Loading '.nd2' file into Matlab

Overview

This is a small tool to load '.nd2' image stack into Matlab with 'ND2SDK' Windows (The Linux version is provided here). The SDK is provided here by Laboratory Imaging.

For now, only uint16 mono-color .nd2 file is supported. The reader can get the infomation of channel infomation and loops infomation of the file.

Knowing issues:

1. Now we cannot get metadata from some spesific .nd2 file with the ND2SDK. For these files we use the information in TextInfo to get the channel and loop information.
2. For the files that show empty metadata in the script, we can neither get the frame metadata. Therefore, the SeqInfo funtion do not work for these files.
3. By comparing the information in TextInfo and ExperimentInfo, I found that there are some difference in loop information if you set multipoints in xp position loop but not check all the choice box during capturing. ExperimentInfo record all the points you set in nd2capture while TextInfo only record the points that you really captured during the expriment.

Usage

Load the library

Matlab provides function loadlibrary to load C / C++ libraries into Matlab. You need to configure for C language compilation in your Matlab.

mex -setup

If you do not have any compiler, you can add 'MinGW64 Compiler' in 'HOME' > 'Add-Ons' follow the image below.

Once you set up the C compiler, you should able to sucessfully loading the library by command,

loadlibrary('Nd2ReadSdk', 'Nd2ReadSdk.h')

Noticed that you should add the 'SDK' (both 'include' and 'windows' folder) into Matlab 'Path'.

Read the infomation in '.nd2' file

You can run the command below to get the information from the file. FileName should contain the full path and extension name, such as 'D:\Data\Test.nd2'

[ImageInfo] = ND2Info(FileName)

The function will runture a structure ImageInfo contains the width, height, number of images and also channel numbers. Also the experiment details are recorded in ImageInfo.metadata and ImageInfo.Experiment.

The infomation will be print on the command window such as,

There are 3608 images in 2 channel(s).
The No. 1 channel is: Phase Contrast 100ms
The No. 2 channel is: GFP 100ms
Images are captured in 2 layer(s) of loops
The No. 0 layer is: 164 TimeLoop
The No. 1 layer is: 11 XYPosLoop

The total images number should be same as the product of loops number in each layer and also the channels number. These images are organised as the figure below.

Read image.

If you want to read a specific image in a '.nd2' file. You can use the command below to read the image with the specific sequence index (not the image index) Num.

[Image] = ND2ReadSingle(FileName, Num)

If your '.nd' file has multiple channels, i.e. Components, these channels would be stored in different cells in Image as Image{1}, Image{2},....

If you want to read out multiple images at once, you can specify the image index as Num = [1,2,3...]. The result Image will be in a 3D array in Matlab. Additionally, multiple channels images will be stored in different cells and each cell contains the 3D matrix of the image stack.

If you haven't spicified the image index Num. [Image] = ND2ReadSingle(FileName) will read all the images in the file into Image.

Here, Single means store all the data into a single variable.

TIF stack output

The ND2TIF function can save the .nd2 stack into .tif stacks based on loops and channels.

ND2TIF(FileName)

Running the above command will seprated your image into .tif stacks of the highest level loops (layer 0, see 2.2 above).

You can also control the output .tif stack with Parameter as below. You can find the corresponding parameters in 3.2 below.

ND2TIF(FileName, 'Parameter', value)

Advanced usage

Read images with a loop

If your '.nd2' file is very huge and not able to read all the data into the memory. You can also read the image with a for loop. But, do not use ND2ReadSingle in a loop as each calling will open and close the file pointer once.

You would better create a file pointer, read the images you need, and then close the pointer. Here is an example.

[FilePointer, ImagePointer, ImageReadOut] = ND2Open(FileName);
Num = 2:2:100;
for i =1:size(Num, 2)
    Image = ND2Read(FilePointer, ImagePointer, ImageReadOut, Num(i));
end
ND2Close(FilePointer)

Select .tif output channel and merge montage stack.

You can control output .tif stack length by setting ..., 'Layer0', Index such as,

ND2TIF(FileName, 'Layer0', 2:2:100)

Each output .tif stacks will only contain the 2:2:100 of the layer0 loop.

You can choose the specific stack or channel by setting ..., 'Layer1', Index and ..., 'Channel', Index

ND2TIF(FileName, 'Layer1', [2, 3, 4], 'ChannelIndex', 2)

This will only generate 3 .tif stacks, i.e., the 2nd channel of No. 2, 3, 4 in layer 1 loops.

You can also generate a montage image by setting 'Montage', 'on'. You can make montage in layer 1 loop and also channels. You can arrange the output montage image with a matrix index.

ND2TIF(FileName, 'Montage', 'on', 'Layer1', [2, 3; 4, 5])

This will generated a montage stack in the arrangement below.

The images in different channels will be seprated into different montage stacks. If you want to montage images in differen channel, you can try,

ND2TIF(FileName, 'Montage', 'on', 'Layer1', [2, 3; 4, 5], 'ChannelMontage', 'on')

You can also set the arrangment of each channel by set 'Channel' with a matrix.

Image compress and resize

You can set 'Compress', 'on' to compress the images into 8 bits .tif stacks. The images will be compress to 0-255 with the min and max value of the first image in the stack.

ND2TIF(FileName, 'Compress', 'on')

You can set 'Resize', 1080 to resize the height of output .tif stack to 1080p.

ND2TIF(FileName, 'Resize', 1080)

For montage stacks, the value contols the final output iamge size of the stack. You can set the value to other number as you with, such as, 'Resize', 1024 or 'Resize', 720

Controling parameters

Parameters	Value
Layer0	The index of the layer0 loop, length of the .tif stack. For example, 1:100
Layer1	The index of the layer1 loop. If Montage is 'on', the images will be combined same as the matrix. For example, [1,2;3,4]
Channel	The index of the channels. If ChannelMontage is 'on', the images will be combined same as the matrix. For example, [1,2]
Montage	Control if combined the images in layer1 loop into one stack. For example, 'on', 'off'.
ChannelMontage	Control if combined (side by side) the images in different channels into one stack. For example, 'on', 'off'.
ChannelConnect	Control if link the images in different channels into one stack. For example, 'on', 'off'.
Compress	Control if compress the images into 8 bits. For example, 'on', 'off'.
Resize	Control if resize the images. For example, 1080, 'off'.

For now, set 'ChannelMontage', 'on' will automatically set 'Montage', 'on' and 'Compress', 'on'； set 'Resize', 1080 （or other number） will automatically set 'Compress', 'on'; set 'Resize', 'on' will automatically set 'Resize', 1080.

Sequence infomation

You can get the time and position information of each frame in the sequence by

[SeqTime,SeqPosition] = SeqInfo(FileName,Num)

If your .nd2 file contains multiple channels. The infomation will be stored into cells as Seqtime{i}, SeqPosition{i}.