Step by step example of rendering a two-channel image
This tutorial explains how to use Free Sfp, the FreeWare version of the Huygens Sfp Renderer. The basic rendering concepts apply also to the full version, in which you can also make animation frames.
The example data set
The demo data set is a two-channel subregion of a deconvolved 3D widefield image of a macrophage. The macrophage was fluorescently stained for actin (red) and tubulin (green). Recorded by Dr. James Evans, Whitehead Institute, MIT Boston MA, USA.
When launching freeSFP the object will be shown in the preview window on the top right-hand side of the window.
The effect of moving any of the sliders will cause an immediate update of the preview image.
Press the Render button to display the scene in the large window. The text 'Render' in the button will be shown in red when any of the render parameters are changed.
Pressing 'Render' to render the data set to the large window. When the rendering is done the text color of the button changes to black.
The sample image as shown here has two channels: Ch0 is shown in red and Ch1 shown in green. Both channels cast a shadow on the grey table. The direction of the light is controlled through the 'Light dir' widget.
The 'Twist' and 'Tilt' sliders allow you to change the viewpoint. 'Zoom' controls the zoom factor.
Adjusting the Twist or Tilt sliders causes the entire scene to be rotated, including the light source.
The view direction is always towards the center of the dataset.
The transparency controls are located in the bottom-right panel. To adjust the transparency of a particular channel, first select the channel with the channel selector below the 'Channel parameters' header, and then adjust the sliders. In this way you can adjust the transparencies of each channel independently.
The channel selector is not shown in the case of a single channel image.
In the example on the right 'Ch0' is selected. By setting the transparency for the excitation and the emission to 1.0 the red channel is removed from the scene, including its shadow.
Excitation and emission
In this example the transparencies for the excitation and emission of Ch0 is set to 1.0 and 0.0 respectively. At an excitation transparency of 0.0 no light is absorbed, the material is not excitated and will therefore also not emit light. However, at the same time it will aborb all light form other objects because its transparency is set to 0.0.
Because a particular material can only be excited by a particular wavelength, re-excitation by light emitted by other channels does not occur. As a result, these transparency settings cause the channel to show up as 'dark matter'.
Fine tuning transparencies
Rendering a particular channel more opaque by reducing its excitation transparency, shown here for the green channel.
At very low excitation transparencies the light is so quickly absorbed that only the outer layer of an object will be excited. As a result parts of objects not oriented towards the light source will emit no light and therefore be dark.
The 'Object size' slider controls the size of objects which will attenuate excitation or emission light by 50%, assuming the object has 100% density and the corresponding transparency is set to 50%. Since this slider acts like a scale factor on all transparencies, it provides a quick way to adapt all rendering parameters to a particular kind of object. For example, the optimal object size for filament-like objects will be lower than for large solid objects.
Often microscopic images have a non-zero background. The contribution of the background in a particular channel to the SFP rendering can be removed with the Soft Threshold slider.
Soft thresholding as opposed to hard thresholding means that values slightly below the threshold are not set to zero but merely attenuated. In this way the introduction of frayed edges and surfaces is avoided (see Soft Threshold). The Option menu allows fine tuning of the transition range of the soft threshold.
In the example on the right the soft threshold level in the top image is 0, causing all low intensity background values to be displayed.
In the middle image the soft threshold level of the red channel is increased.
In the bottom image the soft threshold level of both channels is increased resulting in removal of the background in both channels.
The brightness of each channel can be adjusted independently. In the image to the right, the red channel was made very bright while the green channel was dimmed.
Use the 'Light dir' to change the light direction. Below you can see the light direction has changed compared to the previous image. To find the best light direction rotate the scene to study its effect.
When FreeSFP is started the position of the viewpoint and all parameters are set to defaults. To return to this default view, select 'Go Home'.
The 'Home' postion can be updated with any view and parameter setting by selecting 'Set Home'.
In the Sfp Renderer inside Huygens Essential, this is used also as starting and ending points to generate animations: see Sfp Renderer.
Saving rendered images
To save the rendered image as a RGB Tiff image use 'File->Save'.
- The view point can also be moved by moving the mouse over the large image while pressing the left mouse button.
- The large rendered image can be dragged by moving the mouse while pressing the middle button.
- With the menu 'Options->Compute shadow' you can switch computation of the shadow on or off.
- Selecting the menu 'Options->Small thumbnail' will make the thumbnail in the right upper corner smaller. This can be usefull for increasing speed on slow computers.
- Changing 'Options->Render quality' will alter the quality of the rendered image. Best use 'Fast' mode to find optimal viewing parameters; for the final result use 'Best' quality.
- Changing 'Options->Soft threshold mode' will alter the transition range (see above) of the soft thresholding. Setting it to 'Hard' will cause a sharp cut off of values below the threshold; 'Soft' will make the thresholding more gradual.
On the full version of the SFP rendered present in the Huygens Software you have extra features, like more perspective modes or a movie frames maker. See Sfp Renderer.