TITLE: PSF measurements for ELYRA 7
AUTHORS: Rafael Camacho

[DESCRIPTION]
Protocol for measuring PSFs at the ELYRA 7. This protocol was developed to follow guidelines from ZEISS application specialists. It is therefore, based on the tools and software provided by ZEISS.

As sample you can use the fluorescent beads provided by ZEISS which size is below the diffraction limit.
 

 




[BEFORE_START]
Make sure to read all steps of each section before you try to implement them.

[STEPS]
SECTION: Warming up the microscope
1. Turn on the ELYRA 7
SECTION: Warming up the microscope
1.1. Turn on Main Switch
  
SECTION: Warming up the microscope
2. Let the microscope warmup overnight.
During this process make sure to leave the sample, and the immersion oil inside the chamber, so they also reach the desired temperature.
SECTION: Warming up the microscope
1.2. Turn on Components
  
SECTION: Warming up the microscope
1.3. Turn on Microscope Computer

 

SECTION: Warming up the microscope
1.4. Open ZEN black, and choose start system
  
SECTION: Warming up the microscope
3. Temperature control for increased stability.
SECTION: Warming up the microscope
3.1. Check the temperature of the imaging chamber (incubation XL unit). 
In our case it stabilizes at around 26-27 degrees when the microscope is on.
SECTION: Warming up the microscope
3.2. Taking into account the result of step 3.1, stabilize the temperature of the microscope. In our case we use 27 degrees. This can be done via:
1) Touch screen [Home -> Microscope -> Incubation -> H Unit XL -> Set Temperature -> Set On ->  Click ok] 
  
2) ZEN black [Locate Tab -> Incubation -> Temperature -> H unit XL checkbox]
  
SECTION: Align the stage
4. The stage must be perpendicular to the microscope to avoid imaging artifacts.
Please follow the protocol: 
 

SECTION: Find focus
5. Engage the objective you want to use. In our case Plan-Apochromat 63X / 1.4 Oil 
SECTION: Find focus
6. If necessary put a small drop of immersion fluid on the objective
SECTION: Find focus
7. Place the sample on the holder
SECTION: Find focus
8. Manually move the objective up until it touches the immersion fluid
SECTION: Drift stability test
12. 
 
Reduce your field of view to about 30 micron2 This can be done by cropping the camera chip
SECTION: Drift stability test
13. Generate a Time Series experiment, with a length of 20 minutes and 0.1 fps
SECTION: Find focus
9. You can approximate focus by using the Definite Focus
SECTION: Find focus
9.1. Zen black -> Acquisition Tab
  
SECTION: Find focus
9.2. Acquisition Parameter -> Definite Focus Menu -> Find Surface
  
SECTION: Find focus
10. Set imaging parameters, here we use Laser WF mode. Below an example for the 63X objective:
SECTION: Find focus
10.1. Zen black -> Acquisition Tab
  
SECTION: Find focus
10.2. Select the Laser WF mode by
Setup Manager -> Imaging Setup ->Laser WF
  
SECTION: Find focus
10.3. Laser WF mode settings:
    - 63x / 1.4 oil objective
    - Dichroic: BP 495-550 + BP 570-620
    - Emission beam splitter (Duolink): SBS 490-560 + LP 640
     - Active Camera TV1
   
SECTION: Find focus
10.4. Channel Settings
Acquisition Parameter -> Channels:
    - 488 nm excitation, 4%
    - Illumination mdoe: EPI
    - Exposure time 100-300 ms
  
SECTION: Find focus
11. Go to live mode and fine adjust for focus (for reduced field of view, use Continious instead)
 
  

 
 

SECTION: Drift stability test
12.1. Acquisition Parameter -> Acquisition Mode:
    - Frame size e.g. 512 x 512
Make sure images are 16 bits for better performance during analysis
  
SECTION: Drift stability test
13.2. Activate Time series in Experiment manager
  
SECTION: Drift stability test
13.3. Multidimensional Aquisition -> Time Series
    - Cycles 120
    - Interval 10.0 s
  
SECTION: Drift stability test
13.1. Zen black -> Acquisition Tab
  
SECTION: Drift stability test
13.4. Find focus:   
SECTION: Drift stability test
13.5. Start Experiment via Experiment Manager -> Start experiment
  
SECTION: Drift stability test
14. Review the stability of the drift by looking at your time series. The beads image should not move or get out of focus.
 

 

SECTION: Bleaching test
15. 
 
Reduce your field of view to about 30 micron2 This can be done by cropping the camera chip.
  
SECTION: Bleaching test
16. Check the size of your desired z-stack. Example for 63x 1.4 oil objective:
SECTION: Bleaching test
16.1. Zen black -> Acquisition Tab
  
SECTION: Bleaching test
16.2. Experiment Manager -> Z-stack
  
SECTION: Bleaching test
16.3. It is important that your Z-stack has a resolution of approximately 1/2 times the optimal step.
Multidimensional Acquisition -> Z-stack
    - Keep interval [active]
    - Interval 0.06
    - Set a number of slices that gives you a range >= 4 microns
    - Use Piezo [active]
  
SECTION: Bleaching test
16.4. Take note of the number of Slices, in our example 70, and deactivate the z-stack option via Experiment Manager -> Z-stack
SECTION: Bleaching test
17. Acquire a time series with a number of frames equal to the number of slices determined in    and no time interval (continious imaging)
For a time series example   
Example:
  
SECTION: Bleaching test
18. Review the bleaching of the beads. Below an example using Mean ROI tool in ZEN black:
SECTION: Bleaching test
18.1. ZEN Image Viewer -> Mean ROI
  
SECTION: Bleaching test
18.2. Draw a rectangular ROI around some beads and background area
 
   
SECTION: Bleaching test
18.3. Check that you have no significant bleaching of the beads over the time frame of your experiment

 

SECTION: Measuring the PSF
19. Please note that there are 3 configurations for which we want to measure the PSF
1) Laser WF Tubelens/Optobar 1.0 - Most relevant for SMLM
2) Laser WF Tubelens/Optobar 1.6 - Used as a comparison to the SIM PSF
3) Lattice SIM Tubelens/Optobar 1.6 - Used for Lattice SIM
SECTION: Measuring the PSF
20. Setup Manager -> Laser
Activate the 488 laser

   
SECTION: Measuring the PSF
20. Setup Manager -> Laser
Activate the 488 laser
  

SECTION: Measuring the PSF
21. Imaging Setup -> Laser WF
 
- Objective Plan-Apochromat 63x / 1.4 Oil
- Reflector revolver (dichroic filter): BP 495-550 + BP 570-620
- Tube lens (Optobar): 1x
- Dual Camera Beam Splitter: SBS BP 490-560 + LP 640
- Channel Camera: TV1
  
SECTION: Measuring the PSF
22. Acquisition Parameter -> Acquisition Mode
- Frame size: aim for an area of 32 micron2  this translated to 330 x 330 pixels
- Bit Depth 16
- You might consider averaging or sum in some cases 
- Noise filter: denoising off
Note how the images are taken in the centre of the field of view
  
SECTION: Measuring the PSF
23. 
  
SECTION: Measuring the PSF
24. Setup the Z-stack
SECTION: Measuring the PSF
24.1. Experiment Manager -> Z-stack
  
SECTION: Measuring the PSF
24.2. It is important that your Z-stack has a resolution of approximately 1/2 times the optimal step.
Multidimensional Acquisiton -> Z-stack
    - Keep interval [active]
    - Interval 0.06
    - Set a number of slices that gives you a range >= 4 microns
    - Use Piezo [active]
  
SECTION: Measuring the PSF
25. Go to Continuous mode and adjust focus
  
  
SECTION: Measuring the PSF
26. Multidimensional Acquisition -> Z-stack -> Center mode -> click on center
  

SECTION: Measuring the PSF
27. Start Experiment via Experiment Manager -> Start experiment
  

SECTION: Measuring the PSF
21. Imaging Setup -> Laser WF
 
- Objective Plan-Apochromat 63x / 1.4 Oil
- Reflector revolver (dichroic filter): BP 495-550 + BP 570-620
- Tube lens (Optobar): 1,6x
- Dual Camera Beam Splitter: SBS BP 490-560 + LP 640 
  
SECTION: Measuring the PSF
22. Acquisition Parameter -> Acquisition Mode
- Frame size: aim for an area of 32 micron2  this translated to 512 x 512 pixels
- Bit Depth 16
- You might consider averaging or sum in some cases 
- Noise filter: denoising off
Note how the images are taken in the centre of the field of view
  

SECTION: Measuring the PSF
23. Acquisition Parameter -> Channels
- LUT: Red Green Blue is convenient
- Laser power: 4% shows in our case good signal and low to no bleaching
- Illumination mode: EPI
- Exposure Time: if the drift is stable this value can be large. We use 100-400 ms
  

SECTION: Measuring the PSF
24. Setup the Z-stack
SECTION: Measuring the PSF
24.1. Experiment Manager -> Z-stack
  

SECTION: Measuring the PSF
24.2. It is important that your Z-stack has a resolution of approximately 1/2 times the optimal step.
Multidimensional Acquisiton -> Z-stack
    - Keep interval [active]
    - Interval 0.06
    - Set a number of slices that gives you a range >= 4 microns
    - Use Piezo [active]
  

SECTION: Measuring the PSF
25. Go to Continuous mode and adjust focus
  
  

SECTION: Measuring the PSF
26. Multidimensional Acquisition -> Z-stack -> Center mode -> click on center
  

SECTION: Measuring the PSF
27. Start Experiment via Experiment Manager -> Start experiment
  

SECTION: Measuring the PSF
24. Setup the Z-stack
SECTION: Measuring the PSF
24.1. Experiment Manager -> Z-stack
  

SECTION: Measuring the PSF
24.2. It is important that your Z-stack has a resolution of approximately 1/2 times the optimal step.
Multidimensional Acquisiton -> Z-stack
    - Keep interval [active]
    - Interval 0.06
    - Set a number of slices that gives you a range >= 4 microns
    - Use Piezo [is always active]
   
SECTION: Measuring the PSF
25. Go to Continuous mode and adjust focus
  
  
SECTION: Measuring the PSF
26. Multidimensional Acquisition -> Z-stack -> Center mode -> click on center
  
SECTION: Measuring the PSF
27. Start Experiment via Experiment Manager -> Start experiment
  
SECTION: Measuring the PSF
20. Setup Manager -> Laser
Activate the 488 laser
  

SECTION: Measuring the PSF
21. Imaging Setup -> Lattice SIM
  
- Grating 27.5 micron G5 (for 488nm ex)
- Objective Plan-Apochromat 63x / 1.4 Oil
- Reflector revolver (dichroic filter): BP 495-550 + BP 570-620
- Tube lens (Optobar): 1,6x
- Dual Camera Beam Splitter: SBS BP 490-560 + LP 640 
   
SECTION: Measuring the PSF
22. Acquisition Parameter -> Acquisition Mode
- Frame size: aim for an area of 32 micron2  this translated to 512 x 512 pixels
- Bit Depth 16
- You might consider averaging or sum in some cases 
- Grating: 13 Phases
Note how the images are taken in the centre of the field of view
   
SECTION: Measuring the PSF
23. Acquisition Parameter -> Channels
- LUT: Red Green Blue is convenient
- Laser power: 4% shows in our case good signal and low to no bleaching
- Illumination mode: EPI
- Exposure Time: if the drift is stable this value can be large. We use 100-400 ms
   
SECTION: Analysis of PSF data - Widefield
29. Open the PSF data (z-stack) 
ZEN black -> File -> Open

 

SECTION: Analysis of PSF data - Widefield
30. Go to the Processing tab
  
SECTION: Analysis of PSF data - Widefield
31. Method -> Experimental PSF
  
SECTION: Analysis of PSF data - Widefield
32. Method Parameters -> Select button
  
SECTION: Analysis of PSF data - Widefield
33. Method Paramters -> Change to Adjust ROIs
  
SECTION: Analysis of PSF data - Widefield
34. Parameters: there is no set of parameters that will fit all your experiments. Here we provide some information regarding the parameters. Please consider the numbers as a suggestion, or starting point, not as a fixed value that must be use for all cases.
   
SECTION: Analysis of PSF data - Widefield
34.1. Feature Size: 30
 
Advice: Select a feature size as large as posible. The limitting factor will be the overlap between the beads in the image. We usually work with about 30 pixels for TL 1,6 
SECTION: Analysis of PSF data - Widefield
34.2. Noise Level: 50

 
Advice: It is a good idea to be stringent with this parameter (to use large values). The lower the value the more "initial" detections are considered. If the value is two low even noise spikes are considered as detected spots. We usually work with about 50.
SECTION: Analysis of PSF data - Widefield
34.3. Beads Quality: 2 

 
Advice: We use small numbers (usually 1-3), however if the data set is clean we do not see much effect on this feature.
SECTION: Analysis of PSF data - Widefield
34.5. Display: 

 

SECTION: Analysis of PSF data - Widefield
34.4. Z-range: Selects the number of slides from the z-stack that are used for spot detection. 
Advice: Generally we set this to +-5 from the centre of the z-stack.
SECTION: Analysis of PSF data - Widefield
35. How to overcome hot pixels in the PSF raw data (slat pepper noise)
  
 



SECTION: Analysis of PSF data - Widefield
35.1. Processing tab -> Method -> Filter
  

SECTION: Analysis of PSF data - Widefield
35.2. - Select Input Image (select button)
- Use filter Type Median
- Kernel size:
    Kernel size X: 3
    Kernel size Y: 3
    Kernel size Z: 1
  

SECTION: Analysis of PSF data - Widefield
35.3. Click on Apply
  
This will create a new image document with the filtered result

 


SECTION: Analysis of PSF data - Widefield
35.4. Go to Method -> Experimental PSF
- Select Filtered image via select button
- Adjust ROIs option
- Set appropriate parameters  for advice
  

SECTION: Analysis of PSF data - Widefield
35.5. Go to Image Viewer -> Graphics -> Save button
and save the ROIs
  

SECTION: Analysis of PSF data - Widefield
35.6. Go back to the PSF raw data image window
Go to Method -> Experimental PSF
- Select the raw data via the Select button
- Select manual ROIs
  
- Load saved selections via Image Viewer -> Graphics -> Load button
  
- Click on Apply
  

SECTION: Analysis of PSF data - Widefield
34.7. To apply the method and get the PSF click on apply:
  
SECTION: Analysis of PSF data - Widefield
36. Inspecting PSF output:
- Orthogonal view with gamma correction can help you to find asymmetries in the PSF
- Quantitative results (PSF FWHM) can be found in the output's image info tab

 


SECTION: Analysis of PSF data - Widefield
34.6. PSF sampling: usually 1 or 3

 

SECTION: Analysis of PSF data for WF
29. Open the PSF data (z-stack) 
ZEN black -> File -> Open

 

SECTION: Analysis of PSF data for WF
30. Go to the Processing tab
  
SECTION: Analysis of PSF data for WF
31. Method -> Experimental PSF
  
SECTION: Analysis of PSF data for WF
32. Method Parameters -> Select button
  
SECTION: Analysis of PSF data for WF
33. Method Paramters -> Change to Adjust ROIs
  
SECTION: Analysis of PSF data for WF
34. Parameters: there is no set of parameters that will fit all your experiments. Here we provide some information regarding the parameters. Please consider the numbers as a suggestion, or starting point, not as a fixed value that must be use for all cases.
   
SECTION: Analysis of PSF data for WF
34.1. Feature Size: 30
 
Advice: Select a feature size as large as posible. The limitting factor will be the overlap between the beads in the image. We usually work with about 30 pixels for TL 1,6 
SECTION: Analysis of PSF data for WF
34.2. Noise Level: 50

 
Advice: It is a good idea to be stringent with this parameter (to use large values). The lower the value the more "initial" detections are considered. If the value is two low even noise spikes are considered as detected spots. We usually work with about 50.
SECTION: Analysis of PSF data for WF
34.3. Beads Quality: 2 

 
Advice: We use small numbers (usually 1-3), however if the data set is clean we do not see much effect on this feature.
SECTION: Analysis of PSF data for WF
34.4. Z-range: Selects the number of slides from the z-stack that are used for spot detection. 
Advice: Generally we set this to +-5 from the centre of the z-stack.
SECTION: Analysis of PSF data for WF
34.5. Display: 

 

SECTION: Analysis of PSF data for WF
34.6. PSF sampling: usually 1 or 3

 

SECTION: Analysis of PSF data for WF
34.7. To apply the method and get the PSF click on apply:
  
SECTION: Analysis of PSF data for WF
35. How to overcome hot pixels in the PSF raw data (slat pepper noise)
  
 



SECTION: Analysis of PSF data for WF
35.1. Processing tab -> Method -> Filter
  

SECTION: Analysis of PSF data for WF
35.2. - Select Input Image (select button)
- Use filter Type Median
- Kernel size:
    Kernel size X: 3
    Kernel size Y: 3
    Kernel size Z: 1
  

SECTION: Analysis of PSF data for WF
35.3. Click on Apply
  
This will create a new image document with the filtered result

 


SECTION: Analysis of PSF data for WF
35.4. Go to Method -> Experimental PSF
- Select Filtered image via select button
- Adjust ROIs option
- Set appropriate parameters  for advice
  

SECTION: Analysis of PSF data for WF
35.5. Go to Image Viewer -> Graphics -> Save button
and save the ROIs
  

SECTION: Analysis of PSF data for WF
35.6. Go back to the PSF raw data image window
Go to Method -> Experimental PSF
- Select the raw data via the Select button
- Select manual ROIs
  
- Load saved selections via Image Viewer -> Graphics -> Load button
  
- Click on Apply
  

SECTION: Analysis of PSF data for WF
36. Inspecting PSF output:
- Orthogonal view with gamma correction can help you to find asymmetries in the PSF
- Quantitative results (PSF FWHM) can be found in the output's image info tab

 


SECTION: Analysis of PSF data for Lattice SIM
29. Open the PSF data (z-stack) 
ZEN black -> File -> Open

 

SECTION: Analysis of PSF data for Lattice SIM
31. Go to the Processing tab
  
SECTION: Analysis of PSF data for Lattice SIM
32. Method -> Experimental PSF
  
SECTION: Analysis of PSF data for Lattice SIM
33. Method Parameters -> Select button
  
SECTION: Analysis of PSF data for Lattice SIM
34. Method Paramters -> Change to Adjust ROIs
  
SECTION: Analysis of PSF data for Lattice SIM
35. Parameters: there is no set of parameters that will fit all your experiments. Here we provide some information regarding the parameters. Please consider the numbers as a suggestion, or starting point, not as a fixed value that must be use for all cases.
   
SECTION: Analysis of PSF data for Lattice SIM
35.1. Feature Size: 30
 
Advice: Select a feature size as large as posible. The limitting factor will be the overlap between the beads in the image. We usually work with about 30 pixels for TL 1,6 
SECTION: Analysis of PSF data for Lattice SIM
35.2. Noise Level: 50

 
Advice: It is a good idea to be stringent with this parameter (to use large values). The lower the value the more "initial" detections are considered. If the value is two low even noise spikes are considered as detected spots. We usually work with about 50.
SECTION: Analysis of PSF data for Lattice SIM
35.3. Beads Quality: 2 

 
Advice: We use small numbers (usually 1-3), however if the data set is clean we do not see much effect on this feature.
SECTION: Analysis of PSF data for Lattice SIM
35.4. Z-range: Selects the number of slides from the z-stack that are used for spot detection. 
Advice: Generally we set this to +-5 from the centre of the z-stack.
SECTION: Analysis of PSF data for Lattice SIM
35.5. Display: 

 

SECTION: Analysis of PSF data for Lattice SIM
35.6. PSF sampling: usually 1 or 3

 

SECTION: Analysis of PSF data for Lattice SIM
35.7. To apply the method and get the PSF click on apply:
  
SECTION: Analysis of PSF data for Lattice SIM
36. How to overcome hot pixels in the PSF raw data (slat pepper noise)
  
 



SECTION: Analysis of PSF data for Lattice SIM
36.1. Processing tab -> Method -> Filter
  

SECTION: Analysis of PSF data for Lattice SIM
36.2. - Select Input Image (select button)
- Use filter Type Median
- Kernel size:
    Kernel size X: 3
    Kernel size Y: 3
    Kernel size Z: 1
  

SECTION: Analysis of PSF data for Lattice SIM
36.3. Click on Apply
  
This will create a new image document with the filtered result

 


SECTION: Analysis of PSF data for Lattice SIM
36.4. Go to Method -> Experimental PSF
- Select Filtered image via select button
- Adjust ROIs option
- Set appropriate parameters  for advice
  

SECTION: Analysis of PSF data for Lattice SIM
36.5. Go to Image Viewer -> Graphics -> Save button
and save the ROIs
  

SECTION: Analysis of PSF data for Lattice SIM
36.6. Go back to the PSF raw data image window
Go to Method -> Experimental PSF
- Select the raw data via the Select button
- Select manual ROIs
  
- Load saved selections via Image Viewer -> Graphics -> Load button
  
- Click on Apply
  

SECTION: Analysis of PSF data for Lattice SIM
37. Inspecting PSF output:
- Orthogonal view with gamma correction can help you to find asymmetries in the PSF
- Quantitative results (PSF FWHM) can be found in the output's image info tab

 


SECTION: Analysis of PSF data for Lattice SIM
30. Average the data over the phases

 



SECTION: Analysis of PSF data for Lattice SIM
30.1. Go to the Processing tab
  

SECTION: Analysis of PSF data for Lattice SIM
30.2. Method -> Average
  

SECTION: Analysis of PSF data for Lattice SIM
30.3. Method Parameters:
- Select image
- Select all phases (in our case 13)
  
SECTION: Analysis of PSF data for Lattice SIM
30.4. Click on apply:
  

SECTION: Analysis of PSF data for Lattice SIM
28. Read all steps of this section before you try to implement them
SECTION: Analysis of PSF data for WF
28. Read all steps of this section before you try to implement them
SECTION: Analysis of PSF data - Widefield
28. Read all steps of this section before you try to implement them