Li-Cor Odyssey: How the CLx Imaging System Changed Quantitative Western Blotting

What the LI-COR Odyssey Brings to the Lab

The LI-COR Odyssey CLx near-infrared fluorescent imaging system is built for quantitative Western blotting and multiplexed protein analysis. Unlike traditional chemiluminescence or single-color fluorescence readers, the Odyssey uses two laser excitation sources—685 nm and 785 nm—to detect two separate fluorophore labels in a single scan. This allows researchers to load a housekeeping protein and a target protein on the same membrane and measure both signals without strip-and-reprobe workflows, cutting hands-on time and reducing variability between replicates.

For labs running dozens of blots a week, the elimination of secondary antibody stripping and reprobing is not a minor convenience. It directly improves reproducibility because the same membrane provides both data points under identical transfer and blocking conditions. The system's linear dynamic range, typically cited as spanning over four orders of magnitude, means that both low-abundance signaling proteins and highly expressed structural proteins can be quantified in one pass without signal saturation.

How the Dual-Laser Detection System Works

Near-infrared fluorescence avoids many of the background problems that plague visible-range detection. Biological samples absorb little light between 700 nm and 800 nm, and common laboratory plastics and buffers are largely transparent in this window. The Odyssey CLx exploits this by exciting IRDye 680 and IRDye 780 conjugates independently, then separating the emission signals digitally so that each channel can be quantified without spectral bleed-through.

Laser Sources and Detectors

• 685 nm laser: Excites IRDye 680 and other near-infrared dyes with emission peaks around 700 nm.
• 785 nm laser: Excites IRDye 780 and similar dyes, with emission detected above 800 nm.
• Solid-state detectors: The Odyssey CLx uses non-cooled infrared detectors that remain stable over long acquisition times, reducing warm-up drift and eliminating the noise associated with thermal detectors.

Signal-to-Noise and Dynamic Range

Because the system scans in the near-infrared, autofluorescence from membranes, gels, and blocking agents is minimal. This yields a high signal-to-noise ratio that is especially important when quantifying low-expression targets, such as phosphorylated signaling proteins from limited starting material like microdissected tissue or rare primary cell populations.

Key Features of the Odyssey CLx

The Odyssey CLx occupies a specific niche between older Odyssey models and high-end fluorescence imagers. Its feature set centers on quantitative accuracy and ease of use rather than maximum speed or the broadest dye compatibility.

• Dual-channel scanning: Simultaneous or sequential detection of IRDye 680 and IRDye 780.
• Quantitative analysis software: Image Studio Lite provides lane- and spot-based densitometry with background subtraction and molecular weight calibration.
• Continuous and step scanning modes: Step mode improves resolution for low-abundance bands; continuous mode speeds up scanning of strong signals.
• Membrane and gel compatibility: Supports PVDF, nitrocellulose, and agarose gels up to standard gel casting dimensions.
• Small footprint: The instrument fits on a standard benchtop, which matters for labs where the imaging unit shares space with other equipment.

Common Applications

The Odyssey system is most often used for Western blotting, but its applications extend to any workflow that benefits from quantitative, multiplexed near-infrared fluorescence.

• Relative protein quantification: Comparing target expression across multiple samples normalized to a loading control.
• Post-translational modification analysis: Detecting phosphorylated, ubiquitinated, or acetylated proteins alongside total protein levels.
• Antibody validation: Confirming specificity and linearity of detection for a new antibody pair.
• Multiplexed protein detection: Measuring two or three proteins on one membrane to compare their relative abundance or co-localization.

Odyssey CLx Versus Older Models and Competitors

The CLx replaced the earlier Odyssey Fc and Odyssey CL models in many core facilities. Compared to the Fc, the CLx offers improved detector sensitivity and a more modern software interface, while maintaining compatibility with the same IRDye reagents. Versus chemiluminescence-based systems, the Odyssey trades the broadest dynamic range of some ECL substrates for better inter-lane quantification precision and multiplexing capability. Versus other near-infrared imagers, the CLx balances cost and performance, and its use of LI-COR's proprietary dye chemistry is a consideration when building a reagent purchasing strategy.

Practical Considerations for Labs

Adopting the Odyssey CLx requires matching the instrument to LI-COR's IRDye secondary antibodies and, ideally, fluorescently labeled primary antibodies. The system's quantitative strength depends on consistent blocking, transfer, and antibody incubation protocols; near-infrared fluorescence does not compensate for poor membrane blocking or uneven antibody binding. Labs should also plan for the recurring cost of IRDye-conjugated antibodies, which carry a premium over HRP conjugates but are re-usable in multiplex formats.

Regular calibration using the built-in lane quantification standards helps maintain inter-run consistency, particularly when comparing blots across days or between users. Training on the Image Studio Lite software is minimal, but learning to set appropriate scan intensities and background subtraction parameters is necessary to avoid under- or over-saturation of signals.