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General Recommendations

If there is a file format that saves metadata, use it. Metadata is data about the data, which in this context typically means the acquisition settings. Saving a file with metadata will allow you to automatically record all of those acquisition settings, and reuse them. This is very useful when you are trying to ensure the reproducibility of your experiments.

Never save images as JPEGs. This file format is fine for pictures of loved ones, cute animals and pretty sunsets, not for scientific data. In addition to losing all the metadata detailing how you took your images you will also compress the data in a “lossy” manner, which means it will be irreversibly modified and degraded.

Specific Recommendations for Different Software and Scope Combinations

Zeiss Zen (LSM900, LSM710):

Use the czi format. This retains the metadata about how the image was acquired, lets you reuse the settings, is well read by our deconvolution software (Autoquant), can be opened in FIJI, and its metadata can be read properly by the free version of Zeiss’ software (Zen lite black for LSM710, Zen lite blue for LSM900) in Windows 10. While FIJI can access the metadata of a .lsm or .czi file, it is formatted in a way that makes it cumbersome to read.

Volocity (BX61 – Neville, IX81 – Luna):

Volocity does not save image metadata, so you will need to keep your own record of the conditions you used to acquire data (exposure, gain, offset, binning, channels, ND filters on BX61, LED power on IX81). For more information on what settings you should keep track of, contact the MSL staff or read our guidelines.

Images Acquired with the Retiga Camera (Color)

Save images in the TIFF format. Make sure you click on the “Options…” button and ensure “Convert to RGB for publication” is clicked and “Scale” is unclicked. You must do this each time you save a subset of files, as there is a bug in the software that results in an incorrectly saved image if you don’t do this every time, even if the save options have not been modified. For best results, flatfield your brightfield images you can review some instructions and links to macros to do this.

Images Acquired with the Hamamatsu Camera (Fluorescence)

Save images in the OME-TIFF format, not TIFF. If you will be using these images for measurements in FIJI, and/or for displaying in presentations or papers, we have written Volocity to FIJI conversion macros.

If you are going to deconvolve Z-stacks acquired in Volocity (BX61), it will be easier for you to first convert them into the Imaris file format using the Imaris file converter, and then to open them in Autoquant.

Metamorph (IX81 – Luna, Andor XD – Hagrid):

When you save a file it will save a single TIFF file per channel, timepoint, and location in a multidimensional experiment. If you are doing Z stacks, each of those saved files will be a hyperstack with multiple Z slices. In addition to all of these files, the software will save an “.nd” file every time you press the “acquire” button. This nd file is critical, so make sure you keep it together with its corresponding dataset. If you drag and drop the nd file into FIJI, FIJI will read this file and organize the data from the experiment correctly into hyperstacks with the proper dimensions. In addition, nd files can be read by our deconvolution software (Autoquant, though see note below), as well as by the Imaris File Converter.

If you have many positions and timepoints you may find it easier for subsequent analysis to use a macro to reorganize the data into single hyperstacks (one per position). Please ask MSL staff for help if you need to do this.

To save all of your settings, make sure you save the “state” file when you do a multidimensional acquisition.

Note on deconvolution of Metamorph file: There is a bug in Metamorph such that certain types of acquisitions result in file names that don’t match the nd file information and are not read properly in Autoquant. Please ask MSL staff for scripts we have written that will allow you to work around this problem.

Imspector Pro (UltraMicroscope II – Snape):

Make sure the autosave option is on. Data will be saved in a folder with many OME-TIFF files in it, with one file per channel, Z position, and location. Do not remove any of these files from the folder, and do not put any files into this folder, to ensure proper metadata retention. Note that the metadata will be written to the image files corresponding to the first Z position of each channel and location.

You will need to record all metadata on your own: laser power, light-sheet NA, light-sheet width, channels, light sheet side(s), number of light-sheets, Z spacing, exposure, overlap in tiling positions (if applicable), cropping (if applicable).

If you are doing a large tile and accidentally clicked the “split” setting for the Z setting, in the older version of the software, MSL has written a macro to sort through the resulting metadata mess (each Z position will be interpreted as a different channel if you don’t reorganize the data). Please let us know if you need it.

Fusion (Andor Dragonfly – Hermione):

This software saves files natively in the Imaris format (.ims). Settings from these files can be reused in the Fusion software. Unfortunately, the metadata is not easily accessible in Imaris or FIJI, so you should take manual notes of important settings, including objective, camera, pinhole size, exposure time, laser power, EM Gain (if applicable), timelapse intervals (if applicable), Z stack intervals (if applicable), stitching settings (if applicable), binning settings, cropping settings, etc.

Autoquant (Workstation – Minerva):

Save output in the Bitplane .ims file format. For images taken on the LSM900, Dragonly, IX81, and Hagrid use the 32-bit floating point option; this is absolutely critical for proper intensity comparisons between images. For images taken on the BX61 or LSM710 you can use 32-bit floating point or 16-bit unsigned integer. These formats can be opened by Imaris, and thus are useful for 3D analysis. They can also be read by FIJI, and result in a single file. Other formats result in multiple files, and/or are in less convenient formats.

MSL can provide instructions for using Autoquant in batch mode, which will dramatically speed up deconvolution of many files.

Retired Software and Scope Combinations

NOTE: this microscope was retired in August of 2022. We include its information here only for reference.

Use the czi format. This retains the metadata about how the image was acquired, lets you reuse the settings, is well read by our deconvolution software (Autoquant), can be opened in FIJI, and its metadata can be read properly by the free version of Zeiss’ software (Zen lite black) in Windows 10. While FIJI can access the metadata of a .lsm or .czi file, it is formatted in a way that makes it cumbersome to read.

NOTE: this microscope was retired in July of 2021. We include its information here only for reference.

Save images as OME-TIFFs. If you will be using these images for measurements in FIJI, and/or for displaying in presentations or papers, we have written Volocity to FIJI conversion macros.

NOTE: this microscope was retired in July of 2021. We include its information here only for reference.

Save in .vsi format. Note that this results in a vsi file with metadata, a folder, and an .ets file in that folder.

Metadata is retained as long as you keep everything together, so make sure you don’t move some of the files without the others. To open .vsi files in FIJI, use File/Import/Bio-Formats. Dragging the .vsi file to FIJI will not work. Dragging the .ets file to FIJI will load the file and metadata, but the name will be a generic name (like frame_t_0.ets) that will not be the name you used to save the image file.

NOTE: this microscope was retired in July of 2021. We include its information here only for reference.

When you save a file it will save a single TIFF file per channel, timepoint, and location in a multidimensional experiment. If you are doing Z stacks, each of those saved files will be a hyperstack with multiple Z slices. In addition to all of these files, the software will save an “.nd” file every time you press the “acquire” button. This nd file is critical, so make sure you keep it together with its corresponding dataset. If you drag and drop the nd file into FIJI, FIJI will read this file and organize the data from the experiment correctly into hyperstacks with the proper dimensions. In addition, nd files can be read by our deconvolution software (Autoquant, though see note below), as well as by the Imaris File Converter.

If you have many positions and timepoints you may find it easier for subsequent analysis to use a macro to reorganize the data into single hyperstacks (one per position). Please ask MSL staff for help if you need to do this.

To save all of your settings, make sure you save the “state” file when you do a multidimensional acquisition.

Note on deconvolution of Metamorph file: There is a bug in Metamorph such that certain types of acquisitions result in file names that don’t match the nd file information and are not read properly in Autoquant. Please ask MSL staff for scripts we have written that will allow you to work around this problem.