TITLE:  Monitoring the point spread function for quality control of confocal microscopes
AUTHORS: Glyn Nelson, Ioannis Alexopoulos, Maria Azevedo, Fabio Barachati, Yury Belyaev, Mariana T Carvalho, Yann Cesbron, Aurelien Dauphin, Alexander D Corbett, Ian M Dobbie, Laurent Gelman, Nadia Halidi, Xiang Hao, Hella Hartmann, Rainer Heintzmann, Peter  Hemmerich, Marcel Kirchner, Judith Lacoste, Penghuan Liu, Laure Plantard, Claire A Mitchell, Tobias Müller, Kees van der Oord, Alex L Payne-Dwyer, Santosh Podder, Rachid Rezgui, Sandra Ritz, Stanley Schwartz, Michael J Shaw, Tse-Luen  Wee, Roland Nitschke, Orestis Faklaris

[DESCRIPTION]
This protocol focuses on measuring the microscope's lateral and axial resolution, essential for reporting size measurements of near-resolution limit objects or distances between them. Here resolution is expressed as the Full Width at Half Maximum (FWHM) of a measured Point Spread Function (PSF) of sub-resolution size beads.  PSF is highly related to objective quality and condition, but also depends strongly upon other parameters ranging from sample preparation to signal detection.  Monitoring PSF over time will identify possible aberrations in the system (e.g., damaged, unclean objective, defective or not adapted oil, etc.).  We define test sample preparation, image acquisition, and data analysis protocols for point scanning and spinning disk confocal microscopes.

[BEFORE_START]
Bead sample preparation section- this should be done at least two days before starting imaging to ensure suitable curing times for mountant.

Bead image acquisition section- the system should be warmed up for 1 hour before starting to ensure thermal stability to avoid axial drift during imaging.

[GUIDELINES]
Assumption: This method describes the image capture, assuming you use 175 nm beads and a high NA oil-immersion objective.

Alternative pre-prepared bead samples are available and can be used to obtain reliable PSFs e.g. Tetraspeck (T14792, Thermofisher) and PSFcheck (https://www.psfcheck.com/). We have chosen to prepare fresh bead samples in this protocol to maximise brightness, stability, and availability.  


[STEPS]
SECTION: Bead Slide Preparation
3. The bead slide preparation described below contains steps previously published in the following protocols:
 
as well as another:
 

SECTION: Bead Slide Preparation
2. Bead dilution 


SECTION: Bead Slide Preparation
4. Bead slide mounting
SECTION: Bead Slide Preparation
5. The bead slides can be stored at room temperature for 6-12 months.  
 

 
 









SECTION: Bead image Acquisition
6. Prepare the microscope system.


SECTION: Bead image Acquisition
7. Find the beads.
SECTION: Bead image Acquisition
8.2.  Ensure at least one bead is in or near the centre of the FoV.  

 


 

SECTION: Bead image Acquisition
8.3. 

Set the capture parameters to achieve a voxel size of 40x40x100 nm for objectives with NA>1.3 (see Figure 4). 
 



 

 

 

 

 

 

 

SECTION: Bead image Acquisition
8.5. Using a live image, optimise the laser power (the 488 nm line is recommended for the beads used here) and the detector gain to obtain a reasonable signal with a minimum background such that the detected noise is not clipped at zero. Set image acquisition parameters to capture in single direction mode and use averaging or increased pixel dwell time to improve signal to background ratio (SBR). Adjust the focal plane for the maximum intensity. Ensure that no bleaching of the beads occurs by decreasing the laser power and no pixel saturation, by if needed changing the detector gain.  
 

 

 

SECTION: Bead image Acquisition
8.6. Using the z focus drive (ideally a piezo drive if available), ensure you are at the axial centre of the bead (select for the widest and brightest signals). Set the z stack to capture 5 μm with a stepsize of 100 nm (51 slices) minimum using the centre of the bead as the centre of the stack (See Figure 4 for justification of axial sectioning). 
 

SECTION: Bead image Acquisition
8.7.  Capture images of multiple beads (at least 6, ideally 20).

 
 
 

 


SECTION: Bead Analysis
9. Bead analysis consists in measuring the FWHM of the acquired PSFs.  First one has to choose the analysis software.  
 
 
 

 

 



SECTION: Appendix 3: MetroloJ_QC analysis method
18. Run either the single image PSF calibration (Figure 1, blue arrow) or the batch multi image option (green arrow), which will process all images in a directory.
 

 

SECTION: Appendix 3: MetroloJ_QC analysis method
19.  Running the batch option will ask for a directory- select the directory containing the raw image files.
 



SECTION: Bead Analysis
19. The first found image will open and the window giving an overview of bead image metadata will pop up.  Fill in the metadata fields for the bead images.

 

SECTION: Bead Analysis
21. Once completed, click 'OK' to allow the PSF analysis to run,

 

SECTION: Bead Analysis
22. Upon completion, the batch PSF tool will leave the found bead coordinates from the last image in the directory showing in the Results table. Navigate to the folder of raw images and you will find a new sub-directory structure with summary csv files (.xls), a pdf and individual bead data.
SECTION: Bead Analysis
23. The summary.xls file contains the means, standard deviations and Signal to Background ratios for all three axes, plus the individual bead FWHM and goodness of fit (r2).
 

If individual bead pdf output is selected, the details of each bead are also given.  An example is shown below:
 

SECTION: Appendix 1: Aberrations and PSF
15. Aberrations reduce the resolution and sensitivity of a light microscope. Careful inspection of the collected PSF can help to identify potential aberrations causing poor microscope performance. Examples of common aberrations are shown in Table 1. 

 

 


 
 
  
 
 
 
 
SECTION: Appendix 2: Tutorials
16. MetroloJ_QC manual: 
https://github.com/MontpellierRessourcesImagerie/MetroloJ_QC

PyCalibrate manual and video tutorial: 
https://www.psfcheck.com/psfcheck-processing

PSF characterization video from the microscopy technology network rtmfm (https://rtmfm.cnrs.fr/): 
https://www.youtube.com/watch?v=lFdDLQkNehg
SECTION: Bead Analysis
14. The analysis method is up to the user.  We recommend to at least plot the data as box and whisker plots and to keep track using Excel, Airtable, OMERO, or other suitable software.  KNIME for example, can be used to track output from PSFj over time to monitor a system's performance.

 


SECTION: Bead Slide Preparation
1. Coverslip Preparation 

SECTION: Appendix 3: MetroloJ_QC analysis method
17. Here we provide instructions for running MetroloJ_QC in Fiji, since Fiji comes with the Bioformats plugin automatically installed, allowing easy import of raw images.
Open Fiji and then start the MetroloJ_QC plugin from the Plugins menu.
SECTION: Appendix 3:
24. 
SECTION: Bead Analysis
20. 
SECTION: Appendix 3: MetroloJ_QC analysis method
20. Most of the metadata should be imported correctly from raw microscope files (voxel size), but parameters should be double checked and missing values entered manually, such as the excitation/emission wavelengths, the objective NA, the image bit depth, or the pinhole size.  Several parameters are variable depending on your preferences and dataset, such as whether to discard saturated beads (recommended), the prominence value (important for properly thresholding and taking into account all the beads) and the ROI around the bead (Figure 2). If beads are closer than the given ROI, then the beads are discarded. 


  

SECTION: Appendix 3: MetroloJ_QC analysis method
22. Upon completion, the batch PSF tool will leave the found bead co-ordinates from the last image in the directory showing in the Results table.  Navigate to the folder of raw images and you will find a new sub-directory structure with summary csv files (.xls), a pdf and individual bead data.
  

SECTION: Appendix 3: MetroloJ_QC analysis method
23. The summary.xls file contains the means, SDs and SBRs for all three axes, plus the individual bead FWHM and goodness of fit (r2).
  

SECTION: Appendix 3: MetroloJ_QC analysis method
24. The plugin calculates two QC metrics. The first one is the measured to theoretical FWHM ratio. If the ratio is greater than the value indicated as tolerance value in the metadata and plugin parameters step (step 33) then the value appears in red. If the value is within recommendations, it is highlighted in green. The second parameter is the Lateral Asymmetry Ratio (LAR), defined as the ratio of the minimal to the maximal xy FWHM value.     
  

SECTION: Bead Analysis
13. Here is a common result of an analysis software (MetroloJ_QC) for a FWHM calculation of one bead.
The most important parameter to be calculated is the FWHM along the xyz dimensions. Other information, such as the SBR, the lateral asymmetry ratio (LAR) or the goodness of fit r2 can be provided by the software. 
  

 

SECTION: Bead Analysis
10. Each analysis software may use a different fitting method of the PSF. Most software packages use a 1D or 2D Gaussian fit. Although confocal PSFs can be sufficiently fitted by a Gaussian function, the fitting method is crucial for an accurate measurement of the FWHM and thus the assessment of the objective performance.

 

For monitoring the performance of the objective over time, using the same protocol (sample preparation, acquisition and analysis software) is mandatory. 
 

 

SECTION: Bead Analysis
12. To measure the FWHM, the software package needs to read the image metadata, such as the voxel size. This can be done directly or has to be inserted manually, depending on the software. The software may then need the user to insert other parameters, like the objective numerical aperture, the excitation-emission wavelengths, the pinhole size, the bit depth, among others, for different metrics calculations. The software may then allow a batch analysis of multiple images and multiple acquisitions and provide an average FWHM. 


SECTION: Appendix 3: MetroloJ_QC analysis method
21. The plugin calculates the FWHM along the x, y, and z-axes of all beads in the field of view. FWHM is measured as follows: the maximum intensity pixel within the entire 3D data set is determined. An intensity plot along a straight line through this maximum intensity pixel point is extracted in all dimensions. The curves are fitted by a Gaussian function using the built-in ImageJ curve fitting algorithm to determine the FWHM. After automation of the FWHM measurements on several beads/datasets, average values are extracted in all three dimensions, and standard deviations are calculated. In case the algorithm detects spots that do not originate from the beads, they can be removed using the fitting parameter r2. 
SECTION: Bead Analysis
11. Before analyzing the images visually inspect the acquisitions. Some images need to be cropped to remove saturated beads. An alternative option can be used to automatically discard any saturated beads from the image, if the analysis software offers this option. 
SECTION: Bead Slide Preparation
1.1. Immerse the coverslip in a beaker with 1M HCl and agitate for 30 min at room temperature.
SECTION: Bead Slide Preparation
1.2. Rinse the coverslip twice with 250 mL of distilled water and immerse in 100% Ethanol for storage.
SECTION: Bead Slide Preparation
1.3. Pick up a coverslip with forceps, dab off the excess of Ethanol by carefully blotting onto a lens cleaning paper and let the coverslip air dry completely on a fresh lens cleaning paper in a dust free environment (ideally a laminar flow hood).
SECTION: Bead Slide Preparation
2.1. Centrifuge the bead stock solution for a few seconds in a benchtop centrifuge to collect all suspension in the bottom of the tube and then vortex for 2 to 3 minutes. 
 

 

SECTION: Bead Slide Preparation
2.2. Dilute the fluorescent beads in H2O.
 

 

 

SECTION: Bead Slide Preparation
2.3. Take a cleaned coverslip, allow to air-dry, then dispense 10 μl of the bead solution as mini-droplets across the coverslip, ensuring the drops are as small as possible.
 

 

SECTION: Bead Slide Preparation
2.4. Let the coverslips with droplets air-dry at room temperature (it takes at least one hour) in a dust-free environment (ideally a laminar flow hood, otherwise under cover, e.g. a tip box lid).
 

SECTION: Bead Slide Preparation
4.1. Clean the microscope slides with 100% Ethanol and lens tissue, and allow to air dry.
SECTION: Bead Slide Preparation
4.2. Place a drop of mounting medium in the centre of a microscope slide. For a 22 x 22 mm coverslip, 12 μL of medium is sufficient.
 

 

SECTION: Bead Slide Preparation
4.3. Using forceps, gently take a dried coverslip and place it bead-side down onto the drop of mounting medium in the centre of the microscope slide, lowering down from one side to the other to push the excess medium and potential bubbles towards the opposite edge. 
Remove any bubbles and excess mounting medium by covering with lens tissue and pressing very gently. Remove lens paper immediately, taking care not to remove the coverslip with it! Let the mounting medium cure (if required) to reach its optimal refractive index by storing slides in the dark at room temperature.
 

SECTION: Bead Slide Preparation
4.4. Seal the coverslip edge with Picodent dental silicone or other two component silicone (see Materials), ensuring the resulting seal is not so thick that you will be able to focus with large blunt-nosed objectives. 
 

 

SECTION: Bead image Acquisition
6.4.  Allow the temperature to stabilize.
 

 

SECTION: Bead image Acquisition
7.1. Place your sample on the stage with sufficient immersion medium to just allow coverage of the lens's front element once it has made contact with the coverslip.
SECTION: Bead image Acquisition
7.2. Focus on the beads using epifluorescence and a filter set matching the fluorescence properties of the beads (i.e. the GFP or FITC filter set) - the focal plane is right at the coverslip-mountant interface. The beads will be denser in certain areas. Focussing in a dense area is easier, so moving the stage in x and y directions while focussing helps.
 

SECTION: Bead image Acquisition
7.3.  Move to an area where beads are sparse and ensure you have at least one bead in the centre of the Field of View (FoV), with none too near to each other so that overlapping PSF patterns are avoided. We recommend a spacing at least 4 μm between neighbouring beads.
 

SECTION: Bead image Acquisition
6.2. Use a high magnification, high NA objective, e.g. 63x/1.4 on point scanning confocal microscopes or 100x/1.4 on spinning disk systems. 
 



 

 

SECTION: Bead image Acquisition
6.3. Clean your objective lens.
 

SECTION: Bead image Acquisition
6.1.  Allow the system to warm up, ensuring everything is turned on to allow system stabilisation.
 

SECTION: Bead image Acquisition
8.1. Where possible, set the confocal "zoom" to use the centre of the FoV. 
 

SECTION: Bead image Acquisition
8.  Acquisition settings
SECTION: Bead image Acquisition
8.4. On LSMs, keep the pinhole wide open.