Image Formats

This page contains essential information about image formats, how they function in Image Studio™ Software, and tips for accurate quantification.

The wide range of imaging modalities available in the Odyssey^® M Imager require a wide range of image formats. Empiria Studio^® Software and LI-COR^® Acquisition Software already supported this range of image formats, but support for some formats was introduced in Image Studio version 6.0 to work with the Odyssey M. Given the huge range of functionality in Image Studio and the expanded support for additional image formats, it is worth learning about the new formats and how Image Studio handles them.

Definitions

Image: Within LICORbio software, “image” refers to a visual depiction of a sample in a single channel. Each pixel within the image corresponds to a specific location on the sample.
Channel: Specific wavelength(s) of light used to produce an image. In fluorescence imaging, channels are sometimes referred to by a name that contains both excitation and emission wavelengths, and sometimes they are referred to by a nominal excitation wavelength.
Acquisition: Image(s) from one or more channels grouped with image metadata. Acquisitions are imported/exported between LICORbio software applications using zip files to group the images and data together and to keep them organized.
Pixel: The smallest area unit of an image. Depending on the image format, each pixel in the image will be represented by either A) a single numeric value that is proportional to the intensity of light detected at the pixel’s location or B) the pixel will be represented by a set of numbers that indicate the red, green, and blue components of the light detected at the pixel’s location.
Pixel Intensity: A number that is proportional to the light detected at a pixel’s location.
RGB: RGB is an acronym for “red, green, blue” - the three primary colors that are combined to create full-color images. Each pixel in an RGB image contains a set of numbers - one number that represents the red value for the pixel, one number that represents the green value for the pixel, and one number that represents the blue value for the pixel. Computer software and monitors display the pixel’s color by blending these three components proportionally.
Image format: In this explanation, “format” refers to the format of the image file (e.g., .tif, .png, .jpg).
Image type: In this explanation, “type” refers to the imaging modality that produced the image, e.g., fluorescence, Epi, Trans, RGB.

Image Type Reference

Type 1: Fluorescent and Luminescent Images

Number format for pixel values: Half-float
Number format standard: IEEE 754
Bit depth: 16 bit
Example applications: Fluorescent Western blots, In-Cell Western^™ Assay
Available analyses: All analysis types (Manual, Western, MPX Western, DNA Gel, Plate, Plate Array, Grid, Grid Array, In-Cell Western, Small Animal Image)
Available quantification values: Signal, Total, StdDev, Width, Height, Area, X, Y, Width, Height, Min, Max, Mean, Median, Bkgnd., Bkgnd. StdDev, Trim Bkgnd., Trim Bkgnd. StdDev, Trim Mean, Trim Signal, Trim StdDev; Note: Concentration values (Conc. Std. and Concentration) are computed using a user selection of Signal, Total, Mean, Median, Trim Signal or Trim Mean.

Type 2: Epi-Illumination Images

Number format for pixel values

Unsigned Short Integer

Number format range

0 to 65,535

Bit depth

16 bit

Image format description

Epi images represent reflective light from a sample. Pixels within Epi images are unsigned integer values that range from 0 to 65,535. In versions prior to Image Studio 6.0, third-party images are automatically converted on import from the unsigned integer format to the half-float format. A major new feature of Image Studio 6.0 is support for the unsigned integer format used for both Epi and Trans images types. When an Odyssey Epi or Trans image is imported into Image Studio 6.0, the unsigned integer format is retained and the image is not converted to half-float format. Earlier versions of Image Studio marked values in imported images above 65,504 as infinity, but Image Studio 6.0 does not have this limitation. In Epi images, it is common for the sample area to have lower pixel values than background region. In prior versions of Image Studio, Image Studio used a heuristic to determine when images should be inverted to make sample areas have higher pixel values than the background, but Image Studio 6.0 provides an Invert button so that the analyst can choose when to invert an image.

Example applications

Western blot membranes using colorimetric substrates or stains; Colorimetric MW markers used in chemiluminescent Western blots

Available analyses

All analysis types (Manual, Western, MPX Western, DNA Gel, Plate, Plate Array, Grid, Grid Array, In-Cell Western, Small Animal Image)

Available quantification values

Signal, Total, StdDev, Width, Height, Area, X, Y, Min, Max, Mean, Median, Bkgnd., Bkgnd. StdDev, Trim Bkgnd., Trim Bkgnd. StdDev, Trim Mean, Trim Signal, Trim StdDev; Note: Concentration values (Conc. Std. and Concentration) are computed using a user selection of Signal, Total, Mean, Median, Trim Signal or Trim Mean.

Epi channel images provide the same quantification value types as fluorescent and chemiluminescent channel images, but the values are based on the integer pixels so the range of values will be different. These values are also typically not as linear as fluorescent channels, so some quantification values for Epi channel images should be used with caution.

Type 3: Transillumination (Transmitted Light)

Number format for pixel values

Unsigned Short Integer

Number format range

0 to 65,535

Bit depth

16 bit

Image format description

Pixels within Trans images are unsigned integer values that range from 0 to 65,535. In versions prior to Image Studio 6.0, 3rd party images are automatically converted on import from the unsigned integer format to the half-float format. A major new feature of Image Studio 6.0 is support for the unsigned integer format used for both Epi and Trans images types. When an Odyssey Epi or Trans image is imported into Image Studio 6.0, the unsigned integer format is retained and the image is not converted to half-float format. Earlier versions of Image Studio marked values in imported images above 65,504 as infinity, but Image Studio 6.0 does not have this limitation. In Trans images, it is common for the sample area to have lower pixel values than background region. In prior versions of Image Studio, Image Studio user a heuristic to determine when images should be inverted to make sample areas have higher pixel values than the background, but Image Studio 6 provides an Invert button so that the analyst can choose when to invert an image.

Example applications

Coomassie or silver staining of protein gels; H & E stained tissue sections

Available analyses

All analysis types (Manual, Western, MPX Western, DNA Gel, Plate, Plate Array, Grid, Grid Array, In-Cell Western, Small Animal Image)

Available quantification values

Trans channel images provide the same quantification value types as fluorescent and chemiluminescent channel images, but the values are based on the integer pixels so the range of values will be different. These values are also typically not as linear as fluorescent channels, so some quantification values for Trans channel images should be used with caution.

Type 4: Color Images

Number format for pixel values: Integer
Number format range: 0 to 255
Bit depth: 24 bit (8 bits for each component)
Image format description: Color images use a 24-bit RGB color model (i.e., 8 bits for red, 8 bits for green, and 8 bits for blue) to represent pixels within images. Each color component – red, green, and blue – are represented by 8-bit values that range from 0 to 255. Prior to Image Studio 6.0, 3rd party color images were imported by creating separate channels for each color component (R, G, and B). The pixel values are converted from the original value (between 0 and 255) to a half-float value. Image Studio 6.0 introduces support for RGB. Imported 3rd party color images are imported as native RGB.
Available analyses: All analysis types (Manual, Western, MPX Western, DNA Gel, Plate, Plate Array, Grid, Grid Array, In-Cell Western, Small Animal Image)
Available quantification values: Area, X, Y, Width, Height; Shapes added to RGB Epi and RGB Trans channels will not provide all quantification values that are shown for shapes on other image types. Quantification values that are not supported in Image Studio 6.0 for RGB images will be shown as NaN.