Vendor Odyssey-M-sole-source-features.pdf

Sole Source Justification for the Odyssey® M Imager

The Odyssey M Imager provides reliable data for membranes, plate-based assays, slides, and nucleic acid and protein gels. It is versatile and can capture assays in 18 channels, including white light, luminescence (i.e., bioluminescence and chemiluminescence) and fluorescence (i.e., visible and near-infrared (NIR)). The Odyssey M Imager uses infrared detection and laser excitation and sCMOS-based line imaging for optimal sensitivity; it also offers a wide range of illumination and no imager saturation in biologically relevant samples to overcome common band saturation issues. Together, these features enable the Odyssey M Imager to capture high quality images from a wide range of assays for publishable results.

The Odyssey M Imager pairs with LI-COR® Acquisition Software to capture assay images and Empiria Studio® Software to design experiments and analyze data. Empiria Studio offers step-by-step workflows for analyzing quantitative and qualitative Western blots, multiwell plates (e.g., In-Cell Western™ Assay), protein and nucleic acid gels, and microscope slides. The Odyssey M Imager, LI-COR Acquisition Software, and Empiria Studio Software work together to ensure user-to-user consistency and data replicability for high quality, publishable results.

User-to-User Consistency

Western blot assays are difficult to reproduce. A major contributor to this is user-to-user variability when setting up optical settings (e.g., exposure times and laser intensities) and capturing images.

Stepwise Workflows for Image Capture

The imager must require users to follow identical image capture procedures in order to obtain images of equivalent quality, decrease user-to-user variation and error, and increase data reproducibility. This is achieved with LI-COR Acquisition Software, which allows each user to connect to an Odyssey Imager and scan the assay using pre-designed, step-by-step workflows that help prevent user-to-user inconsistency. Unlike competitors, LI-COR Acquisition Software also automatically tags acquired images with assay type based on the selected analysis workflow. Images are then filtered by assay type when beginning a given assay workflow in Empiria Studio Software, which makes the process easier and faster.

Identical Optical Settings for Assay Replicability

The imager must not require users to adjust exposure times or optical settings (e.g., laser intensity) in order to obtain non-saturated images. All images from the system must be acquired using the same optical settings, which ensures assay replicability. This is only offered by Odyssey Imagers; if competitors claim that they can do this, this is not true and should be challenged.

Addressing User-to-User Inconsistency

To address user-to-user inconsistency due to manual lane finding and background subtraction, the following must be included in the solution.

Adaptive Lane Finding

Patent-pending (Patent-Pending #16/911,898) Adaptive Lane Finding (ALF) finds lanes automatically as opposed to manual lane finding, which can lead to variability. With ALF, user-to-user consistency and data reliability are improved. This is only offered by Empiria Studio® Software and Odyssey Imagers.

Adaptive Background Subtraction

Patented (#10,395,350) Adaptive Background Subtraction (ABS) adapts to an image’s background and automatically subtracts the background. With ABS, user-to-user variability is reduced to approximately 3 percent (1). This is only offered by Empiria Studio Software and Odyssey Imagers; if competitors claim that they can obtain this, this is not true and should be challenged.

Sensitivity

Sensitivity in Western assays is important when measuring expression changes in proteins that are low in abundance. There are three important variables that contribute to sensitivity and are necessary for the solution.

Laser Excitation

Excitation methods other than laser excitation are more prone to light leakage (e.g., LED). This raises the background noise and consequently reduces sensitivity.

Laser excitation is offered by Odyssey Imagers and allows for the detection of very weak and very strong bands alike.

Most competitive instruments do not offer laser excitation.

If they do, this is not sufficient for optimal sensitivity and requires additional variables.

Wide Range of Illumination

The instrument must offer laser excitation across a wide range of illumination to obtain the best sensitivity in all channels. The Odyssey M Imager uses laser excitation across nine channels (Patent-Pending #16/734,731). This allows for uniform illumination and high laser output across the samples and scan area. This results in high signal to noise ratios for highly sensitive detection in visible and NIR regions. Most instruments do not offer laser excitation across a wide range of illumination. If they do, this is still not sufficient for optimal sensitivity and reproducibility.

sCMOS-Based Line Imaging

To efficiently produce high sensitivity across the visible through NIR spectral range, this technology should also combine the best of laser point scanning and sCMOS image sensor technology in a line scanning system that incorporates differential scanning, bi-telecentric imaging, filtering and laser combining (Patent-Pending #16/734,731). This is critical for achieving both highly sensitive detection in visible and NIR channels and consistent image quality across the entire scan surface.

Only the Odyssey® M Imager offers laser excitation across a wide range of illumination in combination with sCMOS-based line imaging for consistent and sensitive imaging.

Saturation of Bands

Reviewers and editors are skeptical of band saturation in Western assays. The imager must have the following to overcome band saturation.

No Imager Saturation

The instrument must offer over 6 logs of dynamic range in a single acquisition—without post-acquisition manipulation—to ensure that the hardware is not contributing to saturation. This is only offered by Odyssey Imagers (Patent #8,410,454) (see specifications in the Odyssey M manual licor.com/m-manual). Competitor imagers claim to do this but cannot accomplish it within a single acquisition or without post-acquisition modifications.

Demonstration of Membrane Saturation

The software must automatically generate a combined linear range chart (Figure 1) to demonstrate the location in the dilution range where saturation begins (2). This is only offered by Empiria Studio® Software. Competitors claim to have this ability but can only accomplish it using multiple software packages—which makes the analysis process time-consuming and prone to error.

Figure 1.

In-Cell Western™ Assay Optimization Resources

The In-Cell Western Assay is an important assay for understanding protein expression within the context of the native cell. Necessary optimization steps require specialized expertise in order to accurately perform the assay. The supplier must demonstrate that they have the following resources to ensure assay optimization.

Antibody and Cell Line Database

The supplier must maintain and provide an updated database of antibodies and cell lines. This database must consist of antibodies and cell lines that have been tested to assess which combinations can be used effectively for the In-Cell Western Assay.

As inventors of this assay in 2005, LI-COR has assembled an extensive database of antibodies and cell lines used by LI-COR and other researchers around the world to accurately measure protein expression. This database includes over 9,000 antibody entries represented in over 2,000 publications and is available to all owners of Odyssey Imagers.

In-Cell Western Assay Software

The supplier must provide a software that assists in the experimental design of the In-Cell Western Assay. This software should include:

▪ Workflow for antibody titration and optimization

▪ Workflow for blocking buffer evaluation and optimization

▪ Workflow for cell stain linearity assessment

▪ Workflow for fixation and permeabilization evaluation and optimization

▪ Workflow for Z'-Factor determination

▪ Workflow for target analysis

Empiria Studio Software provides workflows that are based on industry best practices and guide researchers https://www.licor.com/m-manual through the validation, analysis, replicability, and publication stages of quantitative data analysis. For the In-Cell Western™ Assay and other assays, Empiria Studio® Software offers all six required workflows. No competitive software packages include workflows that standardize key experimental In-Cell Western steps.

Multiwell Plate Experimental Design

Software must be provided that allows researchers to chart experimental multiwell plate design. This decreases user error and simplifies plate preparation. Empiria Studio Software offers the Empiria Studio Experimental Designer, with Plate Templates that allow users to configure their multiwell plates well by well (e.g., positive and negative controls, background, and treatments). Researchers can also place a multiwell plate over the printed template to easily load the wells. Plate Templates are available for In-Cell Western, ELISA, and Cell Analysis assays.

In-Cell Western Assay Development Handbook

The supplier must provide an extensive handbook on the development of In-Cell Western Assays. This handbook should have sections dedicated to:

▪ Cell seeding

▪ Antibody titration

▪ Cell type selection

▪ Experimental controls selection

▪ Fixation and permeabilization evaluation

▪ Normalization strategy selection

▪ Proper replicate techniques

▪ Blocker evaluation

In addition, the handbook must include recommended plate layout templates and full protocols must be available for each step in the assay.

As the inventors of the In-Cell Western Assay, only LI-COR has this foundational resource to effectively run the In-Cell Western Assay. The In-Cell Western Assay Development Handbook can be found at licor.com/icw-handbook.

Online Training Modules for New Lab Members

Training laboratory personnel is an ongoing burden for labs. It introduces variability based on different methods or techniques as well as the skill level of the trainer and trainee. The following solution, offered with Odyssey Imagers, is in complete alignment with publisher best practices and manufacturers.

Online Training

Online learning paths should be provided that are always accessible for the students’ convenience.

Organization

A single, well-organized landing page must allow students to easily discern which learning paths are available.

Teaching Methods

Seminars must be available that present the science and best practices that are easily understood at an undergraduate level. Additionally, in-lab demonstration videos should walk students through the experimental process.

Student Progress

Students must be able to monitor their progress through each course, take quizzes to assess knowledge retention, and print completion certificates.

Publications Database

Existing studies can be valuable to assist with experimental design. Researchers must have access to a database of these studies and be able to quickly find relevant publications. Only LI-COR maintains an extensive list of peer-reviewed publications, including over 14,000 publications that have used Odyssey Imaging Systems.

This database is freely available, and researchers can filter by research area, instrument, application, country, and year to find publications relevant to their experiment.

References

1. LI-COR Biosciences. (2019). Empiria Studio® Software:

Adaptive Western Blot Analysis. http://licor.com/empiria-studio-background

2. LI-COR Biosciences. (2019). Linear Range Determination in Empiria Studio® Software.

https://www.licor.com/documents/ni6ik2p1liv7wz9rjjajzs8 p2ho6r9fe

Patents

Odyssey M Near-Infrared Imaging System

3. U.S. Patent Application No. 16/911,898, Adaptive lane detection systems and methods. Filed June 25, 2020.

4. U.S. Patent No. 10,395,350, Adaptive background detection and signal quantification systems and methods.

Issued August 27, 2019.

5. U.S. Patent No. 8,410,454, High dynamic range scanning with reduced channel cross-talk. Issued April 2, 2013.

https://www.licor.com/icw-handbook https://www.licor.com/icw-handbook http://licor.com/empiria-studio-background http://licor.com/empiria-studio-background http://licor.com/empiria-studio-background https://www.licor.com/documents/ni6ik2p1liv7wz9rjjajzs8p2ho6r9fe https://www.licor.com/documents/ni6ik2p1liv7wz9rjjajzs8p2ho6r9fe https://www.licor.com/documents/ni6ik2p1liv7wz9rjjajzs8p2ho6r9fe https://www.licor.com/documents/ni6ik2p1liv7wz9rjjajzs8p2ho6r9fe

6. U.S. Patent Application No. 16/734,731, Laser line illumination using combined single-mode and multi-mode laser sources. Filed January 6, 2020.

LI-COR Biosciences Regional Offices 4647 Superior Street Lincoln, NE 68504 Phone: +1-402-467-0700 Toll free: 800-645-4267 biosales@licor.com licor.com/bio

LI-COR Biosciences GmbH

Siemensstraße 25A 61352 Bad Homburg Germany Phone: +49 (0) 6172 17 17 771 bio-eu@licor.com

LI-COR Biosciences UK Ltd.

St. John’s Innovation Centre Cowley Road • Cambridge CB4 0WS • United Kingdom Phone: +44 (0) 1223 422104 bio-eu@licor.com

© 2021 LI-COR, Inc. LI-COR, In-Cell Western, Odyssey, and Empiria Studio are trademarks or registered trademarks of LI-COR, Inc. in the United States and other countries. All other trademarks belong to their respective owners.

Doc # 982-19690 05/21 mailto:biosales@licor.com https://www.licor.com/bio mailto:bio-eu@licor.com mailto:bio-eu@licor.com

In‑Cell Western Assay Development Handbook