TITLE: Repeatability of the XY positioning of motorized xy-tables of light microscopes
AUTHORS: Mariana T Carvalho, Hans Fried, Stuart Jarvis, Claire A Mitchell, Roland Nitschke, Kees van der Oord, Arnd Rühl, Stanley Schwartz, Martin Spitaler, Roman Zantl, Arne Seitz

[DESCRIPTION]
The time it takes for the entire microscope system to be in equilibrium with the environment is called the settling time and is highly affected by changes in environmental conditions. Long-term measurements only provide conclusive results of the system if the environmental conditions are stable over time. In particular, temperature fluctuations will influence drift measurements in all axes. Tracking the temperature of the microscope and the room is highly recommended for long-term measurements and to indicate the drift stability. This is particularly important for correctly interpreting the results and accurate measurements. Discussions among the working group experts have shown that allowing the microscope system to adapt and equilibrate to the room/measurement conditions for a specific time is common practice. The time for thermal equilibration with the room conditions can easily reach several hours. It is important to note that it is not always possible to wait such a long time before starting iterative measurement sessions, but at least the microscope system and room should be characterized initially and referred back to as the baseline environment measurement reference parameter. As with other QUAREP-LiMi protocols, a 1 hour equilibrium period should be observed, with minimal deviations in temperature and humidity. 

[BEFORE_START]
Switch on the entire system well in advance before the measurement. Allow the system and stage to reach temperature equilibrium for at least 1 h before starting the measurement.
Ensure a thoroughly cleaned microscopy system. 
Ensure the proper functioning of the entire microscopy system.
Ensure a stable room temperature. The maximal variation in temperature needs to be smaller than 1.0 degrees over the measurement period. If possible, track the temperature next to the stage. Maintain room temperature vs. stage surface temperature data for each calibration session using one data logging room temperature and humidity sensor and another data logging temperature probe located on the stage very near the specimen slide or vessel holder.  
Ensure the system is protected from environmental disturbances (e.g. vibrations, heavy airflow, etc.). Eliminate sources of temperature variation and any vibration sources that affect sub-micron positioning.
If not doable, a run overnight is an alternative.
Ensure that the sample is rigidly mounted in the insert and that the insert is properly fixed to the stage to prevent movements caused by the shift of the sample. Give the sample enough time to be in thermal equilibrium with the room temperature.
Ensure that the microscope room door is not opened/closed many times during the acquisition to minimize any vibrational or temperature-related fluctuations. If needed, put a note on the door saying, 'Do not enter. Imaging in progress.


[GUIDELINES]
The environmental conditions should be 22° and ideally constant temperature (air-conditioned).
A short warm-up of the stage is good, e.g. a short random move over the whole travel range.
If this is not possible, running the stage at least once over the whole travel range is still good. This has the advantage of evenly distributing all the lubricant on the spindle and guides. This also happens every time the stage is initialized.
Regular maintenance by the stage manufacturer should be kept in mind. Depending on the running time, this can be different, as a guideline serves: With max. 8 hours per day at the latest after 5 years (different from stage manufacturer to stage manufacturer). A reference run can also be made occasionally to check the stage performance.

[STEPS]
SECTION: Acquisition
1. When starting the acquisition software, first initialize (if applicable) the motorized stage platform in the XY direction.
SECTION: Acquisition
3. Select a 10x or 20x dry lens and select the XYT acquisition mode of the system control software. Any drift control of the instrument itself must be disabled. 
 

 

5. Position a fluorescent bead in the center of the image. It is acceptable that other beads are within the field of view as well. If necessary, adjust the acquisition parameters. Adjust the exposure time of the detector to avoid saturated pixels within the image as this will impact the subsequent analysis.  For camera-based systems, the maximal pixel intensity of the bead equals roughly 50% of the camera dynamic range, i.e. around 128 grey values for an 8-bit acquisition and 2048 for a 12-bit image. For point-scanning confocal systems, it is recommended to use the entire dynamic range.
It is recommended to use Nyquist sampling for point-scanning confocal systems and no binning for camera-based detection systems.
 

SECTION: Acquisition
6.1. Record/store the position with one bead in the center of the FOV (P1, if possible, mark as “0”) in the acquisition software.
SECTION: Acquisition
6.2. Shift the stage so the bead moves 300 μm to the left (-300 μm in x). Record the position (P2).
 

SECTION: Acquisition
6.3. Return to the initial bead position. Record the position again (P3). 
 

SECTION: Acquisition
6.4. Shift the stage so the bead moves 300 μm to the right (+300 μm in x). Record the position (P4).
SECTION: Acquisition
6.5. Return to the initial position. Record the position again (P5).
SECTION: Acquisition
6.6. Shift the stage so the bead moves 300 μm to the top (+300 μm in y). Record the Position (P6).
SECTION: Acquisition
6.7. Return to initial (center) position. Record the Position again (P7).
SECTION: Acquisition
6.8. Shift the stage so the bead moves 300 μm to the bottom (-300 μm in y). Record the position (P8).
 
 
 

 

SECTION: Acquisition
8. Repeat the whole procedure for a displacement distance of 4000 μm (4mm).
 

SECTION: Analysis
9. The following analyses are based on Fiji. 
Screenshots are made from ImageJ 2.14.0/1.54f; Java 1.8.0_172 [64-bit]. 
SECTION: Analysis
11.1.    

SECTION: Analysis
11.2.   
 
 

 


SECTION: Analysis
13. To report on the performance of the stage, calculate the standard deviation of all X and Y positions. These values can be used to report on the stage performance in the field over time.   

SECTION: Analysis
12.2.      

SECTION: Analysis
12.3.    

SECTION: Analysis
10. Open the obtained image stack e.g. in Fiji and manually move from one image to the next. For a well-calibrated motorized table, it is expected that only minor bead movements are visible. If you encounter larger movements this can either indicate thermal drift and/or an issue with the stage itself in its accepted x,y repeatability/repositioning capability. 
If the bead spot changes shape (diameter) it is showing Z drift of the focus mechanism or thermal drift. 
SECTION: Acquisition
2. Please make sure that the objective turret is positioned (lowered) so that none of the objectives can make contact with the stage platform and be damaged by this initialization procedure. This is an absolute caution step for commercial microscopes and particularly for custom build set-ups.
SECTION: Acquisition
6. Record the stage positions. The set of positions is called one cycle.
 

SECTION: Acquisition
7. Set up a multi-position time-lapse experiment (XYT) with 20 cycles (one cycle corresponding to the nine previously defined positions). Thus the stage travels to each position 20 times (=20 measurement cycles) and acquires an image at each position. The cycles shall be scheduled without any further delay. 
 

SECTION: Acquisition
4. Test that the stage insert as well as the sample is securely fixed. Any sample/insert movement will deteriorate the measurement. 

SECTION: Analysis
10. Find a position in the sample so that the camera field of view contains at least two beads.
SECTION: Analysis
11. Follow the Acquisition protocol.
SECTION: Analysis
12. Crop the image so the resulting window only contains a single bead. Repeat the cropping to obtain a second image stack of another bead.
SECTION: Analysis
13. Analyse the two individual stacks following the Quantitative analysis protocol.
SECTION: Analysis
14.      


SECTION: Analysis
15. The R2 value of a linear regression analysis reports on the correlation of the movement of the two beads. Values below 0.95 indicate that at least one of the beads is not properly attached to the glass surface of the coverslip and is therefore not well suited to report on the performance of the table.
SECTION: Analysis
10. Open image in Fiji.
SECTION: Analysis
10.1. Open image sequence/time lapse of Position 1 in Fiji/ImageJ.
 

SECTION: Analysis
10.2. Check that the image is properly calibrated, i.e., the pixel width and height correspond to the acquisition settings. This information is typically automatically stored in the image's metadata for commercially available systems. The calibration can be checked in the menu
Image-->Properties... in Fiji (see image below)
 


SECTION: Analysis
10.3. Crop the image so the resulting window only contains a single bead, preferentially the most centred one.
 

  

SECTION: Analysis
11. Image Segmentation
SECTION: Analysis
12. Analyse the position of the bead.
SECTION: Analysis
12.1.