Nikon N-STORM - Mode of operation

Microscope controls

To control the microscope stage, you will use the joystick. The central knob controls the X and Y directions, you can change the speed by twisting the top of it. 

The two knobs on the sides of the joystick can be used to lower or raise the objectives and bring the sample into focus. You can also do this by using the two knobs on each side of the microscope itself.

Note: The z height will display on the joystick screen. You want to move the objective up (larger numbers) to focus your sample.

Opening the software

Double click the NIS Elements software.

Based on your experiment, choose your desired laser launch and camera.

• If you will only be using the FRAP laser launch or the FRAP laser launch with epifluorescence, use the camera driver listed under Nikon Ti2.
• If you will be using the TIRF/STORM laser launch at all (including with FRAP), use the camera driver listed under Nikon Ti2 Direct STORM.

• If you would like to use a single Andor (EMCCD) camera, use the iXon 897.
• If you would like to use dual Andor (EMCCD) cameras for use with the TwinCam system, use the Dual iXon 897.
• If you would like to use a single Hamamatsu (sCMOS) camera or the Double Helix optics, use the Quest 2.

Click OK when the dialog box appears saying “Optical Configurations Import successfully finished.”

Overview of the Optical Configuration (OC) panel

The OC Panel (upper right corner of the software) contains the different light source configurations. For each light source, you can select a different fluorescence channel. These channels are a combination of a laser line and an emission filter set. The STORM OC icon will provide the pre-built channels for the TIRF/STORM lasers.

You can confirm that the laser line and the filter set have loaded properly by checking the LUN-F Pad and the Ti2 Pad. The selected laser line and filter set will be outlined in yellow. You can hover the mouse over the filter to get additional information. Not all experiments will necessitate having an emission filter.

 

The light source is controlled by the LUN-F Pad for the TIRF/STORM lasers. In this pad, you can adjust the laser power.

The camera is controlled by the Quest 2 Pad for the Hamamatsu camera. In this pad, you can adjust the binning, exposure, and the camera ROI.

 

Ti2 Pad

The Ti2 Pad allows you to change objective. Every time you click on an objective, the objective plate lowers itself before turning to the next one for safety.

It lets you choose the light path you need and choose to use the PFS (perfect focus system) or not. You can then drive the objective up and down with the Z Drive, adjust the intensity of the transmitted light with the DIA control slider. There is also an option to change the filters.

XYZ Navigation panel

To control the microscope stage and move your sample above the objective you can also use the XYZ navigation panel. This allows you to finely control the steps or set the same position from one experiment to the next.

Align the TIRF laser

TIRF Laser alignment procedure

During your training you practiced aligned the TIRF laser without a sample at first. This is a reminder of the procedure to get the laser going straight up when you position your sample on the microscope. Once you place the sample on the microscope it will only require minor adjustments.

You can control the TIRF laser angle with the Ti2 N-STORM XY-F-Zoom Pad. Click on the mouse cursor button to control the X and Y directions with the mouse wheel. When you click on the mouse cursor button for the focus adjustment you will bring your beam in focus. You can change the mouse wheel speed from coarse (fast) to fine and extra fine (slower).

When you turn on the TIRF laser (using the controls in the OC Panel) it will be misaligned and out of focus, far from the blue tape target on the ceiling straight above the microscope objective. Use the X and Y alignment directions to bring the beam centered on the target on the ceiling and then use the focus button to focus the beam in the center of the target.

IMPORTANT NOTE: to be able to scroll the mouse wheel to control the TIRF laser you MUST click somewhere on the Ti2 N-STORM Pad. If you happen to click somewhere else on the Nikon Elements software, the mouse wheel won’t be connected to the TIRF laser control anymore.

STORM, TIRF, HiLo imaging

At this point in your training it was time to look at the sample. We used the demo sample made of fluorescent beads in water. If you are using the 100x oil immersion objective, start by dropping a drop of oil on the top of the objective, as described in this tutorial video.

You are now ready to delicately place the sample on the sample plate holder.

Bring the sample into focus

1. Press the PFS button on the microscope base. You will hear several beeps and the light will start blinking, indicating that the microscope is searching for the focal plane.
2. Raise the objective slowly using the black knob on the side of the joystick until you hear a beep. The PFS button will be continuously lit when perfect focus is engaged correctly and the LED next to the FOCUS display will be lit. Be careful not to push the objective into your sample - this will damage the objective and lead to costly repairs.
3. Click “Live” in Nikon Elements to see what the sample looks like and adjust the focus using either the external knobs or the controls in the software. The demo sample has many beads floating in water, you will be able to focus on one plane but won’t see all the beads in focus.

Re-align the TIRF laser

Following the same procedure as above, bring the laser straight and focused on the blue target on the ceiling.

Find the TIRF angle

Using the Ti2 N-STORM controls, lower the laser in the direction of the wall until it is almost parallel to the surface of your sample.

While you are lowering the laser beam, keep looking at your sample on the Nikon Elements viewer. As you get close to the critical TIRF angle, the beads will become brighter. When you are just past the critical angle the signal will disappear. You can then adjust the angle using the fine control until you have optimized your signal and it is blinking.

Note: Do NOT stand up to look at the laser coming from the microscope/look at your sample from above before moving the laser down the wall!

Note: This pattern is most visible in aqueous samples that have a considerable amount of liquid. The above patterns will be very hard to see in a low-volume aqueous sample.

Use the Bertrand lens to confirm the TIRF angle

Turn off the lasers using the Stop icon or the Freeze button.

With the lasers off, turn the knob on the side of the eyepiece to the camera icon.

Then, turn the Bertrand lens knob on the front of the microscope base to the left.

Then, using the software, open the TIRF lasers shutter (along the top button bar). Ensure that the upper filter turret is empty and that the lower filter turret is moved to the quad filter (in middle right section).

In the software, click on the observation camera optical configuration (in top right section).

In the observation camera pad (lower left section, tab may need to be selected to see the full pad), click the Start button.

A grey circle should appear with a white spot or ring. Use the inner knob of the Bertrand lens knob on the front of the microscope base to bring it in to focus.

When in focus, the state of the circle should tell you if you are in HiLo, TIRF, or neither.

• The white spot will start in the center and, as you move the TIRF laser down the wall, the white spot will move toward the outside of the grey circle. The white spot should be pretty round.
• As you enter HiLo, you should see two equal-sized spots on either side of the grey circle. Depending on the sample, the spots may be slightly different sizes.
• As you reach TIRF, you should see a pronounced white ring around the grey circle. Depending on the sample, the white spots may entirely blur into the white ring.

After finding the TIRF angle, record the TIRF laser X, Y, and focus so that you can input it into your desired optical configuration.

Click Stop on the Observation Camera Pad, select your desired optical configuration (in top right section), and turn the Bertrand lens back to the top.

Copy the TIRF laser X, Y, and focus numbers that you just recorded into your desired optical configuration. Now you can continue with your imaging! Note: You may need to use the Coarse, Fine, and Extra Fine settings with the mouse wheel to input the correct numbers. Typing the numbers in often changes other numbers in the pad.

Optimizing the image

Understanding the Look Up Table (LUT)

Depending on the camera you are using and how much signal your sample emits with the laser settings that you chose, your image may be very dim to start with, to the point that you don’t see anything. The first thing you should do is adjusting the Look Up Table.

To auto-adjust the lookup tables, clicking on the Auto Scale buttons above the image of interest.

This button will adjust the lookup table for each frame (useful in live view mode)

This button will adjust the lookup table for the current image

You can also set the range of the Look Up Table by dragging the lines on either side of the intensity histogram with the little triangular cursor on the top. If the intensity histogram reaches the end of the range (65535 for the Hamamatsu Fusion which has a 16bits dynamic range) that means your image is saturated and you need to reduce the laser power or the exposure time or both.

Adjusting the exposure and laser power

In the Quest 2 Pad you can adjust the exposure of your image. 

For successful STORM imaging you should pick an exposure time under 100ms. 20ms is typical of many STORM experiments.

You can also choose to bin your image (i.e. clumping the pixels together to make bigger pixels). The scan mode can be adjusted as well to optimized your data. If you don’t see many blinking events you might be scanning your sample too fast.

Adjusting the ROI (i.e Region Of Interest) is a good idea if you do not need to acquire the entire field of view.

In the LUN-F Pad you can adjust the laser power, expressed as a percentage of the maximum power for each laser.

When your image is optimized and you have a good signal with blinking fluorophores it is time to look at the acquisition settings.

Image acquisition and saving

The Nikon Elements software offers many options for acquiring and saving your data in the ND Acquisition panel (middle left of the software).

1. Check the box next to Save to File.
2. Select the folder that you would like to save your image(s) in.
3. Give your image a descriptive name. If you end the filename in "_001" the Nikon software will count up for each acquired image.
4. Set up your imaging parameters. The software will only follow the instructions for tabs and lines whose checkboxes are selected/checked.
5. Click Run now.

 

Acquiring a time-lapse image series

1. Check the box in the Time tab.
2. Add a new time-lapse setting by clicking the checkbox under Phase or clicking the +Add button.
3. Define the interval, the duration, and/or the number of loops.
   

We recommend checking the box next to Use PFS, especially for longer term imaging.

Checking the box next to Close Active Shutter when idle is only recommended when you have extended time between images (multiple seconds).

Acquiring multiple points in (x,y) in the same file

1. Check the box in the XY tab.
2. Add points as you move around your sample by clicking the checkbox under Point Name or by clicking +Add.

We recommend checking the box next to Include Z and next to Use PFS. 

Acquiring a z-stack

1. Check the box in the Z tab.
2. Select a mode to define the z-stack: Define the top and bottom, symmetric around a center, or asymmetric around a "center".
3. Choose either Relative or Home as your center point. Relative works with the PFS. Home sets an absolute z value.
4. Define your step settings by altering the step size, the number of steps, and/or the range. The button next to the step size gives the recommended step size based on the objective.
5. Choose which direction you would like to acquire the steps.

Note: Acquiring a z-stack will remove all 'Use PFS' checkboxes in other tabs.

Acquiring more than one channel

1. Check the box in the λ (wavelengths) tab.
2. Add a new channel by clicking the checkbox under Opt. Conf. or by clicking +Add.
3. If necessary, adjust the channel by clicking the ... in that line.

We recommend checking the box next to Use PFS.

Acquiring a large image

1. Check the box in the Large Image tab.
2. Adjust the number of fields to be stitched together.
3. Ensure that the Overlap is set to at least 10%.

We recommend checking the box next to Use PFS.

Calibrating the FRAP laser

After your sample is in focus, turn off the lasers using the Stop icon or the Freeze button.

Open the upper filter, lower filter, and FRAP shutters.

Make sure the upper filter turret has a filter in the path (not empty) so that you can see the reflection later on.

In the Galvo XY Pad (in lower left section, may need to select the tab for the pad to be visible), select your desired laser line and increase the power. Adjust the dwell time to be at least 100 msec and increase it again later, if necessary. Click the gear icon in the lower right corner.

In the Galvo XY Configuration window, click the Calibrate... button.

In the Galvo XY Calibration window, click somewhere in the XY grid to FRAP that spot. Wait a few seconds for the FRAP spot to appear. With your mouse, move the red crosshair to the center of the FRAP spot.

Note: If a spot does not appear, consider turning up your laser power or dwell time. If spots still do not appear, click closer to the origin of the grid.

In the Galvo XY Calibration window, click the Plus button to add this point.

Repeat this process, adding points that appear in the periphery of your image and adding points that appear in the center of the image. When you're done, click Next.

In the Galvo XY Configuration pad, click OK to save your calibration for this session.