is up to the end user.

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