Laser / Light at the objective: Power and Stability

Method: a suitable power meter is placed in the focal plane of the standard 10x objective on the system (usually air, 0.3-0.4NA). Power for each light source is recorded at a range of intensities with a fixed set of dichroics either with spot scanning or a zoomed small area scan (eg 5x, 256×256).  For widefields, this is done on each filter cube with a range of lamp intensities. Minimum standard of 10 and 100% light source power. 

Justification: this allows monitoring of the light source combined with the whole excitation light path, and will allow comparison of values over time.  This will highlight any drop off in laser/lamp excitation at the specimen, which identifies either light source fault or problems within the light path (eg damaged filters/ dichroics).

Requirements: power meter and sensor covering the wavelengths and power of the system


Stability of Illumination Power (satisfying Confocal ISO test)

Comparison of fluorescence intensity between images requires knowledge that the excitation light sources are stable.  Ideally this should be shown for both short term and long term imaging (Confocal ISO 21073 Section 4.4.2) using an external power sensor.  Slide-based formats are the easiest to mount (e.g. Thorlabs S170C or Exelitas XP750).

Method

  1. Ensure lasers have been warmed up prior to measurement for at least one hour.
  2. A suitable power sensor is placed in the focal plane of the standard 10x objective on the system (usually air, 0.3-0.4NA). Ensure background light is kept to a minimum (room lights off).
  3. Determine a suitable laser power output (usually by % AOTF) that lies between 0.2 to 0.5 mW at the power sensor.
  4. For short term stability testing, set the scan mode to spot (stationary) scanning and record every second for 5 minutes.  Calculate stability (STABshort) as:
    1. STABshort (%) = 100x (1 – ((Pmax – Pmin) / (Pmax + Pmin)))
    2. Where max and min are the maxima and minima recorded during the 5 minutes recording.
  5. For long term stability, spot scanning is performed every 30 seconds for 1 second over 120 minutes, recording the power at each imaging timepoint.  Calculate stability (STABlong) as:
    1. STABlong (%) = 100x (1 – ((Pmax – Pmin) / (Pmax + Pmin)))
    2. Where max and min are the maxima and minima recorded during the 5 minutes recording.

[NOTE: If the software allows, the microscope can be set up to provide alternate laser values  over the same time period, scanning each laser sequentially within the 30 second timelapse.]

  1. These data should be reported along with the laser wavelengths measured and the average laser power measured.