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docs:manual:alignment [2015/06/26 00:09]
Jon Daniels created |
docs:manual:alignment [2020/08/19 02:50] (current)
Jon Daniels marked deprecated and linked to recent version |
| ====== diSPIM Alignment ====== | ====== diSPIM Alignment (deprecated) ====== |
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| | <note important>This is the original version of the manual and users should refer to [[docs:manual#optical_alignment|docs:manual#optical_alignment]] instead</note> |
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| ===== Overview ===== | ===== Overview ===== |
| ===== Beam position and angle: what affects what? ===== | ===== Beam position and angle: what affects what? ===== |
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| We are concerned with getting both the beam position and angle correct in the sample. Fortunately, the adjustments to do so are mostly “orthogonal” or independent of each other. The following table can be derived by understanding the lens system from the fiber input of the scanner to the sample and then through the imaging path to the corresponding camera on the opposite side of the microscope. <tabref what_affects_what> shows how various “knobs” affect the beam position and angle. | We are concerned with getting both the beam position and angle correct in the sample, plus making sure the objectives are looking at the same place. Fortunately, the adjustments to do so are mostly “orthogonal” or independent of each other. The following table can be derived by understanding the lens system from the fiber input of the scanner to the sample and then through the imaging path to the corresponding camera on the opposite side of the microscope. <tabref what_affects_what> shows how various “knobs” affect the beam position and angle. |
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| <tabcaption what_affects_what| How alignment adjustments affect the beam position and angle.> | <tabcaption what_affects_what| How alignment adjustments affect the beam position and angle.> |
| ^ Adjustment ^ Position? ^ Angle? ^ Comments ^ | ^ Adjustment ^ Position? ^ Angle? ^ Comments ^ |
| | Tilt of dichroic mirror | Some | Yes | Position effect varies ((The dichroic tilt affects the angle at | | Tilt of dichroic mirror (Cube-III) | Some | Yes | Position effect varies but usually small ((The dichroic tilt affects the angle at |
| which the beam enters the back focal plane as well as the beam position at the back focal plane. It is possible to | which the beam enters the back focal plane as well as the beam position at the back focal plane. It is possible to |
| compensate by adjusting the scan center but we usually adjust the camera mirrors to compensate instead.)) | | compensate by adjusting the scan center but we usually adjust the camera mirrors to compensate instead.)) | |
| | Tilt of camera mirror | Yes | No | Set during scanner alignment and not after | | | Tilt of camera mirror (Cube-III) | Yes | No | | |
| | Position of upper Z stage | No | No | Angle varies if scanners are not connected to dichroic tubes | | | Z position of SPIM head relative to sample | No | No | | |
| | Tilt of adjustable mirror | Some | Yes | Set during scanner alignment and not after | | | Tilt of adjustable mirror (inside scanner) | Some | Yes | Set during scanner alignment and not after, not user-adjustable | |
| | Tilt of main micro-mirror | Yes| No | Set during scanner alignment and not after | | | Tilt of main micro-mirror (inside scanner) | Yes| No | Set during scanner alignment and not after (not user-adjustable) | |
| </tabcaption> | </tabcaption> |
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| To use a scanner slice position offset, the piezo-scanner cross-calibration explained in Section [sub:Cross-calibrate-piezo] will make the offset appear during imaging, but you should compensate for the offset by moving the imaging mirrors (Section [sub:Adjust-imaging-mirrors]) so that in the middle of the range the epi-spot is centered in the epi camera. Do this by setting the piezo to 0, moving the slice position until the beam is in focus, then adjusting the imaging mirror. Repeat for the other side. This should make it so that the epi spot is overlaid with the beam focus. | To use a scanner slice position offset, the piezo-scanner cross-calibration explained in Section [sub:Cross-calibrate-piezo] will make the offset appear during imaging, but you should compensate for the offset by moving the imaging mirrors (Section [sub:Adjust-imaging-mirrors]) so that in the middle of the range the epi-spot is centered in the epi camera. Do this by setting the piezo to 0, moving the slice position until the beam is in focus, then adjusting the imaging mirror. Repeat for the other side. This should make it so that the epi spot is overlaid with the beam focus. |
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| | ==== Check scanner tilt ==== |
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| | Generate a sheet beam using the Micro-Manger plugin Sheet check box (Navigation or Setup tabs). Adjust the width of the sheet to fill most of the camera field of view. In a dye solution both edges of the sheet should be well focused near the beam waist. Alternatively, you can move the stationary beam from one side of the sheet to the other using the Navigation panel of the plugin. If the focus is not uniform, then the entire scanner needs to be twisted slightly around its optical axis (loosening set screws and then re-tightening afterwards) so that both edges can be equally well-focused. See Figure [fig:Tilted-light-sheet]. Check both scanners. If you have to twist one of them, return to the start of the fine alignment. |
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| | <imgcaption SheetTilt|Tilted light sheet.>{{:docs:manual/SheetTilt.jpg?300|Tilted light sheet.}}</imgcaption> |
| | <imgcaption SheetNice|Corrected by twisting the scanner.>{{:docs:manual/SheetNice.jpg?300|Corrected by twisting the scanner.}}</imgcaption> |
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| | ==== Check camera tilt ==== |
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| | Check that the line generated in the epi view while scanning the beam to create a sheet is exactly vertical in the camera image. Because we have verified in the previous step that the scanner is tilted correctly, any remaining tilt must be in the camera. If the epi line is not vertical, adjust the camera until it is and then return to the start of the fine alignment. The easiest way to twist the camera is to loosen the set screw in ABTS-1013 which holds the SPIM cameras’ tube lens onto the SPIM assembly. Check both scanners. |
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| | ==== Establish coverslip location ==== |
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| | Slowly lower the objective pair into the dye solution until the coverslip becomes apparent where the fluorescent beam stops. See Figure [fi:CameraBeams]. The objectives will touch the bottom of the chamber very near the point where the two beams meet in the center of the camera image. Set the Upper SPIM Z-drive coordinate value to zero somewhere near or slightly above this point. In the future, when changing samples you can use the Navigation tab to move the microscope 25 or 30 millimeters and then quickly get back to the coverslip by pressing the “Go To 0” button. |
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| | <imgcaption ConvergingSheets| Converging light sheets in dye striking the coverslip. Note the epi-beam line of light corresponding to each sheet. Here the objectives are very close to the bottom of the chamber. The light sheets are are barely crossing each other before they disappear when they encounter the coverslip. With the light sheets focuses across the width of the sheet, the epi-beam should be appear parallel to the edge of the camera field.>{{:docs:manual/ConvergingSheets.jpg?300| Converging light sheets in dye striking the coverslip. Note the epi-beam line of light corresponding to each sheet. Here the objectives are very close to the bottom of the chamber. The light sheets are are barely crossing each other before they disappear when they encounter the coverslip. With the light sheets focuses across the width of the sheet, the epi-beam should be appear parallel to the edge of the camera field.}}</imgcaption> |
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| | ==== Align bottom objective with SPIM objectives ==== |
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| | Locate the image of the laser spots/sheet where it intersects the coverslip in the bottom camera. Bring the laser spots to the center of the camera image by adjusting either the CDZ-1000 XY translator that holds the entire SPIM microscope, or use the bottom-side objective adjuster to center the image (RAMM systems). For larger adjustments, you can add shims between the SPIM mount and the CDZ replacement block for front to back adjustments and shift the dovetail on the LS-50 mount for side to side adjustments. Focus the lower objective and set the coordinate for the coverslip as zero in the Micro-Manger Navigation window. When using the bottom objective for sample spotting, it is useful to know the relative positions of the light sheets in XY and Z compared to the bottom camera image. Setting appropriate Z-references helps in this regard. |
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| | <imgcaption BottomCamera| Bottom camera aligned to the centered light sheets shown in Figure [fig:Converging-light-sheets].>{{:docs:manual/BottomCamera.jpg?300| Bottom camera aligned to the centered light sheets shown in Figure [fig:Converging-light-sheets].}}</imgcaption> |
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| | ==== Repeat steps [sub:Co-align-the-objectives] to [sub:Check-collimator-focus] at least once ==== |
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| | ==== Cross calibrate piezo and scanner movement[sub:Cross-calibrate-piezo] ==== |
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| | This process can be accomplished with a either the focused beam in dye, or with a sheet beam using a field of fluorescently labeled objects such as beads on a coverslip or dispersed fluorescent objects in the users sample. We will describe the process using dye as a natural progression from the previous steps. However, when imaging real samples, you may wish to verify this calibration step fairly often (and redo as needed), in which case you can use fluorescent objects in the sample preparation as target objects rather than the focused beam waist described here. |
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| | Use the diSPIM control Setup Path A tab. Establish a focused beam in the dye solution. Set the manual controls so that the left knob control the Imaging Piezo and the right Knob controls the Sheet Beam, Slice Position. Follow the instructions in Section [sub:Setting-piezo-vs-slice-calibration] to calibrate the scanner movement in the slice axis with the piezo movement. |
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