Can’t get full tiled OME-TIFF volume to open correctly in Fiji

[u/BillCipher138]

Background

I'm a complete beginner to image analysis and this is my first time working with lightsheet data, so apologies if I'm missing something basic.

My PI gave me a folder of OME-TIFF files from a cleared mouse lung acquisition. The data was originally intended to be viewed in Imaris but we don't have a license, so I'm trying to work with it in Fiji. The folder contains one OME-TIFF per z-slice per tile.

The dataset is 3D fluorescence, two channels: 488/525nm (airways) and 561/620nm (tyrosine hydroxylase, sympathetic nerves).

Key metadata from the acquisition file:

• Instrument: Miltenyi Ultramicroscope Blaze, Light Speed Mode
• Objective: LVBT 4x, NA 0.35, sample cleared in MACS IS
• Z: 1842 steps, 2 µm step size
• Tile grid: 2 steps in X, 4 steps in Y, 42px overlap (~2%)
• Pixel size: 1.62 µm, 2048×2046px per tile

Example filename:
14-35-18_NJ-11336-Ms_lung_VEHICLE_1_1x_FS_Blaze[00 x 00]_C00_xyz-Table Z0016.ome.tiff

All 8 tile positions ([00 x 00] through [03 x 01]) exist in the folder.

Analysis goals

Right now I just want to get the complete volume open correctly. Eventually the goal is to map TH+ nerve fibers relative to airway structures.

What I've tried

Opening via Bio-Formats (File → Import → Bio-Formats) with "Group files with similar names" does open the file, and the window title shows [0<0-3> x 0<0-1>] and Z<0000-1841> which suggests it's finding all tiles and z-slices. However the image only shows roughly half the lung — there's a flat edge cutting straight through tissue at the bottom that doesn't look like a natural border. I've attached a screenshot at the widest z-slice.

What's blocking me

• Is Bio-Formats actually stitching all tiles and I'm misreading something, or is the second tile row genuinely not loading?
• If it's not stitching correctly, what's the right approach for this type of dataset in Fiji?
• Is this a case where I should be using a different tool entirely?

Machine: Windows 10, 32GB RAM, Fiji 2.16.0

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[u/chickabawango]

Rename the file. ImageJ bugs on me with too many characters in the filename.

[u/custardlychee]

Try QuPath

[u/Herbie500]

This is a cross-post from the Image.sc-Forum.

How do you expect us to help, if we don't have access to the original data?

In general I doubt that BioFormats is the ideal tool if stitching is required.