Hardware reference
Mach3Manual: Editing Hardware Reference
The dOPM system described in this manual is based on an Eclipse Ti2-E which is the motorised version of the Eclipse Ti2-E series.
In particular each system comes with a hardcopy of the Nikon Eclipse Ti2-E manual. Also check the online resources:
https://www.microscope.healthcare.nikon.com/products/inverted-microscopes/eclipse-ti2-series https://www.microscope.healthcare.nikon.com/resources
| Photo of system at IRB in 2022 | Photo of dOPM optics at IRB in 2022 |
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| Click Device Manager in Device Tab | Device Manager |
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Diagram from NIS-Elements showing how the hardware components are connected.
Comment TS: Could you maybe include how to get to this panel in the software?
Each dOPM system should be using the same laser lines and emission filters and dichroics. Bear in mind that the Ti2 frame may have some extra dichroics in the upper and lower turrets.
To confirm which filters are in use, short of physically checking the labels on the filters, you can check the configuration of filter wheels in NIS-elements. When the system was first installed the filters should have been manually entered into the hardware configuration definition within NIS-elements.
From the screenshot you can see an upper turret, lower turret. Also, 'Filter A' which refers to the filters that are in the Sutter filter wheel use in the dOPM detection path.
Comment TS: a brief explanation about the function of the lower and upper filter would be nice at this section (or a hyperlink to the respective page)
For the Nikon frame there is a lower and upper filter cube turret. For future upgrades there is a mirror in the upper turret. For the lower turret one of the following filter cubes is removed (probably the DAPI - check system setup) to allow clear path to dOPM optics.
Upper and lower turrets for filter wheel - we only use the lower one in the current implementation. For the upper turret there should be no filters or mirrors in the current implementation.
Called the 'Stage up kit' the version of the Nikon Eclipse Ti2-E frame we have has this which allows for an additional filter cube wheel to combine more light-paths. For our purposes this is for future upgrades and not currently being used.
https://downloads.microscope.healthcare.nikon.com/phase4/literature/Brochures/Ti2_2CE-MPOH-1.pdf
Comment TS: is it possible to make a table here instead to provide an easier overview?
| Lower Filter Cube Turret | |
|---|---|
| Cube name | Description |
| MXR00303 DAPI-5060C Filter Cube | Excitation: EX 377/50 Dicroic Mirror DM 409 Barrier Filter: BA 447/60 |
| MXR00304 CFP-2432C Filter Cube | Excitation: EX 377/50 Dicroic Mirror DM 409 Barrier Filter: BA 447/60 |
| MXR00306 GFP-1828A Filter Cube | Excitation: EX 377/50 Dicroic Mirror DM 409 Barrier Filter: BA 447/60 |
| MXR00310 YFP-2427B Filter Cube | Excitation: EX 377/50 Dicroic Mirror DM 409 Barrier Filter: BA 447/60 |
| MXR00313 TXRED-4040C Filter Cube | Excitation: EX 377/50 Dicroic Mirror DM 409 Barrier Filter: BA 447/60 |
| MXR00315 Cy5-4040C Filter Cube | Excitation: EX 377/50 Dicroic Mirror DM 409 Barrier Filter: BA 447/60 |
| Upper Filter Cube Turret | |
| 1CRP8610 | P861/100/254, High quality 100% mirror for switching optical pathways (25x40mm) |
As of 2023/02/26 here is a snapshot of filters that should be in each systems Sutter filter wheel.
Note that each system might have extra or different filters and these can be accessed via the nis elements software as shown in the screenshot above.
| Imperial | |
|---|---|
| Filter Matched Ex (nm) | Em (nm) |
| 445 | FF02-483/32 |
| 488 | FF03-525/50 |
| 515 | FF01-542/20 |
| 561 | FF01-600/52 |
| 642 | FF01-698/70 |
| Edinburgh | |
| Filter Matched Ex (nm) | Em (nm) |
| 445 | FF01-483/32 |
| 445 | FF01-482/18 |
| 488 | FF01-525/45 |
| 515 | FF01-542/20 |
| 561 | FF01-609/54 |
| 642 | FF01-697/58 |
| Crick | |
| Filter Matched Ex (nm) | Em (nm) |
| 445 | FF01-483/32 |
| 445 | FF01-482/18 |
| 488 | FF01-525/45 |
| 515 | FF01-542/20 |
| 561 | FF01-609/54 |
| 642 | FF01-697/58 |
| 642 | FF01-698/70 |
| IRB | |
| Filter Matched Ex (nm) | Em (nm) |
| 445 | FF01-483/32 |
| 445 | FF01-482/18 |
| 488 | FF01-525/45 |
| 515 | FF01-542/20 |
| 561 | FF01-609/54 |
| 642 | FF01-697/58 |
| ICR | |
| Filter Matched Ex (nm) | Em (nm) |
| 445 | FF01-483/32 |
| 445 | FF01-482/18 |
| 488 | FF01-525/45 |
| 515 | FF01-542/20 |
| 561 | FF01-609/54 |
| 642 | FF01-697/58 |
- cameras, turn on dOPM from power brick outside dOPM enclosure
- dOPM camera water cooling, check reservoir
- lasers, turn on at Omicron software, check actually on before assuming they are warming up
- water immersion pump, check reservoir
- CO2, regulator at wall/cylinder, regulator at input to okolabs mixer
- air pump for 5% CO2
- microscope frame
- CoolLED epifluorescence box
- okolab enclosure environment control
- sutter filter wheel
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normally always left on:
- remote refocus objective heater
- picomotors for controlling light-sheet mirrors
- pimag linear actuator for scanning dOPM remote refocus mirrors
- microscope control box
- XY stage
| Step | Hardware | Photo | Notes |
|---|---|---|---|
| 1 | Microscope control box |  |
This should be left on even when not using the microscope - see Nikon manual/consult Nikon |
| 2 | Microscope |  |
This is where we power the microscope on and off - see Nikon manual/consult Nikon |
| 3 | Microscope enclosure temp control |  |
This controls the Okolab enclosure temperature - see Okolab environment control manuals. Normally we’d run this at 37 degrees for live imaging |
| 4 | Microscope enclosure humidity control |  |
This is the humidifier for the mixed gas feed, typically 5% CO2 - see Okolab environment control manuals. Normally we’d check this at the start of the experiment to make sure it is topped up. |
| 5 | USB peripherals |  |
These UBS expand the number of ports available on the acquisition computer in order to communicate with all of the peripherals |
| 6 | dOPM lasers |  |
There are 5 laser control boxes for the dOPM system. They need to be enabled before turning on the lasers in the Omicron control software on the computer. See laser manuals for details of control boxes. |
| 7 | sCMOS Hamamatsu Fusion cameras |  |
There are 5 laser control boxes for the dOPM system. They need to be enabled before turning on the lasers in the Omicron control software on the computer. See laser manuals for details of control boxes. |
| 8 | dOPM sCMOS Hamamatsu Fusion camera water cooling |  |
There are 5 laser control boxes for the dOPM system. They need to be enabled before turning on the lasers in the Omicron control software on the computer. See laser manuals for details of control boxes. |
| 9 | dOPM remote refocus scanning mirror actuator control box |  |
Power/control box for the PImag. This is normally always left on. See manual for details. In most systems this is inside the dOPM enclosure. |
| 10 | dOPM remote refocus scanning mirror actuator |  |
Power/control box for the PImag. This is normally always left on. See manual for details. In most systems this is inside the dOPM enclosure. |
| 11 | dOPM filter wheel |  |
Filter wheel for the dOPM emission path. This can be switched off/on for each experiment. |
| 12 | XY Stage |  |
Control box for XY stage should be left on. If turned off, sample holders need to be recalibrated in nis-elements. |
| 12 | Peristaltic pump for water immersion |  |
dOPM system works with the 60x water immersion objective only. Water immersion system consists of a 50 ml syringe as a reservoir for ultra pure type one water - the purest form of water to avoid damaging the microscope objective. The peristaltic pump draws water from the syringe. See manual for details of operation. This should be turned on/off for each experiment. Warning - risk of flooding the microscope objective turret if not monitored. |
| 13 | Peristaltic pump for water immersion |  |
dOPM system works with the 60x water immersion objective only. Water immersion system consists of a 50 ml syringe as a reservoir for ultra pure type one water - the purest form of water to avoid damaging the microscope objective. The peristaltic pump draws water from the syringe. See manual for details of operation. This should be turned on/off for each experiment. Warning - risk of flooding the microscope objective turret if not monitored. |
| 13 | LED box for epi-illumination |  |
LED light source box for wide field epi-illumination imaging. This should be turned on/off for each experiment. |
| 14 | Objective O2 heater |  |
Heater for objective #2 in dOPM enclosure - see dOPM optics diagram for context. This heater needs to be on at least an hour before alignment and imaging to ensure that it has stabilised. In general, always leaving this turned on. The top image is the type of controller for the heater used at ICR, IGC, Imperial and Crick. The bottom image is for the type used at IRB which looks the same as the Okolabs enclosure control box above. |
| 15 | Objective O2 heater @ IRB |  |
Heater for objective #2 in dOPM enclosure - see dOPM optics diagram for context. This heater needs to be on at least an hour before alignment and imaging to ensure that it has stabilised. In general, always leaving this turned on. The top image is the type of controller for the heater used at ICR, IGC, Imperial and Crick. The bottom image is for the type used at IRB which looks the same as the Okolabs enclosure control box above. |
| 16 | air pump for CO2 mixer |  |
Top image shows the air pump used for mixing the gas. Next image shows the gas regulator positioned before the analog gas mixer. Next image shows the analog gas mixer. Imperial and IGC use premixed 5% C02 gas and direct it into one of the inlets of the gas mixer. The air pump is not used in this case. At ICR, IRB, Crick and the room is supplied with pure CO2 and needs to be mixed with air. The air pump is used in this case. See oko labs gas mixer manual for details. |
| 17 | CO2 pressure regulator |  |
is not used in this case. At ICR, IRB, Crick and the room is supplied with pure CO2 and needs to be mixed with air. The air pump is used in this case. See oko labs gas mixer manual for details. |
| 18 | CO2/Air mixer front |  |
Next image shows how the mixer has the CO2 inlet blocked for only using the air inlet with premixed 5%CO2 gas - as used at Imperial and IGC. The block is made with a rubber bung with a whole for a pipe in it connected to the mixer and the other end connected to a 15 ml Falcon tube. There are many ways to seal this inlet. |
| 19 | CO2/Air mixer behind |  |
Next image shows how the mixer has the CO2 inlet blocked for only using the air inlet with premixed 5%CO2 gas - as used at Imperial and IGC. The block is made with a rubber bung with a whole for a pipe in it connected to the mixer and the other end connected to a 15 ml Falcon tube. There are many ways to seal this inlet. |
| 20 | USB temp monitor |  |
This can be used for monitoring the temperature inside the dOPM optics enclosure in case optical alignment seems to be instable |