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You use the Chemiluminescence Setup window to select the chemiluminescence parameters to use with the sample. The following sections describe how to use the Chemiluminescence Setup window (figure 12-4).

Figure 12-4. The Chemiluminescence Setup window contains parameters for scanning a chemiluminescent sample.

12.3.1 Displaying the Chemiluminescence Setup window

To display the Chemiluminescence Setup window, make sure

Chemiluminescence is the selected acquisition mode, and then click the Setup button in the Acquisition Mode area in the Scanner Control window (figure 12-1).

12.3.2 Selecting the sensitivity (limit of detection)

You use the sensitivity parameters to help improve the limit of detection of the Typhoon instrument. The sensitivity parameter controls how long the Typhoon instrument collects data from each pixel. The Sensitivity parameters are—

• Normal (default)—Collects data from each pixel once. In general, you

should use the Normal parameter.

• Medium—Collects data from each pixel four times and averages the

results. Using the Medium parameter to detect weak signals improves the sensitivity by reducing the background noise. Selecting Medium increases the scanning time but does not increase the size of the image file. Check the Scanning Information area in the Scanner Control window for the time required to complete the scan.

(Typhoon 8610, 9210, and 9410 only) For the 10-µm pixel size, the system collects data from each pixel twice and then averages the values.)

• High—Collects data from each pixel eight times and averages the results.

Using the High parameter to detect weak signals improves the sensitivity by reducing the background noise. Selecting High will increase the scanning time but does not increase the size of the image file. Check the Scanning Information area in the Scanner Control window for the time required to complete the scan. Note: This parameter is not available for the 10- and 25-µm pixel sizes.

12.3.3 Selecting a beamsplitter for a multichannel scan

The Typhoon instrument contains three standard beamsplitters and has a slot for one additional custom beamsplitter. Table 10-1 lists the three standard beamsplitters.

If you want to scan the chemiluminescent sample and create a multichannel image, you must select a beamsplitter from the Beamsplitter list. The default selection is None, which is the correct parameter for a single-channel chemiluminescent scan.

12.3.4 Setting the PMT voltage

For chemiluminescent samples, you can alter the PMT voltage to improve the signal collection capabilities of the Typhoon instrument.

Guidelines for changing the PMT voltage

For accurate quantitation of high-intensity samples, the pixel intensities in your scanned image should be within a specific range. To determine the correct PMT voltage, scan a test sample and display the image in ImageQuant. Use the Gray/Color Adjust feature to display the intensity values of the image. For more information, see the ImageQuant User’s Guide or Help within the software.

• If the pixel values are more than 1 count and less than 100 000 counts, all

spots in your image are within the dynamic range of the instrument.

• If some of the pixel values are equal to or greater than 100 000 counts, part

of your image is at or near saturation. Lower the PMT voltage, rescan, and confirm that the image is not saturated.

• For high-intensity samples that saturate the system, decrease the PMT

voltage. This brings high-intensity signals into the linear range of the instrument. The image might lose the sensitivity and quantitation accuracy at the lower end of the signal range. If you decrease the PMT voltage below 400 V, the image might lose linearity.

Important Increasing the PMT voltage can cause the signal-to-noise ratio to deteriorate and lower the sensitivity. For the best sensitivity, use the High sensitivity parameter with a lower PMT voltage.

If you change your sample matrix, you should test the PMT values between 400 V and 1 000 V to determine which voltage provides an acceptable background. As a suggested starting point, use 600 V.

Procedure for changing the PMT voltage

To set the voltage in the PMT Voltage box—

• Click the box to select it, and then type the value you want. The maximum

value is 1 000 V.

• Use the up or down arrows to change the value in 5-V increments. To

approximately double the signal intensity, increase the PMT voltage by 50 V.

If you are using a beamsplitter to perform a multichannel scan, you can select a PMT voltage for each scan.

12.3.5 Closing the Chemiluminescence Setup window

After you finish selecting the chemiluminescence parameters, click OK to save the parameters and close the Chemiluminescence Setup window. If you do not want to save the parameters, click Cancel to return to the Scanner Control window.

The Scanning Information area in the Scanner Control window displays the parameters you selected in the Chemiluminescence Setup window.

12.4 Starting a scan and checking the progress

Warning Do not attempt to defeat the safety interlocks on the sample lid. These interlocks are designed to protect you from laser light exposure.

Caution Never turn off the Typhoon instrument or disconnect the Ethernet cable while scanning. You can damage the internal mechanism of the instrument. Important Keep the sample lid closed during the scan. Opening the lid causes Scanner

Control to abort the scan and save the data already collected.

During the scanning process, the chemiluminescent sample emits light, which is collected by either one or two PMTs.

Scanner Control creates a single image file or a set of files and automatically adds the correct file extensions. The image file extensions are—

• Data File (.gel)—A single image file.

• Dataset File (.ds)—The image file required for viewing a multichannel

image. If you are scanning the sample using a beamsplitter, Scanner Control adds this extension.

If you select Dataset File (.ds), Scanner Control creates a Directory folder (.dir) in the Data folder (or the location you selected) and names the .dir folder using the same name as the .ds file. The .dir folder contains a backup .ds file and one .gel file for each channel of the multichannel image. Note: If you select or type the wrong file extension, Scanner Control changes the file type back to the correct extension.

12.4.1 Starting a single-channel scan of a single sample or

scan area

If you are scanning a single sample or included multiple samples in a single scan area, you create one image file.

To start a scan—

1. Make sure that the sample is in place, the sample lid is shut, and all the parameters are correct in the Scanner Control and Chemiluminescence Setup windows.

2. In the Scanner Control window, click the Scan button. The Save as window