# The Huygens Ortho Slicer tutorial for Microscopic images

## Introduction

The Orthogonal Slicer is useful to study small objects in 3D and shows the same point in 3D space from 3 orthogonal directions;
• axial or xy (top left);
• frontal or xz (bottom left);
• transverse or yz (bottom right).

Each view is enclosed by a colored frame which color indicates to which slices in the other views it corresponds. For example, the yz view has a red frame and its borders are defined by the red sliders in the other two views. The size of the views can be adjusted by placing the cursor at the edges of the views so that a becomes a double-headed arrow. If you move one of the slices, the others will follow to make sure that the center of each of the slices intersect in the same point in space.

The histogram (top right) shows the number of pixels in the complete image plotted against the intensity value. The complete image is the image that is selected in the main window.
Only the active channels (defined under the Colors tab) are shown as overlays. The colors of the histograms correspond to the channel color settings under the tab Colors.

If you move the colored sliders in one of the views, the corresponding ones in the other views will follow to make sure that the center of each of the slides intersects in the same point in space. This behavior makes the Ortho Slicer a useful tool to study small objects in 3D.

## The (crosshair) cursor

The position of your mouse is projected on all views. The value besides the center of the cross-hairs gives the distance of the mouse position to this center. If this number is positive, it means that real pointer is more towards you (in front of your screen). If you click with your left mouse button on a position in one of the views, the slide-borders (indicated with colored lines) will be centered to that specific position.

## Projection regions

New in this Orthogonal Slicer version is the possibility set the projection mode to Slice, MIP, or Sum. The colored sliders in the different views define the borders of the projected region. The start screen of the OrthoSlicer has the Projection tab at the bottom active, and shows the geometry of the projected area with two values (origin and span) for each view. If you select Slice as projection mode, the span is set to one, as just one slice is shown. ((MIP) maximum intensity projection) and Sum show you a range of slices. The MIP shows parallel rays traced from the viewpoint to the plane of projection and only displays the maximum intensity of each ray, whereas the Sum shows you the sum of the voxel intensities along each ray.
If Auto-update is checked, Huygens will update the views in real-time when the position of the sliders is changed. If unchecked, views will only be updated when the mouse button is released.

Use the view menu to show or hide image properties and guides. These are:
• Pointer coordinates. The position of the mouse pointer in microns or in voxel coordinates.
• Time. The time for the current slice in seconds or frame numbers.
• Intensity values. The intensity values for all channels on the current pointer location.
• Zoom. The zoom value in screen-pixels per micron. A magnification factor is displayed as well; using the pixel density for your monitor, this value gives an estimation for the absolute magnification.
• Drop shadows. Enhance the contrast for the overlayed lines and text by showing drop shadows.
• Wireframe box. Show or hide the wireframe box, which shows the data volume (red), the cutting plane (green), and the displayed slice (gray).
• Large crosshair. Show or hide the large crosshairs cursor.
• Graphics. Shows the overlayed lines, sliders and text.
• Auto update. Updating views in realtime or after releasing the mouse left button when changing position of the sliders.

## Panning

Click and hold the middle mouse button on the slice to move it around. Pressing 'c' centers the slice.

## Changing time frames

Click the 'Time frame' tab key at the bottom to view the time frame panel. Drag the slider to change the time frame or press the play button to animate the time series. The time frame that is actually displayed can be indicated in seconds (s) or as a frame number through the 'View' menu.

## Zooming

Use the scroll wheel to zoom in or out on the location of the mouse pointer. Click the 'Zoom' tab key at the bottom to view the zoom panel. The four buttons respectively zoom out, zoom in, zoom 1:1 (the x-sample distance matches 1 pixel), and view all.

## Changing display colors

Click the 'Colors' tab key at the bottom to view the color settings panel. The buttons behind 'Active channels' can be used to enable or disable channels.
In the 'Color scheme' drop down menu, the preferred color scheme can be chosen:
• Greyscale. The image is displayed in grey teints. For single channel images, this gives a higher contrast than the emission or RGB colors.
• Emission colors. If the the emission wavelenghts are set correctly, this gives the most intuitive view.
• Global colors. Can be set as a preference under Edit > Preferences. Either the RGB or CBF, which are colors that can also be distinguised by colorblinds, can be selected. See also here.
• False colors. A false color is given to each intensity value. This view gives a high contrast and makes it easy to spot iso-intensity areas.
• Custom colors. Use the color picker to manually select a color for each channel.

## Tuning the brightness and contrast

Click the 'Contrast' tab key at the bottom to view the brightness and contrast settings panel. The brightness can be changed per channel, or for all channels at once.
The 'Gamma' drop down menu provides you with a linear and some non-linear ways of mapping data values to pixel intensities. If the 'Link channels' box is checked, this means that the way of mapping data values to pixel intensities is the same for all channels; if not, the range is automatically adjusted for the minimum and maximum in each channel.
This range can either be adjusted globally (the complete data set will be taken into account), or for each screen view.

## Automatic panning, slicing and zooming

When you click the right mouse button, a menu will appear with the following options:
zoom in brightest spot. The image will zoom in on the brightest spot in a 3D area close to the cursor.
Fit in canvas. Fits the view in the canvas
Show actual image size. Fits the image with the size of your screen pixels. In other words. the real sizes of the object(s) is shown on your monitor.
Center image. Centers the image in the canvas.
Set marker. Sets a marker that shows the corresponding voxel coordinates and the voxel intensity of each channel.

## Measure distances

To overlay a ruler on one of the slices, hold the left mouse button and drag. The length of the line in microns is displayed besides it. Like the mouse cursor, a projection of the ruler is shown on the other slices.

You may move the end points or the whole ruler by dragging respectively the ends or the middle of the line holding the left mouse button. Hold the ctrl key while dragging to drag the end points parallel to the line or the whole ruler in perpendicular direction.
Just click somewhere else in the image to create a new line. Lines can remove by selecting it with the left mouse button and using the delete button.

## Measure intensity profiles

When a ruler is drawn, the histogram will be replaced by a plot window. This plot shows the intensity profile along the ruler for the different channels of the original data, i.e., the image that is selected within the main window. The values for the disabled channels will not be shown.
The Data Plotter can save the data in CSV format, which can be imported in e.g. Microsoft Excel, and it can export the whole plot to Post Script, which can be read by e.g. Adobe Acrobat or GhostView.