Tips and Tricks (Engl.) | Tipps und Tricks (Deutsch)
Short reference (Engl.) | Kurzreferenz (Deutsch)
How to ...
How to take a focus stack of images
How to add images to the list
Select File | Add images or press Ctrl-A. Select the folder with your image stack. (You should copy a single stack to each folder, only.) Then press Ctrl-A again to select all files, or click on the first image, keep the shift-key pressed, and click on the last image of a stack. By pressing the Ctrl-key and clicking on single images you can select or unselect them. After clicking on 'Open' the selected images will be added to the listbox and sorted alphabetically. The first image will be shown in the left image window. The added images are marked as indicated by [X]. To unmark or mark an image, double-click on its name in the listbox. Only marked images will be included in further image processing.
Under File | Image list there are several options to change all marks, remove files from the list, rename, or delete files.
How to start the stacking procedure
First select the images to be stacked via 'File | Add images' (or Ctrl-A). The simplest way of starting the stacking procedure is pressing Ctrl-F1. Alternatively, you can use 'Stack operations | Set stacking parameters | Go'. This will start the routine applying the current parameters. A little window indicating the progress (= number of images processed) will show up. Finally, the generated stacked image and the corresponding depth map will be saved in the same folder as the image stack, and will be added to the image list (unmarked). The new filenames start with 'p#' and contain the parameters used like 'p#sharp_min1_en.jpg' or 'p#mapsharp_min1_en.jpg', 'min1' indicating that the stacking routine applied a minimum contrast of 1, and 'en' indicating that the automatic image enhancement was used.
How to find perfect stacking parameters
There are several adjustable parameters you can use to obtain the perfect stacking result. Here they are shortly explained. Select Stacking operations | Set stacking parameters or press F2 to open the panel.
How to align an image stack
Reasons making it necessary to align the images of your stack are
How to browse an image stack at constant position and size
After adding, the selected files are shown in the listbox and the first image is displayed. To display another image just click on its name. You can zoom in and out ([+] and [-] on the image window. As indicated by the '[X]' at the left, the images are marked. To unmark or mark an image, double-click its name. Only marked images will be regarded during stack operations.
Note: All pictures should have the same dimensions (width and height). You can reverse the order under 'Image list' depending on whether you started with the uppermost or lowermost layer. PICOLAY assumes that the first images shows the uppermost layer and requires the image names in the corresponding alphabetical order. PICOLAY will not change the original files. However, automatically generated files will overwrite older versions. Next time you start PICOLAY the last image directory will be remembered.
Most images are at full resolution bigger than your screen. While normally image viewers show the upper left corner of an new image or resize it to fit the screen, PICOLAY will keep a selected position a full size when you click through the marked image list or display the list as slide show. Thus, you can watch how details of a specimen are represented in your stack.
How to display an image stack as slide show
Just add images to the list, and press Image list | Start slide show or use the F12-key. Stop the show by pressing F12 again or clicking into an image. Please set unter Options | Slide show features how long (in millisec) each image should be displayed, and whether at the end of the list the show should run backwards or start again with the first image. To generate a single file from your show use the function Image list | Generate animated gif image.
How to generate an animated GIF file
Marked images of your list (resp. the slide show) can be transformed to an animated gif image by selecting Image list | Generate animated GIF image or by pressing or Ctrl-G. Before doing this set unter Options | Slide show features how long (in millisec) each image shall be displayed, and whether at the end of the list the show should run backwards or jump to the first image. You are asked for a name of the gif file, which is saved in the current folder.
How to generate mpo files
MPO (multi-picture object) files are containers harbouring the left and right view of a 3D object. This file format is used by several image processing programs and 3D TVs. To generate them, first produce the left and right view as jpg files using the [R]+[L] mode on the PICOLAY 3D display panel. Take care that these files are the first marked images in your image list. Then select Image List | Generate MPO file, enter a name and click on Go. The MPO file will be saved in you current folder.
How to handle very large images
The PICOLAY stacking routine might have problems with very large images (bigger than 6000 x 4000 pixels), giving an 'Out of memory' error even with computers having sufficient RAM. To avoid this problem you might either resize your images or crop the relevant part prior to stacking them. Please regard that for stacking a higher number of images (layers) is often more relevant than the number of pixels per image.
How to resize images
Marked images of your list can be resized by means of the Enhance function on the left image window. Activation of the 'Apply to all marked images' feature may be used to transform a whole list of images.
How to crop images
To crop images and cut off superfluous edges, draw a rectangle into the image. Then select Edit | Crop selection (for a single image) or Edit | Crop all marked images for a set of images. PICOLAY will generate new files named 'clip#..' (+ original name). The file format of these can be set under Options | Save as.
How to rename images
To rename images, add them to your list and take care that they are marked. Then select Image list | Rename marked files or press Ctrl-N. Type the characters to be overwritten in the left and the new characters in the right text field and Go.
How to change the file format of images
To change the file format of images, select the new format under Options | Save as. Add the images to your list and select Enhance on the image window. On the new panel you might disable all changes or activate some, and select Apply to all marked images if you want and click on Go.
How to delete selected images from disk
CAUTION: PICOLAY deletes files completely, and does not keep them in the bin!
Files in the list generated by PICOLAY, whose names are beginning with 'p#...' can be deleted by pressing Ctrl-X or Image list | Delete all p#-files, resp. Delete marked p#-files. To delete other image files, mark them in your list, select Image list | Delete all marked files and confirm the irreversible deletion.
How to change image parameters like brightness, contrast, sharpness etc.
PICOLAY allows for changing many image parameters using the Enhance function on the image window. A new panel opens that allows to adjust about 20 parameters. Check your settings clicking on Test until you see the perfect result. Then click on Apply, eventually using the option Apply to all marked images.
How to paint on an image
The mouse tool can have different functions to be used on the left image window. Select [Mouse=] Paintbrush. Adjust Brush width and select the colour either by clicking with the RIGHT mouse button into the image or typing in the RGB values. There is an Undo function for one step only. You may save intermediate or final steps with a new name or Overwrite the current image (name indicated on the panel).
How to sharpen or blur details
Select [Mouse=] Sharpen or [Mouse=] Blur and adjust Brush width. There is an Undo function for one step only. You may Save intermediate or final steps with a new name or Overwrite the current image (name indicated on the panel).
How to clone (= copy areas) within an image
Select [Mouse tool=] | Clone within image, set Brush width, RIGHT-click into the image to define the target area and then LEFT-click to define the source area. Now move the mouse with the left button pressed down to clone from source to target area. There is an Undo function for one step. You may save intermediate or final steps with a new name or Overwrite the current image (name indicated on the panel).
How to clone areas of original images into the stacked result
Select [Mouse tool=]Edit | Clone to result image and set Brush width. Now move the mouse with the left button pressed down to clone from source to target image. Using the Synopsis feature (on top of the right image) helps to display the same areas on both images. There is an Undo function for one step. If you save the image it will get a new name with 'clone...'. If you use this function on a stacked image the map will be cloned as well, thus keeping the layer from which the source pixels are derived. You should not use the 'Auto-enhance' function for the stacking routine, when you want to clone from original to the stacked image, as the stacked image will not match the originals perfectly.
How to write text on images
Select [Mouse tool=] Insert text, choose a character font, size and colour. Define whether writing should be horizontal or vertical, and type in the text to be inserted. Then click on the left image to place the text on it. There is an Undo function for one step. Finally, you save the image with a new name 'p#...'. You may also write on all marked images, if you check the corresponding box.
How to draw a scale bar
Select [Mouse tool=] Draw scale bar, define Width and Length of the bar and choose a colour then click on the left image to place the bar on it. There is an Undo function for one step. Finally, you save the image with a new name 'p#...'. You may also write on all marked images, if you check the corresponding box.
How to average images
Use Stack operations | Average marked images or F4 to average images. A new file 'p#mean...' is generated and added to the list.
How to add or subtract images from one another
Take care that the image to be added or subtracted from the others is the first marked image in the list. Then use Stack operations | Add or subtract 1st image or F9 to start the routine. New files 'p#..' (+ old name) are generated and added to the list.
How to insert intermediate images betweeen the images of a stack
Use Stack operations | Insert intermediate images or F5 for this purpose. The new files generated will have an additional 'i' in their names. Please regard that it is better to have more original images in your stack than using this routine, which might give a smoother stacking result but does not contribute any new information.
How to perform colour-based stacking
A colour-based stack analysis might be helpful, if you want to localize structures showing a certain target colour (e.g., a fluorescent dye) in your stack. Use Stack operations | Colour-based stacking or F3 for this purpose. The target colour is selected by clicking with the RIGHT mouse button into the image window or be entering the RGB values. Using black or white as target colours can be helpful to detect the darkest and brightest structures, or to get rid of halos in phase contrast images etc.
How to harmonise image brightness in your stack
To compensate brightness variations in your image stack use Stack operations | Auto-adjust brightness or F7. New files 'b#..' (+ old name) are generated and added to the list.
How to set the white balance of one or all images in your stack
To correct the white balance of marked images in your stack use Stack operations | Set white balance or F8. Define the target colour (the one that should be changed to grey/white) by clicking with the RIGHT mouse button into the image. New files 'p#..' (+ old name) are generated and added to the list.
How to set a smooth background (flat field) by removal of disturbing items from your stack
If you have hot spots on your camera chip, dirt in your optical pathway or a disturbing brightness gradient affecting all images of your stack, this can be corrected by the PICOLAY flat-field correction. First, take a picture above and/or below the level where anything of your specimen is sharp. You might also average these unsharp images. Then take care that the unsharp image is marked as the first image in the list and use Stack operations | Set background/flat-field or F10. Define the target background colour by clicking with the RIGHT mouse button into the image and Go. New files 'p#...' (+ old name) are generated and added to the list.
How to divide RGB values of images by those of the first image in the list
To do this use Stack operations | Divide by 1st image or F11 and Go. New files 'div#..' (+ old name) are generated and added to the list.
How to generate a depth map of your stack
A depth map is automatically generated during the stacking routine. It is saved as 'p#map...' (+ applied parameters information) in your list. The depth map shows colours from yellow (top) over green (middle) to blue (bottom). Grey indicates areas without detected structures and no depth localisation. The map can be used to produce rotations or stereoscopic 3D images. The depth map does not contain information about the absolute height or thickness of the specimen. This parameter (length of Z axis) has to be defined when you generate a 3D view.
How to set correct 3D parameters
When you have defined a sharp image and a depth map (e.g., simply by performing the stacking procedure) you click on 3D view on the right result image to get a new panel with the 3D parameters. At the same time the depth map is displayed. Here a short summary of the adjustable parameters and their effects:
How to generate a 3D hologram view to display hidden structural details
The stacking procedure detects the sharpest structure in a focus stack, ignoring other details with lower score. The latter will be lost in the resulting image. With mono-layered specimen this is no problem. With multi-layered object, that often occur in microscopic analyses, you loose valuable information. To overcome this issue, PICOLAY allows for performing the so-called 'hologram stacking'. For this you define a minimum score value for the details to be displayed. Furthermore, it is usually recommended to rotate the object virtually so that the fine structures are not hidden underneath the maximum score pixels. Finally the stack has to be analysed again to generate the 'hologram'.
How to generate 3D images from depth map and stacked image
One can generate 3D images from a previously saved image + the corresponding depth map. Add any sharp image and the corresponding depth map to the list. Take care that both of them have the same size and show the same position. Click on the map and copy it to the depth map image: Edit | Copy to depth map, similarly with the sharp image: Edit | Copy to result window. Now click on '3D view' on the (right) result window and set the required parameters and display mode.
How to perform stacking in multiple subfolders with a single click
First, make sure that you have set suitable stacking parameters. Then select File | Stack images in subfolders. Use Change folder to find the folder above the image folders you want to analyse. Select all folders by Toggle marks or select single ones by double-clicking on them. Press Go and wait... If you have checked the corresponding button, the computer will be turned off automatically when the job is finished.
How to perform video-stacking
How can you do focus stacking on a (slowly) moving amoeba or a snail? Many cameras produce high-resolution videos that can be used to perform video-stacking on the macro- or micro-scale. This technique can also be used for still objects, as it gives very fine focus steps. Mount your camera in a fixed position. Illumination should be sufficient for 1/60 sec exposure time. Now take short videos, while you either turn the focus ring of your camera or move the specimen over the whole depth range within 3 to 5 seconds seconds. The next step is extraction of the frames from the video. This can be done by freeware like xmedia recode and virtualdub. Finally, select the frames covering the specimen depth and perform the stacking routine.