🔬📂 Three things to know about microscopy image files

‍The proper colocalization experiments’ execution depends on having a comprehensive understanding of the image files involved. Being familiar with the contents, organization, and use of these files is essential for achieving reliable and reproducible results. Here are three key aspects to consider regarding microscopy image files:

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‍1. Contents

‍Microscopy image files consist of two main components: pixel data and metadata.

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‍Pixel data:

• Consists of one or more frames of pixels (XY, XZ).
• Each frame typically represents a two-dimensional array of pixel values.
• Pixel values can indicate light intensity, an array of intensities, or an array of fluorescence lifetimes.
• Properties of the frames in stacked images include channel type (lifetime, color, etc.), Z-stack information, XY plate position, and time.

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‍Metadata:

• Specifies the image type (e.g., TIFF, LSM, LIF).
• Includes pixel dimensions (size, focus position, time point, etc.).
• Contains hardware settings, such as the objective lens used, the source of excitation light, and emission settings.

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‍Important data not stored in your image file:

• Sample details, including tissue types and methods of collection.
• Sample preparation steps, including fixation techniques and visualization procedures.
• Information regarding labeling protocols, such as the types of fluorescent proteins or antibodies and their concentrations.
• Types of mounting media or antifade agents utilized.

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‍2. Formats

‍Microscopy image files are available in various formats, which can categorized into general image file formats and proprietary image file formats.

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‍General image file formats:

• TIFF (Tagged Image File Format) — this format is relatively large, but uses lossless compression, preserving all important image information.
• PNG (Portable Network Graphic) — this format has an intermediate size and also uses lossless compression.
• JPEG (Joint Graphics Expert Group) — this format is smaller in size, but employs lossy compression, which can reduce image quality.

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‍Proprietary image file formats:

• Major microscope manufacturers have developed specific (proprietary) microscopy file formats to organize and store image data.
• The most common proprietary formats include .LSM (Zeiss), .LIF (Leica), and .OIF (Olympus). 
• These formats are typically designed to restrict image data usage to the manufacturer’s software and hardware.

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‍3. Size

‍The sizes of microscopy image files vary greatly depending on the type of microscopes used for their acquisition and the acquisition technique. On average, a single confocal microscopy image is usually 2-3 MB, while a single widefield microscopy image is 5-8 MB. The image size grows for XYZ stacks images, depending on the number of stacks, ranging from 50-500 MB. The size of single PALM/STORM images is usually in the GB range.

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‍Conclusion

‍Microscopy image files contain a wealth of information; however, important details concerning tissue preparation and sample labeling are often excluded. The most suitable format for microscopy image files is uncompressed TIFF. It is also important to note that the size of files increases dramatically when capturing image stacks.

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