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Lightpaths and OCs

Optical Configurations#

Optical Configurations (OCs) are grouped microscope settings. Through OCs, you can tell Elements to "open this shutter; use these filters, set phasers to stun, etc."

Eyepiece-EPI-OCs

Left: Lightpath window. Right: Acquisition window. Animation of switching between OCs to show changing filters and Sola power. The upper turret shutter can also be controlled.

When you acquire images, existing OCs can be modified. The most common modifications to OCs are:

  • Camera exposure time
  • Camera Bit Depth
  • Laser power

Lightpaths in NIS-Elements#

Lightpaths in NIS-Elements are a way to group microscope devices/settings and simplify the Acquisition window by hiding irrelevant components and OCs. If your goal is to look through the eyepieces, there's no need to control the camera or confocal lasers.

Eyepieces#

When looking through the eyepieces, you'll be on the Eyepieces - DIA or Eyepieces - EPI lightpath and choose an OC.

  • DIA refers to "diascopic" which corresponds here to transmitted light / brightfield
  • EPI refers to "episcopic" which corresponds here to reflected light / epi-fluorescence

Eyepieces - EPI (FITC)

Eyepieces - DIA (DIC)

Note

Note how the cameras and spinning disk confocal components are greyed out and inactive in the lightpaths for Eyepieces.

Eyepiece - DIA and Eyepiece - EPI lightpaths are common to both single camera and dual camera modes.

Single camera#

Because one priority for this scope was dual-camera imaging, the emission filters for single-camera imaging are located in a fast filter wheel right before the "Front Fusion" camera. It contains four single emission filters and has an empty position for brightfield.

SC-405

SC-488

SC-561

SC-640

Note

Both brightfield and fluorescence imaging will be "confocal"1 because the spinning disk can't be moved out of the light path. Light throughput for brightfield is drastically reduced, and the quality of DIC is also impaired.

Dual camera#

A long pass filter2, which will reflect light below 570nm towards the "Back Fusion BT", is inserted into the TwinCam unit. The back filter wheel has DAPI and FITC emission filters, but the main use-case is simultaneous imaging of green and red fluorophores.

Concerns when using dual camera mode:

  1. Spectral bleed-through
  2. Registration between cameras
  3. Slight (5%) loss in light due to imperfect transmission of the TwinCam DM 2.

SC-405

SC-488

SC-561

SC-640

  1. The spinning disk pattern is optimized for high NA objectives ↩

  2. Semrock Di02-R561 ↩↩