N-SIM guide NIKON IMAGING CENTRE @ KING S COLLEGE LONDON
Starting-up / Shut-down The NSIM hardware is calibrated after system warm-up occurs. It is recommended that you turn-on the system for at least 30 minutes prior to use for optimal performance. A digital thermometer attached to the microscope stand can be used to gauge when the system is ready the temperature of the microscope will rise by approximately 2 o C when all the system components and PC are switched on. To switch on the system: 1. Switch on the laser bed (see figures 2-4) and select either multi or single to choose the appropriate optical fibre depending on whether you are doing 2D/3D (multi) or TIRF- SIM (single). 2. Switched on the XY stage, transmitted light and 561nm laser (by turning the key) as shown in figure 5. 3. Switch on the remaining lasers (by turning the keys) and Intensilight unit as shown in figure 6. 4. Switch on the PC; doing so switches on the SIM illuminator and EMCCD camera. 5. The microscope stand (Ti) should already by powered on as should be the piezo z drive (see figure 5). Figure 1 To shut-down the system: Power 1. Close NIS Elements. 2. Shut-down the PC. 3. Turn-off the lasers and lamps. Figure 2 Figure 3 1 @
561nm laser XY-stage Transmitted light 405, 488, 640nm lasers Fluorescence lamp Figure 3 Figure 4 Figure 5 Figure 6 2 @
A note about user rights This NSIM system is driven through the use of optical configurations to enable new users to get up and running quickly and to make day-to-day use of the system straight-forward. In order to prevent modifications to the base set of configurations each new user will be given common privileges in NIS-Elements. These privileges are restricted so that shared optical configurations cannot be modified. However, common users can still make new configurations and mark them as private so that other users cannot change the configuration. To enable laser power and camera settings to be saved into the configuration (required when using ND Acquisition) it is also necessary to make a change to the General settings for each user. This change is made as follows: 1. Click the cog icon or go to Edit Options. 2. In General change Optical Configuration to save all camera settings to optical configuration automatically. Each time you change the laser power and camera settings these values will automatically get saved to the optical configuration. Note these settings are not applied globally they are saved only in your instance of the optical configuration. 3 @
Viewing your sample through the eye-pieces Since the N-SIM system is essentially a wide-field microscope we choose to drive the system using the Optical configuration panel ( OC panel ). The OC panel is used to hold shortcuts to microscope configurations for performing a specific task. The configurations are grouped on the panel according to mode of operation they control. To view your sample through the eye-pieces use the Eyes group of configurations. These configurations: 1. Sets the Light Path to E100 (eyes) 2. Sets turret 2 to the empty position. 3. Sets turret1 to the appropriate filter set. 4. Use the Intensilight remote control (see figure 6) to open the lamp shutter. 5. Also not that the laser interlock is engaged when the light path is set to E100 which prevents control of the lasers on the N-SIM pad. Switching to an optical configuration which uses the camera (R100) releases the interlock. Figure 7 Figure 8 4 @
Image acquisition SIM acquisitions are possible with both the 60x water and 100x oil immersion objectives. The choice between the two is driven by how thick or how deep into your sample you need to image. A depth of approximately 20µm and 7µm is possible with the 60x and 100x objectives respectively. If you plan to use the TIRF- SIM mode you will need the 100x objective. The optimal resolution is achieved with thin samples (low out-of-focus light) and the 100x objective. The microscope stand (Ti-E) includes two filter turrets (see the Ti Pad); turret 2 is used when performing SIM acquisitions while an empty position (no filter cube) is selected in turret 1. The filter blocks in turret 2 are specially designed and factory aligned filter sets to minimised chromatic misalignment for multi-channel images. The example shown here is the Ti Pad configuration for a 488nm SIM acquisition. SIM acquisitions are performed using the N-SIM pad. This is a plugin for NIS- Elements that drives both acquisitions and image reconstruction. Optical configurations have been created for each channel of each type of acquisition. 3D-SIM, single channel, single z-plane 1. Select the appropriate optical configuration (3D-SIM 405, 3D-SIM 488, 3D-SIM 561, 3D-SIM 640) depending on the channel you want to acquire. 2. Make sure the appropriate grating is selected in the N-SIM pad (100 EX V-R for the 100x objective lens). 3. Make sure to select the correct fibre input Multi for multi-mode fibre. 4. Make sure the correct fibre is selected on the N-SIM illuminator. 5. Set a laser power (for the appropriate laser wavelength) and camera exposure time (on the DU-897 Settings ) to obtain an appropriate signal level. This will be ~4000 ADU with the camera in 14-bit mode and ~12,000 ADU with the camera in 16-bit mode (recommended see DU-897 Settings ). It is important for final image quality that you do not exceed these values. 6. Make sure to select 3D-SIM from the drop-down menu below the Capture button. 7. Press the Capture button to acquire a single z-plane. 3D-SIM ND Acquisition ND acquisition is used to acquire multi-dimensional data, for example a z-stack in multiple channels. Select the appropriate tab(s) to add dimensions to the dataset. Here we will describe how to acquire a z-stack in two channels. Figure 9 Figure 10 Figure 11 5 @
1. Check the λ (lambda) tab to add optical configurations to the acquisition. Simply click the checkbox on the first line and then select the optical configuration from the dropdown menu. Note that acquisition is sequential. 2. To add another channel, check the box on the next line and select a configuration from the dropdown menu. Here we have selected 3D-SIM 488 and 3D-SIM 561. 3. If you require more than one z-slice, check the Z tab and choose one of three acquisition modes: a. Top and bottom b. Symmetric (selected in the figure) c. Asymmetric 4. Choose a step size of at least 120nm (or multiples thereof) ignoring the suggested step size of 200nm and set the range or number of steps. 5. Make sure the appropriate grating is selected in the N-SIM pad (100 EX V-R for the 100x objective lens). 6. Make sure to select the correct fibre input Multi for multi-mode fibre. 7. Make sure the correct fibre is selected on the N-SIM illuminator. 8. Set a laser power (for the appropriate laser wavelength) and camera exposure time (on the DU-897 Settings ) to obtain an appropriate signal level in each channel to be recorded. This will be ~4000 ADU with the camera in 14-bit mode and ~12,000 ADU with the camera in 16-bit mode (recommended see DU-897 Settings ). It is important for final image quality that you do not exceed these values. 9. Make sure to select 3D-SIM from the drop-down menu below the Capture button. 10. Press Run now. Figure 12 Figure 13 6 @
3D-SIM image reconstruction During 3D-SIM acquisition a total of fifteen images are captured, 5 images with varying grating phase at 3 different grating rotations (separated by 120 o ). This of course limits the rate at which frames can be recorded which is an important consideration for live cell imaging. For 3D-SIM 1 frame per second is possible while for 2D- and TIRF-SIM a slightly faster rate of around 1.2 frames per second is possible since a total of 9 images are recorded in these modes. Figure 14 Image reconstruction works best when the recommended number of grey levels are collected and when good grating contrast is achieved. The final can however be improved by adjusting the image reconstruction parameters. These are accessed on the N-SIM pad; choose between Preview, Slice, Stack and Batch reconstruction. See the Advanced section of this document for a description of the reconstruction parameters. To reconstruct: 1. Tune the reconstruction parameters by clicking the Param button below the Reconstruct Slice button on the N-SIM pad. 2. Adjust the Illumination modulation contrast. Use larger values for situations where the grating contrast is poor: for example for thick samples with lots of out-of-focus light. Start with a value of 0.5 when the grating lines are clearly visible (see the example opposite). 3. Adjust the High resolution noise suppression to remove any high frequency reconstruction artefacts. This can occur for samples with lots of out-of-focus light. Start with a value of 0.25 for samples with good grating contrast. Use of large values of this parameter will compromise the resolution of fine structure in the image. Figure 15 7 @
4. Adjust the Out of focus blur suppression parameter. This is effectively optical sectioning and can help to remove any outof-focus light from the image. Start with a value of 0.3; larger values suppress out-of-focus light to a greater extent. Note that this parameter is unavailable for 2D-SIM, TIRF-SIM and Stack reconstruction. 5. If reconstructing ND acquisition data you can tune the parameters for each channel by ticking Use different settings for each Channel and select each channel from the dropdown list. Tick Use best K-Vectors found in: and choose All Frames from the drop-down list if you have multiple z-slices. 6. Press Reconstruct to perform image reconstruction. 7. Press Apply to store these parameters enabling them to be used with the Preview Slice and Reconstruct Slice buttons on the N-SIM pad. Figure 16 After image reconstruction the final image will typically have a large baseline offset. To remove this offset you will need to adjust the black level on the LUT for the image. Click the right mouse button and select LUTs from the Visualisation controls in the resultant context menu. Figure 17 8 @
Figure 18 9 @
Capturing a widefield image It is possible to reconstruct widefield images from the 15 phase images captured during a SIM acquisition but this requires splitting the raw data using a macro. The easier method is to record a new image with the microscope in its widefield mode. In this mode only the zero order of the illumination grating is transmitted through the microscope resulting in epi-like excitation of the sample. A single z plane can be captured use the SIM Full Illumination configurations on the OC panel (click the button corresponding to your channel of choice either Full 405, Full 488, Full 561 or Full 640 ). This will configure the N-SIM pad as follows: 1. On the N-SIM pad the Widefield option is selected from the dropdown menu under the Live button. 2. The Widefield option under the capture button is selected. You should then set the exposure time ( DU-897 settings ) and laser power to achieve the same number of ADUs as captured in the SIM data (either 4,000 or 12,000 for 14- or 16-bit modes respectively). Pressing the Capture button on the N-SIM pad captures and image at the current z- plane and with the current laser power and camera settings. Figure 19 Multi-channel widefield images can be captured using ND Acquisition. Simply select the λ (lambda) tab, click the checkbox on the first row and select the SIM Full Illumination configuration corresponding to your channel of choice. Continue to add channels by clicking the checkboxes on subsequent rows. Synchronizing SIM and widefield images The Synchronizer is a tool that allows you to compare two images by locking the pan and zoom. The widefield image is as captured and has a format of 512x512 pixels whereas the SIM image has extra pixels introduced during the image reconstruction and is 1024x1024. So, before using the synchronizer you will need to resize the widefield image to match the SIM image. On the NIS Elements toolbar go to Image Size resize to set the widefield image to 1024x1024. Click the right mouse button and select Synchronizer from the Visualisation controls. Add both images to the Synchronizer to lock the pan and zoom. Figure 20 Figure 21 10 @
Notes about image reconstruction The intention of this section of the notes is to give a brief (non-mathematical) description of the process of SIM image reconstruction and the parameters used. A more in-depth discussion of SIM can be found in the Structured Illumination Methods chapter of the Handbook of confocal biological microscopy by James Pawley. The two most important concepts relating to SIM are spatial frequency and the modulation transfer function (MTF). Images with small details have high spatial frequency and conversely large details (and out-of-focus light) have large spatial frequencies. The spatial frequency content of an image can be examined by performing a Fourier transform (FFT) into frequency space. Low frequency components lie at the centre of the FFT image (see examples in figure 22). Figure22 In a microscope the resolution and performance can be characterised by the MTF which is a measurement of how specimen contrast is transferred through the optical system to the image plane. The microscope objective lens is the most important component contributing to the MTF and forms a pass band for the microscope with a cut-off at a spatial frequency defined by the numerical aperture (NA) of the lens. The example shows that when an image of a periodic structure is transmitted through the microscope the high frequency details get blurred and there is a resultant loss of contrast. The purpose of SIM is to restore the high frequency detail that gets lost when transmitted through a microscope objective lens. Figure23 This is achieved by illuminating the sample with patterned or structured light and relying on the Moire effect to transmit high frequency information through the objective lens. The Moire effect is a simple pseudo-interference effect where two overlapping, slightly mismatched, patterns produces a low frequency fringes. The key concept is that the low frequency fringes are unique to the overlap between the two patterns (see figure 24). In SIM the spatial frequency of the illumination pattern is arranged to be just on the cut-off frequency of the objective lens while the Moire fringes produced by the overlap of the sample and the illumination pattern has a low enough frequency to be transmitted by the lens. By adjusting the position of the illumination pattern in real space (3 rotations and 5 lateral shifts or phases for each rotation for 3D Figure 24 SIM) it is possible to extend the cut-off limit of the objective lens by almost a factor of 2. Knowledge of the rotation and the phase shift allows the pass band captured in each image to be moved to its true position in frequency space (as illustrated in figure 25). A reverse Fourier transform then produces an image with the high frequency information restored. 11 @
Figure 25 Image reconstruction parameters The following is adapted from the N-SIM manual. Illumination modulation contrast This parameter allows adjustment of the relative amplitudes of the MTF for the different phase images captured during a SIM acquisition. Matching the separate image components captured at each grating rotation and phase in frequency space is dependent of the contrast of the illumination pattern. The purpose of this parameter is to improve image reconstruction when the contrast of the illumination pattern is low (i.e. when stripe visibility is low). This can happen in highly scattering samples or where there is a large amount of out-of-focus light. Conventional High frequency noise suppression During image reconstruction image filtering and apodization takes place. The first is to remove noise from the image and the second is remove artefacts that can be introduced during the filtering process. The High frequency noise suppression parameter can be tuned in order to remove these high frequency artefacts but at the expense of high frequency information in the image. Large value MTF Super resolution 2D SIM 3D SIM Spatial frequency Small value MTF Spatial frequency Small value MTF Spatial frequency Figure 26 Large value MTF Spatial frequency Figure 27 12 @
Out of focus blur suppression Out-of-focus light can be removed during image reconstruction by tuning this parameter. Put simply larger values produce more optical sectioning. Small value Z Large value Z X,Y X,Y Spread of intensity distribution of luminescent point in real space Figure 28 Recommended starting parameters Figure 29 13 @