Feb 14, 2023
PSF measurements for ELYRA 7
This protocol is a draft, published without a DOI.
- 1Centre for Cellular Imaging, Core Facilities, Gothenburg University
Protocol Citation: Rafael Camacho 2023. PSF measurements for ELYRA 7. protocols.io https://protocols.io/view/psf-measurements-for-elyra-7-cm8su9we
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 24, 2023
Last Modified: February 14, 2023
Protocol Integer ID: 75762
Keywords: PSF, ELYRA 7
Funders Acknowledgement:
National Microscopy Infrastructure of Sweden
Grant ID: NMI (VR-RFI 2019-00217)
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Abstract
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.
Equipment
ELYRA 7
NAME
Microscope
TYPE
ELYRA
BRAND
431014-9903-000
SKU
Software
ZEN black
NAME
ZEISS
DEVELOPER
Before start
Make sure to read all steps of each section before you try to implement them.
Warming up the microscope
Warming up the microscope
Turn on the ELYRA 7
Turn on Main Switch
Turn on Components
Turn on Microscope Computer
Note
In the ELYRA 7 if the microscope computer is not on, then the microscope's touch screen will not light up.
Open ZEN black, and choose start system
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.
Temperature control for increased stability.
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.
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]
Align the stage
Align the stage
The stage must be perpendicular to the microscope to avoid imaging artifacts.
Please follow the protocol:
Protocol
NAME
Insert Alignment ELYRA7CREATED BY
Rafael Camacho
Find focus
Find focus
Engage the objective you want to use. In our case Plan-Apochromat 63X / 1.4 Oil
If necessary put a small drop of immersion fluid on the objective
Place the sample on the holder
Manually move the objective up until it touches the immersion fluid
You can approximate focus by using the Definite Focus
Zen black -> Acquisition Tab
Acquisition Parameter -> Definite Focus Menu -> Find Surface
Set imaging parameters, here we use Laser WF mode. Below an example for the 63X objective:
Zen black -> Acquisition Tab
Select the Laser WF mode by
Setup Manager -> Imaging Setup ->Laser WF
In following sub-steps you will find detailed information about our experiment configuration. We recommend that you store these settings via the experiment manager. In our case we stored this as "488PSF"
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
Channel Settings
Acquisition Parameter -> Channels:
- 488 nm excitation, 4%
- Illumination mdoe: EPI
- Exposure time 100-300 ms
Go to live mode and fine adjust for focus (for reduced field of view, use Continious instead)
Live button
Live active
Expected result
Drift stability test
Drift stability test
Note
PSF measurements take time. Thus we want to make sure that there is no sample drift before starting. Drift will induce elongations of the output PSF.
Reduce your field of view to about 30 micron2 This can be done by cropping the camera chip
Acquisition Parameter -> Acquisition Mode:
- Frame size e.g. 512 x 512
Make sure images are 16 bits for better performance during analysis
Generate a Time Series experiment, with a length of 20 minutes and 0.1 fps
Zen black -> Acquisition Tab
Activate Time series in Experiment manager
Multidimensional Aquisition -> Time Series
- Cycles 120
- Interval 10.0 s
Find focus: go to step #5 if you need help
Start Experiment via Experiment Manager -> Start experiment
Review the stability of the drift by looking at your time series. The beads image should not move or get out of focus.
Note
It can take up to 20 minutes for a sample to stabilize.
Expected result
Sample should not drift within the time frame of your experiments (usually a few minutes).
Bleaching test
Bleaching test
Note
During PSF measurements we acquire a Z-stack. Therefore, the sample should not significantly bleach during a time series experiment of the same length as your Z-stack. If not your PSF shape will be deformed due to bleaching.
Reduce your field of view to about 30 micron2 This can be done by cropping the camera chip.
go to step #12.1 for details
Check the size of your desired z-stack. Example for 63x 1.4 oil objective:
Zen black -> Acquisition Tab
Experiment Manager -> Z-stack
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]
Take note of the number of Slices, in our example 70, and deactivate the z-stack option via Experiment Manager -> Z-stack
Acquire a time series with a number of frames equal to the number of slices determined in go to step #16.4 and no time interval (continious imaging)
For a time series example go to step #13.1
Example:
Review the bleaching of the beads. Below an example using Mean ROI tool in ZEN black:
ZEN Image Viewer -> Mean ROI
Draw a rectangular ROI around some beads and background area
Drawing tools
Example of selection
Check that you have no significant bleaching of the beads over the time frame of your experiment
Expected result
Here an example:
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
Laser WF 1.0 TL32 steps
Setup Manager -> Laser
Activate the 488 laser
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
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
Setup the Z-stack
Experiment Manager -> Z-stack
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]
Go to Continuous mode and adjust focus
Multidimensional Acquisition -> Z-stack -> Center mode -> click on center
Start Experiment via Experiment Manager -> Start experiment
Analysis of PSF data - Widefield
Analysis of PSF data - Widefield
Read all steps of this section before you try to implement them
Open the PSF data (z-stack)
ZEN black -> File -> Open
Expected result
Go to the Processing tab
Method -> Experimental PSF
Method Parameters -> Select button
Method Paramters -> Change to Adjust ROIs
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.
Feature Size: 30
Note
From ZEN help: "sets the mask size that is used to cut out the bead and to create the Experimental PSF. The extensions are displayed in the Preview tab of the image container as circles."
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
Noise Level: 50
Note
From ZEN help: "sets the threshold of the intensity of the sub-resolution beads over background according to I – M > SD x NL (I = intensity of bead; M = mean intensity of image frame; SD = standard deviation of image frame; NL = noise level). Lowest number means that everything (even noise) is taken into account; highest number means only the brightest beads will be used."
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.
Beads Quality: 2
Note
From ZEN help: "evaluates two characteristics, the intensity (first moment) and the size (second moment). These two moments are calculated for every localized bead. Then the beads are sorted according to these characteristics, resulting in two sorted lists of beads. The beads that are closer to the medians in both orderings are assumed to have higher quality. The Beads quality defines how far the beads characteristics (intensity and size) may be away from the median value. The smaller the threshold, the closer beads should be to the median."
Advice: We use small numbers (usually 1-3), however if the data set is clean we do not see much effect on this feature.
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.
Display:
Note
From ZEN help: "All sub-resolution beads that will be taken into account will be circled by a white line. Those that do overlap with their circles or are considered unsuitable (beads beyond the quality thresholds) are outlined in red and will not be taken into account."
PSF sampling: usually 1 or 3
Note
From ZEN help: "The PSF sampling pull down menu allows you to interpolate the result to different degrees. Since the resulting PSF is an average of many PSFs corresponding to the localized beads and every bead is localized with the sub-pixel accuracy, one can obtain after averaging a PSF with higher resolution"
To apply the method and get the PSF click on apply:
How to overcome hot pixels in the PSF raw data (slat pepper noise)
Note
Strategy: Removing hot pixels via median filter. Then use the filtered image to detect the spots of interest and save these selections. Finally load the selections manually for analysing the raw data to avoid the loss of resolution due to the filtering.
Processing tab -> Method -> Filter
- Select Input Image (select button)
- Use filter Type Median
- Kernel size:
Kernel size X: 3
Kernel size Y: 3
Kernel size Z: 1
Click on Apply
This will create a new image document with the filtered result
Expected result
Go to Method -> Experimental PSF
- Select Filtered image via select button
- Adjust ROIs option
- Set appropriate parametersgo to step #34 for advice
Go to Image Viewer -> Graphics -> Save button
and save the ROIs
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
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
Expected result
Orthogonal views
PSF FWHM information