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docs:mm_dispim_plugin_user_guide [2019/07/03 00:03]
Jon Daniels [Acquisition Mode] 		docs:mm_dispim_plugin_user_guide [2020/09/23 18:29]
Jon Daniels [Volume Settings] fixed axial resolution
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 Distinguishing two nearby point sources of light is a classic problem in optics. If a camera’s pixels are so large that both points are read by the same pixel, you won't be able to tell them apart, regardless of how good the optics. The camera resolution must be at least twice the optical resolution to avoid being the limiting factor; essentially, a dark pixel must be between the two bright pixels. This is a manifestation of the Nyquist sampling theorem, which describes a fundamental mathematical relationship between continuous (or analog) and discrete (or digital) signals. Distinguishing two nearby point sources of light is a classic problem in optics. If a camera’s pixels are so large that both points are read by the same pixel, you won't be able to tell them apart, regardless of how good the optics. The camera resolution must be at least twice the optical resolution to avoid being the limiting factor; essentially, a dark pixel must be between the two bright pixels. This is a manifestation of the Nyquist sampling theorem, which describes a fundamental mathematical relationship between continuous (or analog) and discrete (or digital) signals.
-The standard formula for the optical lateral resolution is the Rayleigh criterion, a distance given by the formula: 0.61*lambda/NA (where lambda is the wavelength of light). For a 40X 0.8NA objective with 500 nm light, the lateral resolution is ~381 nm. That objective with a camera sensor that has a 6.5 um pixel pitch, is spatially sampling at (6.5 um/40X) ~162.5 nm pixel size, so we meet the Nyquist criteria (because 381 nm/162.5 nm = 2.34), but we wouldn't if we were using 400 nm light. + 
-Normally, for axial resolution limited by optics, the Z-axis step must be smaller than twice (per Nyquist) the optical axial resolution. Optical axial resolution, or depth of field, is usually taken to be lambda/(NA^2); for the 40X 0.8NA objective at 500 nm, it is ~781 nm. Therefore, the Z-step should be <0.39 nm.+The standard formula for the optical lateral resolution is the Rayleigh criterion, a distance given by the formula: 0.61*lambda/NA (where lambda is the wavelength of light). For a 40X 0.8NA objective with 500 nm light, the lateral resolution is ~381 nm. That objective with a camera sensor that has a 6.5 um pixel pitch, is spatially sampling at (6.5 um/40x) ~162.5 nm pixel size, so we meet the Nyquist criteria (because 381 nm/162.5 nm = 2.34), but we wouldn't if we were using 400 nm light. 
 +Normally, for axial resolution limited by optics, the Z-axis step must be smaller than twice (per Nyquist) the optical axial resolution. Optical axial resolution, or depth of field, is usually taken to be 2*lambda*RI/(NA^2); for the 40x 0.8 NA water objective at 500 nm, it is ~2.1 um. Therefore, the Z-step should be < 1 um. 
 For diSPIM, we have two views that can be merged computationally. The axial perspective from each objective is a lateral perspective (with higher resolution) from the other, so we can undersample in Z to a certain extent, which is advantageous from a speed perspective. However, giving up too much axial resolution the registration of the two views will suffer; in an extreme case your Z-step could be large enough to completely skip over a point source. For this reason, we recommend the Z-step be at least as small as the objective's depth of field. (With Fiji MVR and bead datasets, it's easy to register datasets with 0.5 um Z-step spacing but not with 1 um Z-step spacing.) For diSPIM, we have two views that can be merged computationally. The axial perspective from each objective is a lateral perspective (with higher resolution) from the other, so we can undersample in Z to a certain extent, which is advantageous from a speed perspective. However, giving up too much axial resolution the registration of the two views will suffer; in an extreme case your Z-step could be large enough to completely skip over a point source. For this reason, we recommend the Z-step be at least as small as the objective's depth of field. (With Fiji MVR and bead datasets, it's easy to register datasets with 0.5 um Z-step spacing but not with 1 um Z-step spacing.)
 </WRAP> </WRAP>
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 ==== Default Timing ====  ==== Default Timing ==== 
  
-Specify ​the **Sample ​exposure [ms]** in Slice Settings, ​and either define ​the slice period or let the plugin minimize the slice period automatically. The timing depends on the reset and readout time of the cameras; plugin code specific ​to each supported camera computes timings for the user-specified ​ROI using information provided by the manufacturer (usually either via their detailed camera documentation or read-only ​Micro-Manager ​properties). ​  Any extra time in the slice is placed before the camera trigger to allow maximum time for piezo settling. The values computed by the default timing mode are shown in the slice timing settings and can be subsequently modified manually.+Specify ​the **Sample ​exposure [ms]** in Slice Settings, ​and either define ​the slice period or let the plugin minimize the slice period automatically. The timing depends on the reset and readout time of the cameras as well as the trigger mode set on the [[docs:mm_dispim_plugin_user_guide#cameras_tab|Cameras tab]] (e.g. "Overlap/synchronous" trigger mode performs readout and reset at the same time whereas they are sequential in "Edge" mode).  Plugin code specific ​to each supported camera computes timings for the user-specified ​ROI using information provided by the manufacturer (usually either via their detailed camera documentation or read-only ​Micro-Manager ​properties). ​  Any extra time in the slice is placed before the camera trigger to allow maximum time for piezo settling. The values computed by the default timing mode are shown in the slice timing settings and can be subsequently modified manually.
  
 <WRAP center round tip 60%> <WRAP center round tip 60%>
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 Based on the angle specified on the settings tab "Path A stage/objective angle", the pixel size defined in Micro-Manager’s “Pixel Size Calibration”, and slice spacing then the plugin runs the deskew.  If empirically it is off, check the "Invert direction" checkbox and try again.  Deskew fudge factor is an additional scale factor on the deskew and should generally be 1. Based on the angle specified on the settings tab "Path A stage/objective angle", the pixel size defined in Micro-Manager’s “Pixel Size Calibration”, and slice spacing then the plugin runs the deskew.  If empirically it is off, check the "Invert direction" checkbox and try again.  Deskew fudge factor is an additional scale factor on the deskew and should generally be 1.
  
 +
 +===== Synthetic Stage =====
 +
 +Sometimes it is handy to create a synthetic stage to move the sample relative to the optic axis, e.g. to move deeper into the sample along the imaging axis by moving the SPIM head and the X axis of the XY stage together.  In the hardware config wizard add an instance of the Utilities > Multi Stage device which creates a synthetic 1D stage out of 2 real 1D stages.  Specify which stages to use and the scale factors in the properties in the System-Startup group in your hardware config.  The scale factors should be +/- 1 for 45 degrees, +/- 1 and +/- 2 for oSPIM at 60 degrees, and so forth.
 +
 +In order to use one axis of an XY stage as a 1D stage for the Multi Stage device you can use the Utilities > Single Axis Stage device.  Set its properties in the System-Startup group as well.
 +
 +Once set up, it is likely best to use the new Multi Stage device via the Stage Control plugin rather than assigning it as a stage in the Micro-Manager plugin.

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