Video script: STEVE
This tutorial video provides an introduction STEVE, the microscopic imaging management desktop application. STEVE controls the 3D Cell Explorer tomographic microscope and allows viewing, organizing, analyzing, and sharing your images. With STEVE, you can add digital stains, explore your sample in 3D, and watch time-lapse videos of cellular processes.
The interface of STEVE consists of two main areas: The three panels on the top and the control strips below. The panel on the left is the 2D view that shows a horizontal slice of your sample. The panel in the center shows the digital stains. The panel on the right is the 3D viewer that allows exploring your data in three dimensions.
Below the three panels are the control buttons and sliders. The four sliders allow you to control the image. The ‘overlay’ button allows to move between the unaltered refractive index and stained data. The ‘slices’ slider allows you to move through the horizontal slices of your sample. The ‘opacity’ slider sets the opacity of the currently selected stain. The ‘Edge softness’ allows making the stain’s edges soft and avoid solarized images in 2D.
Below the four sliders is the digital staining area. You can load an existing digital stain or create your own.
Below the staining area, you see the controls for time-lapse videos.
On the lower left, you find the microscope controls that allow you to switch from whitelight to 3D and 4D acquisition modes. Below the microscope controls are the buttons to open, save, and share your file. For more details about how conduct an acquisition with the 3D Cell Explorer, have a look at the ‘first acquisition video’. You’ll find the link below.
Load new file
Now let’s open an existing digital file. Click on the ‘load file’ symbol. [Reference to sample file]
2D view panel
The unstained file can now be explored in the 2D viewer panel. The image shows a horizontal slice of the sample’s refractive index. The 3D Cell Explorer captures 96 slices of a sample. Starting from the central plane at ‘0’, you can go 48 slices up or 48 slices down. You can move through the slices either by moving the ‘Slices’ slider or by moving your mouse up/down while pressing the left mouse key. The number on the ‘slices’ slider shows you which slice you are currently looking at, for example slice ‘+12’ or ‘-4’.
You can zoom in on an area of interest by using the wheel on your mouse.
You can pan by moving the mouse while pressing the left mouse key and holding the ‘shift’ key.
You can take a snapshot of the current slice by clicking on the camera symbol in the top right corner of the panel.
Now, we’ll measure our sample. You can easily get the dimensions of your sample and its substructures. Click on the ‘ruler’ symbol in the top right corner on the image. Click on the two end points in the image to see the distance in micrometers. Click on the ruler symbol again to unselect this function.
To determine the height of your sample, move the ‘Slices’ slider until you reach the bottom of the sample. Take note of which slice it is. You can see the slice number on the slider. Then move the slider until you reach the top of the cell. Now you can calculate the height. Find the height of a slice by clicking on ‘File’ -> ‘Properties’ -> ‘Z-resolution’ [This is the thickness of each slice in micrometers. Therefore, the height of the cell (in micrometers) = # of slices * slice thickness (Z- resolution).
Next to the ruler symbol is the crosshair symbol. This allows you to learn about the refractive index of a particular pixel.
Now that we explored the unstained image, it is time to add some digital stains.
First, find an area of interest by panning, moving through the slices, and zooming in. Let’s zoom in on the nucleus. In the digital staining area, click on one of the circles with the plus sign to set up your first stain. Each circle represents a different stain. Now pick a color and select a small area you want to stain. Just a few pixels are enough to select that particular refractive index value. If other areas have the same refractive index, they’ll also be highlighted in this color. You can label that stain in the text box above the color picker. You can also choose a different color for this stain by clicking on a different color. Stains can be hidden by clicking on the eye symbol or deleted by clicking on the trashcan symbol. Clicking on the eye symbol shows the stain properties and allows setting the background definition. It is recommended to use different colors to stain different parts of your sample.
You can also open an existing staining file. To open existing staining files, click on ‘Load Panel’. Alternatively, you can also drag’n drop the staining file right into STEVE.
You will now see your digital stain represented as a box in the middle panel. In the staining panel, the x-axis represents the refractive index and the y-axis the index gradient. You can now adjust your digital stain to get the desired result. Move the overlay slider to the middle to see the stained areas. There are two ways to adjust your digital stain:
First, you can change the position of the stain box. Click in the center of the stain box, which will be highlighted in white. You can now move the box around while pressing the left mouse button. Moving the box left or right lowers or increases the refractive index of this stain. Moving the box up or down changes the index gradient range.
Second, you can change the shape of the stain box. Grab a line of the inner frame with the mouse. The line will be highlighted in white. While holding the left mouse button pressed, you can now move this edge to make the box larger or smaller. You’ll see the effects in the panel on the left in real-time. Once you let go of the mouse button, the changes will show in the 3D panel on the right as well.
To stain all pixels with a specific refractive index value, create a stain box as narrow as possible (with a small RI range) but as high as possible. Additionally, set ‘Opacity’ and ‘Edge softness’ to maximum levels. This is often useful when staining membranes.
You can save your digital stains by clicking on the ‘Save Panel’ button. You can reuse the same staining file for other samples of the same kind.
To get a larger view of the 2D and 3D panels, click either on the downwards arrow in the lower right corner of the 2D panel or choose ‘viewer’ in the top menu bar under ‘mode’.
3D viewing panel
Next, we can explore the sample in 3D by looking at the panel on the right. This panel shows a 3D reconstruction of the sample. You can rotate the view by grabbing the grid while holding down the left mouse key. You can zoom in and out with the mouse wheel. To pan, hold the shift key and the left mouse key. You can also change the background color of the 3D image by clicking on the small circle below the right-hand panel. For example, you can choose a white or black background.
When rotating the grid to the side, you can also estimate the height of your sample by looking at the 3D representation. Each horizontal grid layer is 5 micrometers thick.
When looking at your digitally stained sample in 3D, you might notice a mirror image of your sample. This is an image artefact caused by overlaps of the stain with the background region. To resolve this issue, you can move the stain box up a bit along the Index Gradient axis.
You can turn the grid lines and captions on and off with the buttons ‘Hide Axes and Cube’ and ‘Hide caption’.
To take a snapshot of the 3D reconstruction, click on the photo camera symbol in the top right of the panel.
To take a video of manipulating the 3D reconstruction, click on the video camera symbol in the top right corner. Indicate where you’d like to store the video file and then recording will begin – as indicated by the red dot. To stop and save the video, click on the red dot again.
4D time-lapse controls
Now we’ll go from 3D to 4D. With STEVE, you can explore time-lapse videos.
The controls at the bottom allow you to explore your data on a timeline. You can see the current frame number and the acquisition time of the current frame. Using the controls, you can jump to the beginning or the end. You can play it at normal speed (1x) or accelerate up to 32x.
To extract and save a specific section of the time-lapse video, set the markers ‘A’ and ‘B’ and the click on ‘Export’ on the left. Choose ‘4D’, select ‘indexed’ and format ‘Vol’.
The ‘Export’ button gives you additional options to export your data. For example, you can export a staining file for 3D printing.
You can find a detailed description of best staining practices in the ‘User manual for the 3D Cell Explorer’ document and the ‘best staining’ tutorial video. Click on ‘help’ in the top menu bar to access the user manual.
For more information on the 3D Cell Explorer, visit our website www.nanolive.ch.