In order to generate video, it is required to have FFMPEG installed on the computer. Instructions on how to install FFMPEG can be found on Internet (e.g. MacOS)

Installation using PyPi

To install microImage using a terminal and PyPi, simply use the following command

pip install microImage

Installation using the setup.py script

Download the module folder on the github repo

Installation in a Terminal-based environment
1. Open a terminal in the module folder
2. (Opt.) Start your virtualenv if you use one
3. Type and run the command
```
> python3 setup.py install
```
Installation in an Anaconda environment
1. Open Anaconda and go to your Environments
2. Select the environment you want to install the module into
3. Click on the arrow next to the name of the environment and select Open a Terminal
4. Navigate to the module folder
5. Type and run the command
```
python setup.py install
```

How-to use the module

The examples/ folder provides a .py script as well as a Jupyter notebook to load and display an image using MicroImage.

Basic Input/Output functions

Opening an image

To load an image, simply use the openImage() function:

from microImage import openImage

imageArray = openImage('./path/to/folder/or/image.image_extension')

The imageArray output is a NumPy array of shape (frames, Y pixel, X pixel). To open a folder containing multiple frames, just type the path to the folder.

Saving an image on the computer

To save an array as an image, you can use the saveImage() function:

from microImage import saveImage

saveImage(imageArray, './path/to/new/file.tif', bit_depth=16, rescale=True)

The function will automatically detect if you are trying to save a single frame or a stack/animation.

You can specify the bit depth of the output image (8 or 16 bits, 8 bits only for gif animations) using the bit_depth argument. The function will always try to cover the full bit depth with the given pixel values; this can be prevented with the argument rescale=False.

If an extension is not specified, in the path of the file to create, the default extension .tif will be used. This can be changed by using the argument default to specify another default format (e.g default='.png')

Video generation

It is also possible to save the array as a .mp4 video using the saveVideo() function:

from microImage import saveVideo

saveVideo(imageArray, './path/to/new/file.mp4', fps=25)

Having ffmpeg installed on the computer is required to use this function.

All arrays will be converted to 8-bits prior to be saved. The framerate of the video can be selected using the fps= argument.

It is possible to select a specific video codec for the output video using the video_codec= argument. The default video codec is libx264.

Image correction and modification

Background correction

Assuming the input array is a stack of image (> 1 frame), microImage can be used to remove the static background of the stack using the function backgroundCorrection()

from microImage import backgroundCorrection

correctedArray = backgroundCorrection(imageArray, signed_bits=True, average='median', correction='division')

If the images come from a signed bit array, it should be specified using the argument signed_bits.

The type of average used can be selected between mean or median, and the type of correction has to be picked between division and subtraction, using their respective arguments.

Contrast correction

The contrast of the image contained in the array can be modified with the function contrastCorrection()

from microImage import contrastCorrection

correctedArray = contrastCorrection(imageArray, min=None, max=None, percentile=10, percentile_min=None, rescale=True)

The user can specify either the min and max pixel value, or can ask the function to calculate it based on percentiles. The min value is calculated based on percentile_min and the max based on percentile (from 100%, so 10 corresponds to 90%). If percentile_min is not given, the value of percentile will be used.

The output can be rescaled to the full bit depth with rescale=True. If left False, the scale will be based on the old min and max pixel values.

Displaying the pixel value distribution

It is possible to display the pixel value distribution of the image array, along with the position of the min and max values calculated by the contrastCorrection() function. This can be done with the showPVD() function.

from microImage import showPVD

showPVD(imageArray, n_bins=10000, min=None, max=None, percentile=10, percentile_min=None, log_scale=None)

The function will display the pixel value distribution as an histogram. The number of bins can be controlled by the n_bins= argument (default is 1000). The scale of the histogram can be switched to log using the log_scale= argument. If left to None, both axes will be shown as normal scales. "x" will use log scale for the X-axis, "y" for the Y-axis, and "xy" (or "yx") will use log scale for both axis.

All other arguments are similar to the one of the function contrastCorrection(), besides the rescale= which cannot be used here.

Crop the image

The image can be cropped using the cropImage() function

from microImage import cropImage

croppedArray = cropImage(imageArray, top_left=(200,200), bottom_right=(800,800))

Each limit should be given as (X,Y) coordinates. If left to default, top_left will be equal to (0,0) and bottom_right to (Xmax, Ymax).

Generate a montage

A stack of images can be turned into a montage of individual frames in a single image. This is done by the function makeMontage()

from microImage import makeMontage

montageArray = makeMontage(imageArray, frames=5, column=None, row=None, margin=20, white_margin=False)

The arguments frames= define the frames that has to be added in the montage. If an integer is given, the function will save every N frames of the stack. If a list is given, the list will be used as list of incides of the frame to save.

The general shape of the montage can be selected using the arguments column= and row=, respectively the number of columns and rows making the montage table. If any or both of these arguments are set to None, the function will automatically calculate what is required.

The arguments margin= allows for drawing margin between each pictures in the montage. If different from 0, it will be used as the thickness in pixels. The color of the margin is selected via the argument white_margin=.

Writing labels on images

The module microImage includes some simple tool to quicky write labels on the images

Scale bar

It is possible to add automatically a simple scale bar on the image using the addBar() function

from microImage import addBar

modifiedArray = addBar(imageArray, scale_length=5, thickness=50, padding=20, white_bar=True, space_unit='µm', space_scale=46.21, add_text=True)

The scale and space units can be specified using the arguments space_scale= and space_unit= respectively. If None are given, the function will assume a scale in pixel. The length of the bar (in the given space scale and unit) can be selected using scale_length=. The thickness and the padding surrouding the bar car be selected with their eponymous arguments. User can select between white and black bars using the white_bar= boolean argument.

It is possible to add text on top of the bar with the add_text= boolean argument. User can select the font= .ttf file to use (default: Arial.ttf) and the font size with the font_size= argument. If let empty, the function will automatically the font size to match the scale bar length.

Time stamps

To add time stamps on an image stack, one can use the function addTime()

from microImage import addTime

modifiedArray = addTime(imageArray, position='top', padding=20, white_text=True, time_unit='s', time_scale=1/200)

Position of the stamp can be selected between top and bottom using the position= argument. The scale and time units can be specified using the arguments time_scale= and time_unit= respectively. The color of the text is selected using the argument white_text=.

The text options are the same as the one for the addBar() function.

Using the ImageStack class

It is possible to extract the images in an ImageStack object rather than in an array. Using an ImageStack object will reduce the versatility as compared to an array, but make the image processing easier to perform.

Loading the image in the class

To load an image and generate an object, we use the function loadImage()

from microImage import loadImage

image = loadImage('./path/to/folder/or/image.image_extension')

Additionally, the ImageStack class can be loaded with an array instead of an image file using the loadArray() function

from microImage import loadArray

image = loadArray(imageArray, name='test array')

It is essential that the imageArray object here is a 3-D array of dimension (number of frame, Y, X)

The element returned by each of these functions is an object with the following attributes:

Name	Type	Description
`image.name`	String	Name of the image stack (usually taken from the path)
`image.n_frames`	Int	Total number of frame in the stack (1 if single image)
`image.size`	2-D Tuple	Size of the array in each dimension (Y,X)
`image.source`	3-D Array	Array used to generate the class instance. Is used to reset all corrections. Dimensions are (t,Y,X)
`image.array`	3-D Array	Originally copy of `image.source`, all modifications and corrections are applied to this array only (except crop). Dimensions are (t,Y,X)
`image.frame`	ImageFrame object	Instance of the class ImageFrame used to handle single frame images. See below
`image.frame_nbr`	Int	Index of the current frame being loaded in `image.frame`

To display the frames, the ImageStack class is calling another class named ImageFrame. This class has the following attributes

Name	Type	Description
`frame.raw`	2-D Array	Non-contrast corrected version of the frame being displayed. Used for calculation. Dimensions are (Y,X)
`frame.corrected`	2-D Array	Array containing the frame being displayed, eventually with the selected contrast correction. Dimensions are (Y,X)

Navigate in the frames and display them

The frame loaded in the ImageFrame class for image processing can be selected using the command .setFrame()

image.setFrame(12)

To dislay the frame (using the matplotlib library), just call the .show() command.

image.show(show_raw=False, cmap='gray', title=True)

The user can select whether the raw or contrast corrected image can be displayed, the color map and if the title should be displayed or not on the image.

Duplicate and modify the image

It is possible to create a copy of the ImageStack object anytime by using the command .duplicate()

new_copy = image.duplicate()

All previous modification made on the initial object will be pasted into the copy.

The image can be cropped to a much smaller size with .crop().

image.crop(top_left=(200,200), bottom_right=(800,800))

The change will affect all image arrays in the ImageStack object, but also in the ImageFrame one. Check the cropImage function for details on the arguments of the function.

In the case of a stack of several frames, the stack can be reduced to a subrange of frames with the command .reducedRange()

image.reducedRange(first=10, last=20)

If not specified, the first and last frame of the new subrange will be calculated respectively as the first and the last frame of the image.source array.

Apply a correction on the image

Background correction similar to the one of the backgroundCorrection() function can be applied with the .backgroundCorrection() command

image.backgroundCorrection(imageArray, signed_bits=True, average='median', correction='division')

Contrast correction similar to the one of the contrastCorrection() function can be applied with the .contrastCorrection() command

image.contrastCorrection(imageArray, min=None, max=None, percentile=10, percentile_min=None, rescale=True)

The effect of the contrastCorrection on the pixel value distribution can be assessed with the .showPVD() command. Check the documentation on the showPVD() function above for more details.

image.showPVD(min=None, max=None, percentile=10, percentile_min=None, n_bins=1000, log_scale='xy')

All modifications can be reset anytime using the .reset() command.

image.reset()

This will cancel any background and contrast correction, but not modifications made by the .crop() and .reducedRange() commands.

Modify the space and time scale of the image

Set the scales using the setScale() function

image.setScale(time_scale=1/200, time_unit='s', space_scale=46.21, space_unit='µm')

The time_scale= should be given in unit/frame and the space_scale= in unit/pixel. Only the called arguments will be modified, the one set to None or not added will be left unchanged.

Add a scale bar on the image(s) using the scaleBar() function

image.scaleBar(frame=0, scale_length=4, add_text=False)

The argument frame= can used to select on which specific frame the scale bar should be added. If set to None, the scale bar will be added on all frames.

Refer to the function addBar() above for description of other arguments.

Add time stamps on an image stack using the timeStamps() function

image.timeStamps(white_text=True)

Refer to the function addTime() above for description of all arguments.

Save the image(s) in a file

The command .saveStack() will save the whole stack saved in the ImageStack instance.

image.saveStack('./path/to/new/file.tif', bit_depth=16, rescale=True, save_raw=False)

The save_raw argument allows the user to select if the corrected or raw array should be save. Check the saveImage function for details on the arguments of the function.

To only save the current frame in the ImageFrame instance, the command .saveFrame() can be used.

image.saveFrame('./path/to/new/file.tif', bit_depth=16, rescale=True, save_raw=False)

To save a montage of a selection of frame, the command .makeMontage() can be used

image.makeMontage(name='./path/to/montage.tif', frames=[0,2,4,10], bit_depth=16, rescale=True)

Refer to the function makeMontage() above for description of all montage-related arguments of the command.

To save a video file, the command .saveVideo() cam be used

image.saveVideo(name='./path/to/montage.mp4', fps=25)

Refer to the function saveVideo() above for description of all video generation arguments of the command.

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