ND Acquisition

The ND Acquisition window within NIS-Elements allows users to configure N-Dimensional imaging.

There are five tabs: Time, XY (Multipoint), Large Image (for tiling/stitching), \(\lambda\) (for multiple channels/OCs), and Z.

When multiple dimensions are required, NIS-Elements will default to the fastest order of experiment, but the "Order of Experiment" can be changed.

Options for "Order of Experiment" with the fastest selected by default. Here, a z-stack will be acquired before switching to different channels and finally a large image will be acquired.

Time

The Time tab allows for (complex) timelapse imaging.

Tip

• Close Active Shutter when idle and Switch Transmitted Illuminator off when Idle (0.01 s) should nearly always be enabled.
• The 1 time loop button is very handy for checking multipoints.

In the figure below, note the two phases: the first is an intentional delay, and the second leads to acquisitions every five seconds for two minutes (25 timepoints/"loops" total).

Time tab in ND Acquisition with two phases.

XY (Multipoint)

The XY tab allows for the sequential imaging of multiple points. This can be a great way to increase throughput during timelapse acquisitions. Each location can have its own Z and PFS values.

Warning

• There is increased light dosage, so the risk of phototoxicity is higher.¹
• If using immersion objectives, the likelihood of stage jitter increases.²

Tip

• If dealing with an uneven stage (as evidenced by multiple points having PFS values off by 1000s), enabling Include Z should help.
• Nikon also recommended giving PFS additional time for each point via Wait(0.200000);
• NIS-Elements can Optimize the order of the points for you to reduce the amount of stage travel
• Split Multipoints is highly recommended to avoid dealing with one very large .nd2 file.

XY tab in ND Acquisition with three different points and configured for an uneven stage.

Large Image (tiling/stitching)

The Large Image tab allows for tiling/stitching when what you want to capture requires a larger FOV. Note: there are (better) alternatives that allow for more interactive and flexible ROIs.

Channels

The \(\lambda\) or Channels tab allows for multiple OCs to be used in an experiment. An example of a four-color experiment would include imaging DAPI, AF488, AF555, and AF647. Regular brightfield and DIC are also options.

Note

• Some objectives have a focus (axial) offset between colors. NIS-Elements allows you to correct for this by setting a Focus Offset within the \(\lambda\) tab.

Animation of setting up channels in a preferred order and setting Focus Offsets

Z

The Z tab allows for 3-D acquisition/"z-stacks".

Tip

• The speed of a z-piezo is much greater than the microscope body's focus drive.
• This scope is equipped with a z-piezo with a travel range of 450 \(\mu\)m

Using absolute limits

Z-stack limits using absolute limits

Using symmetric mode

You may be forced away from using absolute limits because you're using focus offsets or PFS.

Z-stack limits using symmetric mode defined by range (when previously defined using absolute limits)

Using asymmetric mode

Asymmetric mode can be helpful if you know you want a set thickness and can set a limit easily. For example, you only want the first X\(\mu\)m from the coverslip or the thickness of tissue sections are known.

Z-stack limits with asymmetric mode defined by range

Z device

• I do not recommend using the Focus Drive as the Z Device because it will be much slower than using the piezo.
• NIDAQ Piezo Z should be slightly more accurate than Triggered NIDAQ Piezo Z at the slight expense of speed.

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1. Even if your multipoints are far from each other, remember that your media can become phototoxic ↩

2. Oil has inertia. ↩