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docs:mm_dispim_plugin_user_guide [2019/10/26 00:07]
Jon Daniels [Data Analysis Tab] |
docs:mm_dispim_plugin_user_guide [2021/04/14 20:28]
Jon Daniels [Channels] |
| - Click the green-bordered **Use these!** button to compute the slope and offset of the calibration relationship. | - Click the green-bordered **Use these!** button to compute the slope and offset of the calibration relationship. |
| - Check the computed calibration using the up and down arrows in the upper right of the Setup tab (you may need to increase the step size). | - Check the computed calibration using the up and down arrows in the upper right of the Setup tab (you may need to increase the step size). |
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| | For stage-scanning, the value of the calibration slope only matters if you want the value of the offset as measured in microns to match the physical world. Normally you can just leave it at its default value. |
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| Generally the calibration slope will remain relatively constant but the offset can change slightly. It is easy to update the offset without changing the slope (e.g. when introducing a new sample): | Generally the calibration slope will remain relatively constant but the offset can change slightly. It is easy to update the offset without changing the slope (e.g. when introducing a new sample): |
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| The channels feature is patterned after that of Micro-Manager’s [[https://micro-manager.org/wiki/Micro-Manager_User%27s_Guide#Multi-dimensional_acquisition|Multi-Dimensional Acquisition]]). It is most commonly used to take volumes with different illumination wavelengths (e.g. 488 nm and 561 nm for samples labeled with GFP and mCherry). Before using this feature, you must create and configure a group of presets containing the settings for that channel. | The channels feature is patterned after that of Micro-Manager’s [[https://micro-manager.org/wiki/Micro-Manager_User%27s_Guide#Multi-dimensional_acquisition|Multi-Dimensional Acquisition]]). It is most commonly used to take volumes with different illumination wavelengths (e.g. 488 nm and 561 nm for samples labeled with GFP and mCherry). Before using this feature, you must create and configure a group of presets containing the settings for that channel. |
| Simple per-volume channel switching is implemented unless a Programmable Logic Card (PLC) is present in the controller; this mode is generic and can be used with any Micro-Manager group. PLC-based channel switching is hardware-based, requiring extra setup time but then no delay during acquisition. Using the PLC, channel switching is possible on both a per-volume and per-slice basis. In this case the channel group must contain the property “Output Channel” which is used to select the correct laser to trigger; other properties in the channel group, if any, are ignored. | |
| | Simple per-volume channel switching is generic and can be used with any Micro-Manager configuration group. Control of the acquisitions reverts to Micro-Manager between volumes so that Micro-Manager can e.g. move filter wheels to the specified positions, and then the ASI controller is re-triggered by Micro-Manager to collect the next volume. The plugin requires a certain amount of time between finishing one volume and beginning the next so that the extra communication and control change can happen. |
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| | Alternatively there are two hardware-based channel switching modes -- "Every volume (PLogic)" and "Every slice (PLogic)" -- which which leverage the PLC so that TTL triggering which happens within each volume continues across all volumes and control of the acquisition never returns to the host PC. These hardware-based channel switching modes require a bit of extra setup time but no delay during acquisition. The main limitation of the hardware-based channel modes is that they will only change the channel via the PLC property "Output Channel" which is used to select the correct laser to trigger (other properties in the channel group, if any, are ignored). Mode "Every slice (PLogic)" interleaves the channels within the volume whereas "Every volume (PLogic)" changes channels between volumes. |
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| ==== Volume Settings ==== | ==== Volume Settings ==== |
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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> |
| ==== Default Timing ==== | ==== Default Timing ==== |
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| 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. |
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| <WRAP center round tip 60%> | <WRAP center round tip 60%> |
