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. The excel file provide a template that can be filled in Maximum Illumination Power Output_Template.xlsx

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)).

The excel file provides a template that can be filled in Illumination Stability_Template.xlsx

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 (%). The excel file provides a template that can be filled in Illumination Power Linearity_Template.xlsx

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.

Objectives and cubes transmittance

Since we are using a power meter we can easily assess the transmittance of the objectives and the filter cubes. This measure compares the power output when different objectives and cubes are in the light path. It evaluates the transmittance of each objective and compares it with the manufacturer specifications. It can detect defects or dirt on objectives.

Objectives transmittance

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 100%
   2. Record the power output for each objective as well as without any objective
5. Repeat for Multiple Light Sources
   1. Repeat steps 3 to 4 for each light source you

Results

For each objective plot the measured power output (mW) function of the wavelength (nm). The excel file provides a template that can be filled in Objective and cube transmittance_Template.xlsx

Calculate the relative transmittance: Relative Transmittance = Power/PowerNoObjective and plot the Relative Transmittance (%) function of the wavelength (nm).

Calculate the average transmittance for each objective.

Compare the average transmittance to the specification provided by the manufacturer.

Here we see that the measurements are close to the specification at the exception of the 63x-1.4 objective. This is expected because the 63x objective has a smaller back aperture which reduces the amount of light received.

Conclusion

The objectives are transmitting light properly.

Cubes transmittance

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 100%
   2. Record the power output for each filter cube
5. Repeat for Multiple Light Sources
   1. Repeat steps 3 to 4 for each light source you

Results

For each cube plot the measured power output (mW) function of the wavelength (nm). The excel file provides a template that can be filled in Objective and cube transmittance_Template.xlsx

Calculate the relative transmittance: Relative Transmittance = Power/PowerObjective and plot the Relative Transmittance (%) function of the wavelength (nm).

Report the transmittance of the appropriate wavelenth for each filter cube.

• The DAPI cube only transmits 14% of the excitation light compared to the Quad Band Pass DAPI/GFP/Cy3/Cy5. It is usable but will provide a low signal. This likely because of the excitation filter within the cube is not properly matching the light source. This filter could be removed since an excitation filter is already included within the light source.
• The GFP and DsRed cubes transmit 47% of the excitation light compared to the Quad Band Pass DAPI/GFP/Cy3/Cy5 transmits. It works properly.
• The DHE cube does not transmit any light from the colibri. This cube could be removed and stored.
• The Cy5 cube transmit 84% compared to the Quad Band Pass DAPI/GFP/Cy3/Cy5. It works properly.

Conclusion

Actions have to be taken for the DAPI and DHE.

XYZ Drift

This experiment evaluates how stable is the system in XY and Z at 4 different timescales:

• Real-time drift: Continuous recording for 1-5 min.
• Short-term drift: Every 10 seconds for 15 min. This represents the duration of a z-stack acquisition
  
• Mid-term drift: Every 30 seconds for 1-2 hours. This represents the duration of a typical acquisition session or short time-lapse experiments.
  
• Long-term drift: Every 10 min overnight (6-8 hours). This is required for long time-lapse experiments.

XYZ drift warmup kinetic

As mentioned earlier, 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. Place 1 um diameter fluorescent beads (TetraSpec Fluorescent Microspheres Size Kit mounted on slide) on the stage.
2. Center the sample under a high NA dry objective.

3. Choose an imaging channel (exemple Cy5)

4. Acquire a large Z-stack every minute for 24 h

Results

Use TrackMate Plugin for FIJI to detect the spot and tack it over time. DoG Spot detection with 1 um object detection, 20 quality threshold with sub-pixel localization. Detected spots coordinates were exported and displacement from the initial image was calculated in nm. The excel file provides a template to measure XYZ Drift over time XYZ Drift Kinetic_Template.xlsx

Calculate the relative displacement in X, Y and Z: Relative Displacement = Position - PositionInitial and plot the relative displacement over time.

We observe an initial drift that stabilizes over time in X (+3.5 um), Y (+1 um) and Z (-12 um).

Calculate the velocity in X, Y and Z. Velocity = (Pos2-Pos1) /(Time2-Time1)

We observe the system stabilizing after 300 minutes.

Conclusion

The warmup time for this specific instrument is quite long 5 hours !

XYZ drift real-time

XYZ drift short-term

XYZ drift mid-term

XYZ drift long-term

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

What need to be assessed?

Resolution

Fiel Illumination Uniformity

Channel alignement (Co-registration)

Stage drift

Stage repositioning accuracy

Detector Noise