1. Before you start

• You must already have had your super-user introduction. The first solo session is by appointment.
• Book the slot in Calira and stick to the booking rules.
• Prepare your sample on a #1 coverslip (0.17 mm). Single-molecule buffers (dSTORM, DNA-PAINT) need to be made fresh.
• Decide which path you will use: Abbelight SAFe-360 (widefield, HiLO, TIRF, SMLM), Nikon A1 (confocal), or both. Both can run on the same Ti2 body but only one camera path acquires at a time.
• Two PCs share the bench through a KVM switch. Light 1 = Abbelight PC, Light 2 = Nikon A1 PC. Press Select on the KVM to switch.
• Data routing: the Abbelight PC is on the TU/e Intranet, transfer via the network drive \\store04.tue.nl\mbx_exchange\common (do not plug a USB stick into this PC). The Nikon A1 PC is offline, transfer via a virus-checked external USB drive (D, E, F drives are local storage).
• The instrument is at Flux 4.102.

2. Power-up sequence

Combined sessions (Abbelight + Nikon A1). Turn on the Nikon A1 first, then the Abbelight. The A1 interlock cable must be connected to the back of the microscope body before either path will arm.

2a. Abbelight SAFe-360 path (blue stickers, KVM → “Abbelight”)

1. Confirm the laser switch on the electricity pillar is on. Turn the steel laser-interlock key on the Nexus unit to “on” and flip the black switch to “Laser on”.
2. Press the red button on the Optospin controller to power the filter wheel.
3. Turn on the camera using the small button on its side.
4. Turn on the stage.
5. Turn on the microscope body. Verify the Abbelight interlock cable is plugged into the Interlock port at the back. Without it, the lasers will not arm.

Safety: The interlock cable must be plugged into the Interlock port at the back of the microscope body. Without it the lasers will not switch on. Opening the chamber lid during acquisition automatically disables the lasers.

6. Press the silver button on the Nexus unit to boot the Abbelight PC.
7. For acquisition press the Camera L button on the microscope body. It should turn green, confirming the camera-to-PC link.
8. Verify the controller box is on. It should always be on.

Safety: Never turn off the controller box. It controls the microscope body, and power-cycling it can damage components and forces a long re-initialisation (vendor service call may be required).

2b. Nikon A1 path (orange stickers, KVM → “Nikon”)

1. Turn on the laser switch on the electricity pillar.
2. Confirm the A1 interlock cable is connected to the Interlock port at the back of the microscope.
3. Turn on the microscope body.
4. Turn on the confocal box. Press the rear button first, then the side button.
5. Verify the controller box is on (always on).
6. Switch on the laser combiner box LU4.
7. Turn the keys for the 638/647 nm and 405 nm lasers. The 638/647 line uses software-selectable filters.
8. For the 561 nm line, switch on the back button first, then turn the key.
9. For the 457/488/514 nm line, switch on the front button, turn the key, then engage the discharge pin on the Melles Griot controller. Filter selection is in software.
10. Switch on the stage.
11. Switch on the Nikon A1 PC, set the KVM to “Nikon”.
12. Press the Camera R button on the microscope body to enable acquisition. It should turn green.

2c. Sample loading

1. Place a small drop of immersion oil on the coverslip (#1, 0.17 mm).
2. Insert the sample into the chamber, oil-side down. Ensure the oil contacts the objective.
3. Engage the Perfect Focus System (PFS button on the body).
4. Replace the chamber lid carefully (mind the cables). The lid blocks ceiling-light contamination, removing it auto-disables the lasers.

2d. Live-cell imaging (optional, Okolab Top-stage Incubator)

1. Open the CO₂ valve (counter-clockwise).
2. Switch on the Okolab unit (rear button).
3. Power the controller (round button next to the screen).
4. Connect the gas tube to the metal inlet at the top-left of the sample chamber, lid still on.
5. Set temperature on the touchscreen. Adjust air and CO₂ flow on the Okolab unit. Refer to pages 18 to 19 of the Okolab manual (on the MBx common drive, 03 Labs / 02 Equipment Flux / Microscopy / OKO gas mixer) for gas-percentage targets.

3. NIS-Elements basics (Nikon A1 PC)

Software: NIS-Elements AR 5.21.03, shortcut in the centre of the desktop. Press Ctrl + Alt + Delete, then Enter (no password as of April 2026). Open NIS-Elements. During initialisation all devices connect, you should hear the system calibrating.

Strategy. Find and focus first. If your sample bleaches quickly, configure all acquisition settings (steps 2 to 4 below) before locating the region of interest, then acquire immediately. Use a lower resolution and faster scanner speed during ROI search than during the final acquisition.

Step 1, find the sample (brightfield, lasers off, lid open)

1. Confirm all lasers are off. Open the chamber lid and locate the sample through the eyepiece using the side knob on the body.
2. Once focused, press PFS to lock focus while you navigate with the joystick.
3. Close the lid, switch from ocular view to acquisition mode by pressing Camera L (turns green, PC and camera linked).

Tip: to swap sample or well, press ESC, swap the slide, disable ESC, then re-engage PFS to regain focus.

Step 2, configure acquisition channels

4. Choose acquisition mode (simultaneous or sequential channels).
5. In the A1 Plus Compact GUI panel, click to open the Dye and Spectral Setting window.
6. Pick auto or manual. For each channel, set the dye and emission/excitation wavelength, choose the detector (default Detector DU4), select the lasers, and verify each spectral window matches the expected emission without overlap between channels.

Step 3, set scanning parameters

All scanning parameters live in the A1 Plus Compact GUI. Key knobs:

• Scanner Speed, frames per second. Pixel dwell time follows.
• Size, image size in pixels. With high-resolution lenses, use 1024×1024.
• Pinhole, trade-off between optical sectioning and signal strength.
• HV (Gain), photomultiplier voltage, raise to compensate for low signal.
• Offset, adjust to suppress background.

LU4A Pad checks before scanning:

• Beam switcher set to Confocal (right button under “Beam Switcher”).
• All laser shutters open (yellow circles in the interface).
• Remove the interlock when ready to scan. Red interlock indicator = active = lid-open safety.

Scanning procedure

7. Start with low laser power and moderate HV.
8. Click Scan for live preview. Check there are no saturated pixels and that the dynamic range is well used.
9. If signal is weak, raise laser power incrementally, watch for photobleaching.

Step 4, optimise Z-stack for 3D imaging

10. Scroll Z to find where signal begins and ends.
11. Click Top and Bottom in the Z-stack panel. Add a 1 to 2 µm margin on each side.
12. Set step size. The software suggests an optimum (and the matching number of steps), tweak as needed. Press Run Now to acquire.

Save

Review parameters before final acquisition. Save in .nd2 format, it preserves all acquisition metadata.

4. SAFe Insight / NEO acquisition (Abbelight PC)

The Abbelight PC runs two applications:

• NEO_LiveImaging for acquisition.
• NEO_Analysis for offline reconstruction and analysis.

Manuals are in the “user manual” folder on the Abbelight desktop. Workflow summary:

1. KVM to “Abbelight”. Press Camera L on the body (green), the camera is now bound to the Abbelight PC.
2. Open NEO_LiveImaging. Pick the illumination mode (epi, HiLO, or TIRF) and set the laser angle. The angle is continuously tunable between modes.
3. Select your laser line(s): 488 nm, 532 nm, or 637 nm. Lines can be used simultaneously or individually.
4. Pick the filter cube via the Optospin filter wheel (software control). The wheel changes filters quickly between sequential acquisitions.
5. Set exposure, frame count, and laser power for SMLM (STORM, PALM, DNA-PAINT). Typical SMLM runs are tens of thousands of frames.
6. Acquire to disk. Run reconstruction in NEO_Analysis after the run finishes.

Detailed NEO menu walk-throughs are in the vendor manuals. If you are running a method for the first time, schedule a session with your super-user.

5. Drift correction

Drift correction is technique-dependent and is not covered by SOP v2.1. Ask your super-user or imaging scientist for the recommended NEO settings for your acquisition. As a general rule, long SMLM acquisitions (dSTORM, DNA-PAINT) need either fiducial markers (e.g. 100 nm gold beads or photostable bright beads embedded in the sample) or cross-correlation drift correction in NEO_Analysis. Plan this before you mount the sample.

6. Shutdown

6a. Remove the sample

1. Clean hands. Sand or grit can scratch the objective.
2. Press ESC on the body to lower the objective.
3. Remove the sample. Wipe oil off the coverslip with a lens tissue.
4. Wipe oil off the objective with a fresh lens tissue.
5. Replace the chamber lid.

6b. Abbelight path

1. Turn off the laser box (key, then button).
2. Turn off the Optospin.
3. Turn off the camera.
4. Turn off the stage.
5. Turn off the microscope body.
6. Shut down the Abbelight PC (silver button on the Nexus).

6c. Nikon A1 path

Safety: When shutting down the Nikon A1 path, always turn off the 457/488/514 nm laser first. Switch the discharge pin on the Melles Griot controller off, turn the key, then wait roughly 2 minutes for the cooling fan to stop on its own before pressing the main button. Skipping the cool-down can damage the laser.

1. Switch off the 638/647 nm, 405 nm, and 561 nm laser keys.
2. Switch off the 561 nm button.
3. Switch off the laser box.
4. Switch off the confocal box (side button first, then rear).
5. Switch off the stage.
6. Switch off the microscope body.
7. For the 457/488/514 nm laser, wait until the cooling fan has stopped (about 2 minutes) before pressing the main button to turn it off.

6d. Okolab Top-stage Incubator (live-cell sessions)

1. Long-press the round button on the controller to switch it off (a short press only blanks the screen).
2. Switch off the Okolab unit (rear button).
3. Disconnect the gas tube from the chamber.
4. Close the CO₂ valve (clockwise).

Last step. After everything else is off, switch off the laser button on the electricity pillar.

7. Common errors

Symptom	Likely cause
Saturation, white pixels	Gain or laser power too high, dynamic range clipped in bright regions.
Noisy, grainy image	HV (gain) too high relative to signal.
Sample fades over time	Photobleaching from excessive laser power or repeated scans.
Loss of optical sectioning	Pinhole opened too far.
Image too dark	Pinhole too closed for a weak signal, not enough light reaching the detector.
Poor resolution despite 100× objective	Image format too small (e.g. 512×512), undersampling. Use 1024×1024.
Lasers refuse to arm	Interlock cable not seated, or chamber lid is open.