Absorbance Assay Analysis

Use this workflow to analyze plate images from absorbance assays, including ELISA (enzyme-linked immunosorbent assay) and protein concentration estimation assays.

Overview

  • A Plate Template is required for Absorbance Assay analysis. Plate Templates are created in the Empiria Studio Experiment Designer. In a Plate Template, you designate which wells will contain which samples and controls. During analysis, choose the multiwell plate image to analyze and the Plate Template to use for analysis. Wells on the image that correspond to wells designated on the Plate Template will be quantified. Statistical values will be calculated from the quantification values.

    See Getting Started with Plate Templates for more information about creating Plate Templates. See Signal Analysis Calculations for more information about calculations.

  • Plate Templates are divided into categories. The Plate Template categories match the Absorbance Assay analysis categories. When you start an analysis workflow, choose the category that matches the Plate Template you will be using.

  • The instructions on this page are applicable to all categories of Absorbance Assay.

Name

Name and describe the Experiment for your records.

The selected Acquisition is shown in the Image Information section. Click to add other fields with additional information about the Acquisition.

Select template

A Plate Template is required for analysis.

  • If you have already created a Plate Template, click Select to select it for analysis.

  • If you do not have a Plate Template, click Create New to create one. You will then create a Plate Template in the Experiment Designer workflow.

Select template

A Plate Template is required for analysis.

  • If you have already created a Plate Template, click Select to select it for analysis.

  • If you do not have a Plate Template, click Create New to create one. You will start the Plate Template Creator workflow.

    See Getting Started with Plate Templates for more information about creating a Plate Template.

Quantify wells

On this page, ensure quantification is set up correctly for your plate image.

Image display options are available above the image. Only the image display is changed; the original image file remains the same.

  • Zoom: Click the zoom buttons above the image to make the displayed image smaller or larger . A zoom level, shown as a percentage relative to the size of the software window, is shown next to the zoom buttons.

  • Display Settings: Click Empiria Studio brightness and contrast gear icon to change display settings.

  • Display Channels: Click Empiria Studio channel selection button icon to select the channels you want to display.

  • Display Well IDs: Click to stop showing Well IDs.

  • The plate grid is the grid of well circles inside a rectangular outline overlaying the image. The plate grid is used to specify well location.

  • Only well circles that correspond to the wells designated in the Plate Template are quantified.

  • These well circles are identified by default with a Well ID label.

  • Ensure the well circles being quantified are placed on top of the wells on the image that need to be quantified.

  • Adjust plate grid rotation: Click Empiria Studio rotate counter clockwise icon to rotate the entire plate grid counter-clockwise or Empiria Studio rotate clockwise icon to rotate the entire plate grid clockwise.

  • Adjust plate grid location: Click the rectangular outline of the plate grid. Drag the plate grid so that the well circles in the plate grid cover the appropriate wells on the plate image.

You may not want to quantify all the wells that were designated for quantification in the Plate Template.

  • If there is a well you do not want to quantify, select the well and then click Exclude Well.

  • To quantify a well that had previously been excluded, select the well and then click Include Well.

Click Empiria Studio Analysis Table icon Analysis Table to view well quantification data, including Total, Signal, Background, and Normalized Signal.

Click to export all the data to a spreadsheet.

Review and report

The data in the report is organized into sections. Click to expand a section to view data and export options.

Images

  • Click above an image to export the image. Images can be exported as a high-resolution TIFF for publication in a journal or as a PNG appropriate for slide presentations. Do not use these images for analysis.

  • The units for image size can be set to English or metric on the General Options page.

  • Click to hide shapes. If well circles are hidden, they will not be included in the exported image or PDF Report.

Tables

Click Export above a table to export the data in the table in CSV or Excel (XLSX) format.

Export Menu Options

On the right side of the window, there is a menu with three export options.

  • Click PDF Report to export a PDF of the images and data on the Review and report page.

  • Click Experiment File to export all the images, quantification steps, and data from this Experiment to a single file that can be imported into Empiria Studio on another computer.

  • Click All Report Images to export all images from the Experiment to separate files for use in print publications or digital media (such as a slide presentation). Do not use these images for analysis.

    In the export dialog, click Browse to find a parent folder for the images and enter a name for a new folder in the Folder Name field. The images will be exported into the new folder, which will be inside the parent folder.

Rate Experiment

Rate the Experiment for your records. This is an opportunity to record if the Experiment went "well" or not, depending on the specific requirements for your research.

Figure 245. Your rating will appear in the Experiment list for the Project.

Done

Click Done to mark this Experiment as complete and return to the Experiment List.