It is important to regularly assess the quality and performance of a given microscope. This blog post will gather and describe a practical guide to microscopes quality control.

Illumination Power Warmup Kinetic

When starting an instrument, it takes time to reach a stable steady state. This duration is known as the warmup period. It is critical to record a warmup kinetic at least once to accurately define this period.

Acquisition Protocol

1. Setup the Power Meter
   1. Place a power meter sensor (e.g., Thorlabs S170C) on the stage.
   2. Center the sensor under the objective.
2. Prepare the Measurement
   1. Select the wavelength of the light source you wish to monitor using your power meter controller (e.g., Thorlabs PM400) or software.
   2. Zero the sensor to ensure accurate readings.
3. Record the Warmup Kinetic
   1. Turn on the light source and immediately record the power output over time until it stabilizes.
4. Repeat for Multiple Light Sources
   1. Repeat steps 2 to 3 for each light source you wish to monitor.

Results

For each light source plot the measured power output (mW) over time.

For this instrument the warmup time is virtually null.

Calculate the relative power: Relative Power = Power/MaxPower and plot the Relative Power (%) over time.

The 385nm, 475nm, and 630nm light sources exhibit some variability; however, this is unlikely to impact the measurement.

Conclusion

The warmup time for this specific instrument is virtually null but a 10 minutes warmup period is recommended.

Maximum Illumination Power Output

This measure evaluates the maximum power output of each light source, considering both the quality of the light source and the components along the light path. Over time, we anticipate a gradual decrease in power output, accounting for the aging of the hardware, including the light source and other optical components.

Acquisition Protocol

1. Warm up the system
2. Setup the Power Meter
   1. Place a power meter sensor (e.g., Thorlabs S170C) on the stage.
   2. Center the sensor under a low magnification objective.
3. Prepare the Measurement
   1. Select the wavelength of the light source you wish to monitor using your power meter controller (e.g., Thorlabs PM400) or software.
   2. Zero the sensor to ensure accurate readings.
4. Record the maximum illumination power output
   
   1. Turn on the light source to 100%
   2. Record the average power output.
5. Repeat for Multiple Light Sources
   1. Repeat steps 3 to 4 for each light source you wish to measure.

Results

For each light source plot the measured maximal power output (mW) and compare it to the specifications from the manufacturer. Calculate the relative power: Relative Power = Measured Power / Specifications.

Conclusion

This instrument provides 80% of the power given by the manufacturer specifications. These results are consistent because the manufacturer specifications are using a different objective and likely different dichroic mirrors.

Illumination stability

The light sources used on a microscope should be constant or at least stable over the time scale of an experiment. For this reason power stability is recorded over 4 different time-scale.

This measure compares the power output over time. Four different timescales are measured:

• Real-time illumination stability: Continuous recording for 1 min
• Short-term illumination stability: Every 1-10 seconds for 5-15 min. This represents the duration of a z-stack acquisition
  
• Mid-term illumination stability: Every 10-30 seconds for 1-2 hours. This represent the duration of a typical acquisition session or short time-lapse experiments. For longer time-lapse experiments, longer duration may be used.
  
• Long-term illumination stability: Once a year or more over the lifetime of the instrument (this is measured in the Maximum Power Output section comparing with previous measurements)

 The Stability factor is then calculated S (%) = 100 x (1- (Pmax-Pmin)/(Pmax+Pmin)).

Real-time illumination stability

Acquisition Protocol

1. Warm up the system
2. Setup the Power Meter
   1. Place a power meter sensor (e.g., Thorlabs S170C) on the stage.
   2. Center the sensor under a low magnification objective.
3. Prepare the Measurement
   1. Select the wavelength of the light source you wish to monitor using your power meter controller (e.g., Thorlabs PM400) or software.
   2. Zero the sensor to ensure accurate readings.
4. Record the maximum illumination power output
   
   1. Turn on the light source to 100%
   2. Record the power output as fast as possible for 1 minute
5. Repeat for Multiple Light Sources
   1. Repeat steps 3 to 4 for each light source you wish to measure.

Results

For each light source plot the measured power output (mW) over time.

Calculate the relative power: Relative Power = Power/MaxPower and plot the Relative Power (%) over time.

 Calculate the Stability factor S (%) = 100 x (1- (Pmax-Pmin)/(Pmax+Pmin)).

Conclusion

The light sources are highly stable (>99.9%) during a 1 min period.

Short-term illumination stability

Acquisition Protocol

1. Warm up the system
2. Setup the Power Meter
   1. Place a power meter sensor (e.g., Thorlabs S170C) on the stage.
   2. Center the sensor under a low magnification objective.
3. Prepare the Measurement
   1. Select the wavelength of the light source you wish to monitor using your power meter controller (e.g., Thorlabs PM400) or software.
   2. Zero the sensor to ensure accurate readings.
4. Record the maximum illumination power output
   
   1. Turn on the light source to 100%
   2. Record the power output every 10 seconds for 15 minutes
5. Repeat for Multiple Light Sources
   1. Repeat steps 3 to 4 for each light source you wish to measure.

Results

For each light source plot the measured power output (mW) over time.

Calculate the relative power: Relative Power = Power/MaxPower and plot the Relative Power (%) over time.

 Calculate the Stability factor S (%) = 100 x (1- (Pmax-Pmin)/(Pmax+Pmin)).

Conclusion

The light sources are highly stable (>99.7%) during a 15 min period.

Mid-term illumination stability

Acquisition Protocol

1. Warm up the system
2. Setup the Power Meter
   1. Place a power meter sensor (e.g., Thorlabs S170C) on the stage.
   2. Center the sensor under a low magnification objective.
3. Prepare the Measurement
   1. Select the wavelength of the light source you wish to monitor using your power meter controller (e.g., Thorlabs PM400) or software.
   2. Zero the sensor to ensure accurate readings.
4. Record the maximum illumination power output
   
   1. Turn on the light source to 100%
   2. Record the power output every 10 seconds for 1 h
5. Repeat for Multiple Light Sources
   1. Repeat steps 3 to 4 for each light source you wish to measure.

Results

For each light source plot the measured power output (mW) over time.

Calculate the relative power: Relative Power = Power/MaxPower and plot the Relative Power (%) over time.

 Calculate the Stability factor S (%) = 100 x (1- (Pmax-Pmin)/(Pmax+Pmin)).

Conclusion

The light sources are highly stable (>99.5%) during a 1 h period.

Long-term illumination stability

Long-term illumination stability is intented to measure the power output over the lifetime of the instrument. This is measured in the Maximum Power Output section by comparing with previous measurements.

Illumination stability conclusion

The light sources are highly stable (>99.5%).

Illumination Input-Output Linearity

This measure compares the power output when the input varies. We expect a linear relationship between the input and the power output.

Acquisition Protocol

1. Warm up the system
2. Setup the Power Meter
   1. Place a power meter sensor (e.g., Thorlabs S170C) on the stage.
   2. Center the sensor under a low magnification objective.
3. Prepare the Measurement
   1. Select the wavelength of the light source you wish to monitor using your power meter controller (e.g., Thorlabs PM400) or software.
   2. Zero the sensor to ensure accurate readings.
4. Record the power output
   
   1. Turn on the light source to 0%, 10, 20, 30…, 100%
   2. Record the power output for each input
5. Repeat for Multiple Light Sources
   1. Repeat steps 3 to 4 for each light source you wish to measure.

Results

For each light source plot the measured power output (mW) function of the input (%).

Calculate the relative power: Relative Power = Power/MaxPower and plot the Relative Power (%) function of the input (%).

 Calculate the curve equation (usually a linear equation : Output = K x Input). report the Slope and the R² coefficient of dertermination (closest to 1).

Conclusion

The light sources are highly linear.

References

The information provided here is inspired by the following references:

doi.org/10.17504/protocols.io.5jyl853ndl2w/v2

https://doi.org/10.1083/jcb.202107093

Record a kinetic of light intensity over time to define the optimal warmup time for your light source. Typical values are 10 min for LED and Arc lamps, 1h for lasers.

Place the S170C sensor onto the stage.

Center the sensor on the objective

Select the wavelength on your power meter

Zero the sensor

Measure the power

What need to be assessed?

Resolution

Fiel Illumination Uniformity

Channel alignement (Co-registration)

Illumination stability

Stage drift

Stage repositioning accuracy

Detector Noise