.....................................1 1 Project Folders: Organization of Experiment Files.................................1 2 Steps........................................................................2 2.1 Preview................................................................3 2.2 GSD....................................................................4 2.3 GSD Tools..............................................................8 2.3.1 t-series Analysis.....................................................8 2.3.2 Eventlist Analysis.....................................................9 2.3.3 Eventlist Processing.................................................11 2.3.3.1 Compensate the Image Shift.......................................11 2.3.3.2 Generating a Super-resolution Ground State Depletion Image..........14 3 Creating 2-color SR GSD Images...............................................15 4 Exporting Data...............................................................15
The goal of this wizard is to determine the precise location of molecules using GSD technology. 1 Project Folders: Organization of Experiment Files All images and image series that you acquire and, if applicable, edit are collected into experiments and displayed in a directory structure. Figure 1: Project folder File name Job 1_XXX (xy) SeriesXXX (xyt) SeriesXXX_el_X SeriesXXX_el_X_2D_X (xy) SeriesXXX_TruncatedXXX (xyt) SeriesXXX_TruncatedXXX_el_X SeriesXXX_TruncatedXXX_el_X_2D_X (xy) SeriesXXX_el_X_Filtered_X SeriesXXX_el_X_Filtered_X_2D_X (xy) SeriesXXX_el_X_corrected_X SeriesXXX_el_X_corrected_X_2D_X (xy) Contents Preview images Time series generated during the experiment (GSD raw image) Unfiltered event list; contains the xycoordinates and intensity values from the time series (GSD raw image) High-resolution image from the time series Range cut out from the time series Event list from the range cut out from the time series High-resolution image based on the range cut out from the time series Filtered event list High-resolution image based on the filtered event list Event list after correcting the image shift High-resolution image after correcting the image shift 1
2 Steps The wizard consists of five steps: Preview Acquire preview images as reference images GSD Excite the specimen using high light intensity in order to shift fluorescent molecules into the dark state Record the GSD raw image GSD Tools Process the raw data once again (t-series analysis) Locate molecules, save the results and filter the event list (Eventlist Analysis) Compensate for the image shift, display the high-resolution GSD image (eventlist processing) Process Tools for editing acquired images and image series Quantify Tools for quantitative evaluation of acquired images and image series 2
2.1 Preview Preview images are acquired at conventional resolution and low excitation intensity in this step in order to be able to make comparisons to the high-resolution GSD image later on. Depending on the specimen and requirements, the images can be acquired in epifluorescence mode or in TIRF mode. Follow these steps: Figure 2: Acquiring preview images 1. Adjust the instrument parameters. 2. Click on Live to view the specimen in live mode. 3. Bring the area of the specimen to be examined into the predefined GSD area. This has a size of 180 x 180 pixels. 4. If you would like to acquire an image of the entire specimen, click on Capture Image. 5. If you want to acquire an image of the GSD area, click on Capture ROI. 6. Acquire as many images as you need. The images are saved under Experiments with a Job 1_xxx naming model. 7. If necessary, save your settings for future experiments. 8. Change to the GSD operating step. The image marked under Experiments is transferred into the GSD operating step. 3
2.2 GSD In this step, the specimen is excited using high light intensity (pumping mode) and a GSD raw image is acquired in the form of a time series (acquisition mode). The parameters for pumping mode and for image acquisition have to be configured for this. First, adjust the parameters for pumping mode as follows: Figure 3: Adjusting the pumping mode 1. Click on Pump. 2. Adjust the instrument parameters. 3. Adjust the options for pumping mode under Advanced: Parameter Use Pump Settings for Acquisition Automatically switch to Acquire Table 1: Setting pumping mode options in the GSD step Description The settings for pumping mode are also used for image acquisition Automatically switch to acquisition mode when the value falls below that specified in the correlation parameter under Particle Detection Settings > Frame Correlation 4
4. Under Particle Detection Settings, configure the detection parameters: Parameter Detection Threshold Auto event control Events per image Single molecule detection while pumping Frame correlation Events per frame Pixel Size in Image Table 2: Description Setting detection parameters in the GSD step The photon count per pixel is specified here. Each signal that has a photon count lower than the value specified is treated as noise. Controls event density automatically using backpumping light Number of events per image Mark identified events in the image during pumping Correlation parameter averaged over at least 10 frame pairs Averaged number of events per frame Effective pixel size for the GSD image 5. The camera parameters can be changed depending on your specimen. 6. Set the intensity of the GSD laser to the desired value. Now set the parameters for image acquisition: Figure 4: Setting the acquisition mode 7. Click on Acquire. 5
8. Adjust the instrument parameters. 9. If necessary, save your settings for future experiments. 10. Click on Start Pumping to start pumping mode. The specimen is excited with high laser intensity until the fluorophores are almost completely inactive. In pumping mode, the live image is displayed and the laser intensity can be adjusted during pumping. Fluorescence intensity drops as a result. While general fluorescence intensity decreases, individual molecules spontaneously return to a fluorescent state and give off light until they drop back to the inactive state. This happens simultaneously for many molecules, giving the impression that the image is "flashing". If the option under Acquisition > Particle Detection Settings > Single Molecule Detection while Pumping is set to on, online locating is already taking place and the identified molecules are marked with small circles. Figure 5: Online localization This way you can use the raw data view to determine whether the coordinates determined by the algorithm using the configured parameters makes sense. The parameters, particularly the detection threshold, can be adjusted if necessary. After pumping, most molecules are in a dark state and probability is low that individual molecule events physically overlap. Now the actual acquisition process can begin. 11. Click on Start Acquisition to start acquisition using the configured parameters. The raw images are acquired with continuous excitation below the GSD threshold and saved to the hard disk drive. The extracted position data is already used to generate a GSD raw image during acquisition. 12. You can influence the number of detected events by changing the intensity of the excitation laser or the detection threshold during acquisition. You can also switch on the backpumping laser (405 nm). This reduces dark state of the molecules by shifting the 6
molecules back into the ground state. This makes them available for the fluorescence process again more quickly. Note: Depending on your specimen and the results you would like to achieve, you have to decide when you want to stop excitation with the high light intensity by clicking on Stop Pumping. 13. Once you have extracted enough data, click on Stop Acquisition. Acquisition is ended. The generated GSD raw image is saved under Experiments together with the associated event list: SeriesXXX (xyt) and SeriesXXX_el_X. 7
2.3 GSD Tools The events of each individual molecule are located in all of the raw images and saved in an event list together with all information such as position, brightness, image number, etc. The coordinates of each fluorophore are determined using an algorithm. Evaluation options are available to you in this step. It is divided into three tabs: t-series Analysis Eventlist Analysis Eventlist Processing 2.3.1 t-series Analysis Here you can edit the raw data once again. Follow these steps: Figure 6: Processing raw data 1. Under Experiments, click on the raw data ( SeriesXXX (xyt)) you would like to evaluate once again. 2. You can also limit the evaluation to one or more areas. You can add or delete evaluation ranges using the +/- buttons. 3. You can determine the size of the evaluation range using the sliders in Range Selection or direct input in the Range from - to fields. 4. Click Truncate. The limited evaluation range is stored in a separate file under Experiments: SeriesXXX_TruncatedXXX (xyt). 5. If applicable, change the detection threshold under Detection Threshold. 6. Click Evaluate to configure a new evaluation. Cancel all Jobs aborts the evaluation. 7. A new evaluation list appears under Experiments due to the configured settings. SeriesXXX_TruncatedXXX_el_X. 8
2.3.2 Eventlist Analysis In this tab, the detected events are collected and shown in graphics. There is also an option for filtering the events in the event list, such as limiting them to a specific range or merging or excluding specific events. Particle Intensity Histogram shows the photon statistics of the events from the selected event list in the form of a histogram. The scaling for the histogram can be changed using the context menu (right mouse button) using the Scaling option. Event/Frame-Time Diagram shows the number of events per raw data image averaged over 200 raw data images. Follow these steps: Figure 7: Filtering Event Lists 1. Under Experiments, click on the event list you would like displayed. 2. Select a range in one or both histograms as needed. To do so, right-click on the histogram surface and select the option Range Selection > Add. You can move the range limits using the mouse, as well as define multiple ranges. 3. The following values are shown under Settings: Parameter Total No. of events Avg. Photon Count No. of Events in Range Avg. Photon Count BinSize Table 3: Displayed events Description Total number of events Average photon count Number of events in the defined range Average photon count in the defined range Step size of the depicted histogram (can be changed) 9
4. Configure the filter settings undereventlist Filter Settings : Parameter Merge consecutive events Discard consecutive events Max. Distance from first events Max. number of appearance Max. number of dark events Table 4: Filter settings Description Merge events that are detected in consecutive raw data images within a radius (Max. Distance from first event). The merged events are viewed as one molecule and are only listed once in the event list where they occurred for the first time. The photon count is the sum of all photons from all frames. The position is a weighting from all positions in all frames. Events that occur more often than specified in Merge consecutive events are ignored. Maximum detection radius for merging events Maximum number of consecutive frames for merging events Maximum number of undetected particles in consecutive events that take place in the range specified under Max. number of appearance 5. Click on Start Filtering. Filtering is configured using all programmed filter parameters. Only those events that meet all selected filter parameters are filtered out. The filtered events are saved under Experiments in a separate event list: SeriesXXX_el_X_Filtered_X. After you have filtered the event list according to your needs and do not wish to conduct any further corrections/filterings, you can then display the high-resolution GSD image (see Chapter "Generating a Super-resolution Ground State Depletion Image", page 14) 10
2.3.3 Eventlist Processing In this tab you can compensate for any image shift which may have occurred during acquisition, generate the high-resolution GSD image and combine event lists as needed. 2.3.3.1 Compensate the Image Shift In order to generate a high-resolution complete image that mirrors the fluorescent molecule distribution of the marked structures, several thousands of individual raw data images must be captured. If recording times are long and there is a high number of images, it can occur that the specimen moves relative to the imaging system. The movement can be triggered by multiple factors: mechanical movement of the system, thermal effects, relief of mechanical tension or influence from other forces. Movement of any kind has a negative impact on the localization of the fluorescent molecules in the complete high-resolution image. The Drift Compensation function serves to determine and compensate for the image shift. The wizard for compensation of the image shift guides you through three steps: Prepare Select ROI: Adjust/Finish In this step, the event list to be corrected is selected and the pixel size is set for the image in which the correction is to be carried out. In this step, a rectangular region of interest (ROI) is drawn around a reference point (bead). This reference point is for determining the image shift. In this step, the correction of the image shift is optimized and then carried out. 11
To determine and correct the image shift, follow these steps: Figure 8: Compensating for the Image Shift 1. Under Experiments, click on the event list to be used as the basis for compensating for the image shift. 2. In the Drift Compensation field you can see the selected event list and the number of detected events. 3. Click on Start in the Drift Compensation field. The high-resolution image created on the basis of the event list is displayed in the first half of the display window. This has not yet been corrected with regard to image shift. The wizard jumps to the step Select ROI: Figure 9: Drawing an ROI 4. Draw a rectangular region of interest (ROI) into the still uncorrected image. In doing so, mark the bead introduced during preparation of the specimen and as little other signal as possible. 12
After drawing the region of interest (ROI), the wizard moves to the last step (Adjust/Finish). Figure 10: Correcting the Image Shift In order to carry out the compensation of the image shift, the raw images are collected into blocks. You can influence the result of the compensation by changing the defined number of frames per block or move the region of interest (ROI) in the image. 5. In the No. of Frames/Block field, specify the number of frames per block. The software provides a default value using the programmed parameters, the exposure time and the number of raw images. However, this default value can be adjusted. Each block has to contain exactly as many frames as are needed for it to contain sufficient information about measuring the image shift between two blocks. 6. After making each change, click Perform Correction. The image shift of all blocks is measured and corrected. The correction is made by shifting the determined focal point positions of the detected single molecules. The images created by Perform Correction are temporary, in other words the created image is overwritten upon clicking Perform Correction again with a new corrected image. 7. Repeat this correction step until you are satisfied with the result. 8. When you are satisfied with the result, click Apply and Finish. The corrected event list and the high-resolution image compensated for with regard to image shift appear under Experiments with the nomenclature SeriesXXX_el_X_corrected_X or SeriesXXX_el_X_corrected_X_2D_X. 13
2.3.3.2 Generating a Super-resolution Ground State Depletion Image The event list corrected and, where appropriate, filtered with regard to image shift is taken as the basis for graphically representing the information collected in all the raw images. There are two display methods: Histogram display: The image area is divided into a 2D pixel array with pixels of a specific size, e.g. 15nm at approximately 30nm estimated average resolution (i.e. into something about half the size of the resolution, based on the Nyquist-Shannon criterion from signal processing). The listed events are sorted according to their position in the corresponding pixel. This means if an event in the corresponding pixel is missing, its value is increased by one. The pixel array can then be color-coded as an image, thereby providing an image of the dye distribution. This is a 2D histogram and is the simpler and more efficient method. Gaussian display: The events are not sorted into relatively large pixels, but rather a Gaussian intensity distribution is drawn at the detected position of the dye. The distribution's full width at half maximum corresponds to the location uncertainty. The sum across the Gaussian distribution is normalized to 1. The location uncertainties are to be accommodated according to event brightness this way. Follow these steps: 1. Under Experiments, click on the event list to be used as the basis for creating the SR GSD image. Note: You can also merge multiple event lists under Eventlist Processing>Eventlist Merging. To do so, you have to mark the corresponding lists under Experiments while holding down the Ctrl key and then click on Merge Eventlists. This can be helpful when you are carrying out longer measurements consisting of multiple stacks of raw images. 2. Select the display method for the SR GSD image. 14
3. Under Pixel Size in Image, enter the pixel size for the SR GSD image. 4. Total Number of Events provides the total number of events displayed in the SR GSD image. 5. Click on Create Image. The high-resolution image is built up and saved under Experiments. The acquisition of high-resolution images can take between 2 and 5 minutes. The SR GSD image is composed online so that you have the option of stopping the experiment as soon as you have enough data. 3 Creating 2-color SR GSD Images You also have the option of acquiring 2-color SR GSD images. Both colors are acquired sequentially for this. This results in two separate stacks of data for the same specimen. The extracted high-resolution images for each data stack can be merged using the Merge function from the Process step of LAS AF. The following combinations have good results: AlexaFluor TM 532 and AlexaFluor TM 647 AlexaFluor TM 488 and AlexaFluor TM 647 The fluorophore with the longer wavelength should be acquired first to prevent photobleaching and crosstalk of the other fluorophore. 4 Exporting Data The acquired images and stored event lists can be exported for advanced processing. 1. Under Experiments, click on the data you would like to export. 2. Open the context menu using the right mouse button. Select the Export option. Different options are available depending on which data you would like to export. 15
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