Nikon A1 — NIS-Elements MVP

1. Introduction

The Nikon A1 is Nikon’s older generation of confocal laser scanning microscope, retained at the AMF as the everyday confocal alongside the Abbelight super-resolution platform on the same Ti2 body. Excitation is handled by the LU4 laser combiner, fiber-coupled into the scan head; up to four fluorophores can be acquired in the same sample.

Lasers (LU4 combiner): 405, 488, 561, 640 nm primary excitation channels; the LU4A Pad in NIS-Elements drives the discrete laser lines. Objectives (shared with the Abbelight platform): 60× TIRF and 100× TIRF. The system has an Okolab Top-stage Incubator for live-cell imaging (temperature, humidity, CO₂); see the companion SOP for incubator setup.

2. Practice guide: step by step

After completing the power-up sequence (see the Abbelight SOP, section 2b for the Nikon A1 path), unlock the screen with Ctrl+Alt+Delete then Enter (no password). Open NIS-Elements AR 5.21.03 from the desktop. The system calibrates audibly during initialisation.

Step 1. Find the sample and focus (brightfield)

1. Ensure all lasers are off. Open the chamber lid and locate the sample through the eyepiece using the side knob.
2. Once focused, press PFS (Perfect Focus System) to lock focus while you navigate with the joystick.
3. Close the chamber lid. Switch from ocular view to data acquisition by pressing the Camera L button on the microscope body. It should turn green, confirming the PC-camera link.

Step 2. Configure acquisition channels

1. Set the acquisition mode (simultaneous or sequential channel acquisition).
2. In the A1 Plus Compact GUI panel, click the colour-pad button to open the Dye and Spectral Setting window.
3. For each channel, choose the dye and emission/excitation wavelength, select the detector type (standard: DU4), and verify each spectral window matches the expected emission spectrum without overlap.

Step 3. Set scanning parameters

LU4A Pad configuration

1. The beam switcher is set to Confocal (right button under “Beam Switcher”).
2. All laser-related shutters are open (yellow circles in the interface).
3. Remove the interlock when ready to scan. When the interlock indicator is red, the safety system is active and lasers are disabled when the chamber lid is open.

Scanning

1. Start with low laser power (avoid photobleaching) and moderate HV.
2. Click Scan to begin live scanning. Look for a well-utilised dynamic range: visible signal, no saturated pixels.
3. If signal is weak, increase laser power incrementally; monitor for photobleaching.

Step 4. Optimise Z-stack for 3D imaging

1. Scroll along Z manually to identify where the signal begins and ends.
2. Click Top and Bottom in the Z-stack panel to set the range. Add ±1–2 µm margin.
3. Set the step size. NIS suggests an optimal value; you can override. Press Run Now to start 3D acquisition.

Final acquisition and saving

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

3. Common errors checklist