ScanArray Overview. Principle of Operation. Instrument Components

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1 ScanArray Overview The GSI Lumonics ScanArrayÒ Microarray Analysis System is a scanning laser confocal fluorescence microscope that is used to determine the fluorescence intensity of a two-dimensional array of dots on a bio-chip or microarray. Each dot on the microarray consists of target material that is chemically bound to the substrate. A probe, which is complementary material that is labeled with a fluorophore is preferentially bound to the target spots via a hybridization process to generate a fluorescent compound. The scanner can be used in conjunction with QuantArray TM for quantitation, processing and reporting and storage of data. Principle of Operation The fundamental operation of the ScanArrayÒ Microarray Analysis System involves the measurement of the fluorescence intensity of each dot on the sample. The sample dots are typically µm in diameter and a few µm thick on top of the substrate surface. The microarray is scanned (with a user settable resolution) to generate a map or image which indicates the fluorescence intensity for each point or pixel on the array. Typically, two (or more) different fluorophores are used to determine the concentration of the compounds of interest in each sample. This requires that the substrate is scanned two (or more) times with different lasers to provide different excitation wavelength. Each scan is performed on a sequential basis and the data is stored. Instrument Components The instrument includes an Excitation Source, Emission Optics, Detector and a Sample Entry System. Excitation Source Excitation radiation is provided by 1-4 lasers (depending on the system configuration). Most lasers will be mounted inside the scanner, however, large lasers or lasers with a significant power dissipation may be externally mounted. The excitation source will include appropriate beam shaping optics, an excitation filter, a shutter and beam alignment optics. Excitation Source The bio-chip or microarray is placed on the sample stage, which is scanned to collect data from all pixels within a user-defined area. Scanning is performed in both the X and Y direction. In addition, the Y direction is used to insert and eject the microarray into the unit. Emission Optics The emission optics collect the emitted fluorescence and direct it toward the detector. The optics includes emission filters to purify the light by blocking the reflected and scattered excitation radiation. Detector A photomultipler tube detects the emission radiation and converts it into a voltage. The signal is amplified, filtered, digitized and stored.

2 Manually Acquiring a Scan To acquire a scan with the manual sample loader: 1. Click the Acquire command on the Start menu to open the Acquire Image dialog box. 2. Select the desired protocol (and edit, if necessary). 3. Click the Eject command on the Start menu to eject the slide holder from the instrument. 4. Insert the microarray into the scanning position and retract the slide using the Eject command. 5. Focus the instrument (if necessary). 6. Press the Acquire button. Protocol Files When the Acquire Image dialog box is accessed, the user can select the desired protocol (*.sapr) by selecting the file using the Protocol Files field. The protocols are presented in a standard Windows file tree format and the user should click on the desired protocol to select it. Acquisition Area The Acquisition Area field indicates the area that is to be used for data acquisition and the area to be used for the Auto-Range/Auto-Balance function. The region indicated in color on the slide defines the region that contains the calibration spots for the Auto-Range/Auto-Balance operation, while the region indicated in black defines where data will be collected. The acquisition area can be defined by the cursor or by using the X-Position, Y-Position, Width and Height fields. Note: Editing the Acquisition Area for one fluorophore will change the Acquisition Area for all fluorophores. The X-Position, Y-Position, Width and Height fields are used to define the starting coordinates for the scan and the size of the region to be scanned. It is very important to start spotting 1 mm from the top of the slide and to spot 1.5 mm from each side of the slide so that spots are not placed outside the area to be scanned.. To edit the value for any of these fields, click Enter or Tab to move to the next field and enter the desired value. The region for the Auto-Balance Auto-Range function is selected on the Hardware Settings dialog box.

3 Resolution Resolution refers to the pixel (picture element) size, which is the dimension of each small square element that makes up the electronic image. A larger value (e.g. 50 µm) will result in a rapid scan which presents gross features of the sample while a small value (e.g µm) results in a slower scan that allows for the maximum information from the slide. We recommend a pixel size that is roughly 10-12% of the diameter of the microarray spot. Typically, a Quick Scan is used to preview the slide, determine where spots of interest are located and to set the sensitivity. Once the optimum parameters are determined, then the region of the slide that contains spots of interest are re-scanned using a smaller pixel size (and perhaps a slow scan speed) to collect data for quantitative analysis. To indicate the desired resolution, click on the appropriate radio button. If 30 or 50 µm resolution is desired, place a check mark in the box adjacent to Quick Scan. Scans The Scans field is used to indicate the number of scans or passes that are desired in the present data acquisition protocol. The scan number can be edited by clicking the field, typing the desired value and clicking Enter. The Sum and the Average radio buttons are used to indicate the desired data processing option if more than one scan is selected. Summing will increase the measured signal from dim samples, while averaging will reduce the effect of noise (this feature is not yet active). Scan Rate The Scan Rate is set in the Adjust Hardware Settings dialog box. Fluorophore Selection Field The Fluorophore Selection field lists the fluorophores that will be addressed by the current Acquisition protocol and the order in which the samples will be scanned. If the instrument does not include a filter or a laser that is required for a specific fluorophore, the listing in this field will be grayed out and cannot be included in the acquisition. If the operator clicks the Acquire button to indicate that a scan of the non-supported flurophore is desired, a dialog box will be presented that alerting that the scan will not include data from the indicated fluorophore. The Add button is used to access the Choose a Fluorophore dialog box, which lists the various fluorophores that are defined to ScanArray. Fluorophores that are not supported by the instrument that is attached will be indicated in gray. To select a fluorophore in the Selection field, click on the number to the left of each entry (in the # column). The Delete button in the Fluorophore Selection field is used to remove a fluorophore from the Flurophore Selection field. The fluorophore to be deleted should be highlighted via the mouse before the button is clicked. Deleting a fluorophore merely removes the fluorophore from this field; it does not remove it from the listing in the Choose a Fluorophore dialog box.

4 The Move Down and Move Up buttons are used to change the order of the scanning of the sample for the various fluorophores. To change the order, highlight the desired fluorophore with the mouse and click the appropriate button to lower or raise the fluorophore. Auto-Save Images The Auto-SaveImages check box is used to indicate if the images should be automatically saved at the end of the scan. If the box is not checked, the system will stop after each set of image and the data must be saved manually by accessing the Save dialog box. (Do not use Auto -Save on the BIPL scanner!) Auto Range before Every Scan The Auto Range before every scan check box is used to indicate if the Auto-Range/Auto-Balance feature should be used between each scan. Auto-Range The Auto-Range button is used to perform the Auto-Range/Auto-Balance measurement using the Auto-Range/Auto-Balance (ABAR) settings. Experiment Set Name The Experiment Set Name field is used to indicate the name of the folder in which the acquired images will be stored, if the Auto-save option has been selected. The file name must be Windows compatible (i.e. it cannot contain a slash, a backslash, a dot or any other Windows reserved character). QuantArray Analysis Protocol The QuantArray Analysis Protocol field is used to indicate the QuantArray that should be used to analyze the data. The Browse button is used to access the list of QuantArray analysis protocols, and the desired protocol is selected in the same manner as the acquisition protocol described above. The entire path of the desired QA protocol must be entered, not just the file name. Comments for the Protocol The Comments for the Protocol field is used by the operator to add information that describes the experiment. Any alphanumeric information can be added in this field.

5 The Scan Rate Field The Scan Rate field is used to indicate the speed of scanning of the sample. The radio button selects the desired value. A slow scan (25% or 50 %) will decrease pixel-to-pixel variation (AC noise). We recommend using the maximum speed setting except in unusual cases where the standard deviation or variation of pixel values is critical. Selecting the Auto-Balance, Auto-Range Area The X Position and Y Position fields define the point (in mm) where the Auto-Ranging and Auto- Balancing processes for that fluorophore should begin. The origin reference point is the upper left corner of the slide. The initial X Position and Y Position for Autoranging can be set in the same way as they are set for acquisition. The Width and Height define the size of the region for which Auto-Ranging is desired for that fluorophore. Microarray Spot Size The Microarray Spot Size is selected by adjusting the thumbwheel up or down. The ABAR algorithm uses the spot size to calculate the appropriate quick-scan resolution. If you do not know the diameter of your microarray spots, you can measure them on an image using the Circle cursor tool. Target Intensity The Target Intensity value can be selected by adjusting the thumbwheel or by entering a value. This is the pixel brightness value, which ABAR will attempt to achieve in the brightest part of the ABAR area. This is generally set to 90%, but any other value will work. Adjustment Method Two radio buttons are provided for selecting Adjustment Method. If PMT is selected, the laser power selection will remain fixed and ABAR will adjust the PMT setting to reach the target intensity. If Laser is selected, the PMT Gain selection will remain fixed and ABAR will automatically adjust the laser power to reach the target intensity. The Image File Name Field The Image File Name field defaults to the name of the fluorophore and can be changed (if desired) to simplify reports. Dark Signal Correction Performing a Dark Signal Correction The Dark Signal Correction function is used to compute a dark signal offset so that an appropriate offset adjustment can be made.

6 To perform a Dark Signal Correction on a clean, blank glass slide: 1. Remove the sample (if present) and insert a clean, blank microscope slide in the instrument. 2. Select the x position, y position, width and height to of the region to be used for the measurement of the dark signal. 3. Indicate the Pixel Value Upper Limit, which is the maximum pixel brightness value that will be displayed in the histogram. 4. Click the Dark Signal Correction button to access the Dark Signal Correction dialog box. 5. Press Scan 6. If the dark level, as indicated by the histograms is satisfactory, press OK to accept the correction and return to the Adjust Hardware Settings dialog box. 7. If the dark level is not satisfactory, enter a + or - delta intensity value into the Correction field and scan again. Repeat this process until you are satisfied with the dark level. To perform the dark signal correction with no laser, check the Scan with no Laser check box before initiating the scan. Dark Signal Correction Dialog Box The Dark Signal Correction dialog box is presented when the Dark Signal Correction button is clicked on the Adjust Hardware Settings dialog box. The Dark Signal Correction dialog box contains the following information: The Fluorophores field. The Scan Area field, which is used to define the region for the determination of the dark signal. Select this region by entering the x-position, y-position, width, and height fields by clicking in each box and entering the desired value or by selecting the area on the Preview of Area region with the mouse. The Pixel Value Upper Limit field, which is used to enter the upper limit for the histogram display. The Histogram Scan region presents the data that is obtained when the background scan is run. A histogram with a left-hand tail that goes near zero shows that almost no pixels have a zero intensity value. A histogram that shows a significant population of pixels at zero intensity may indicate that some low-level signals are being hidden by a negative offset to the signal. Your preferences for this setting may depend on the quantitation methods that are used downstream.

7 Role of the Focus/Line Scan Function Note: It is recommended that the Focusing operation is performed at a low laser power and a higher PMT voltage than might normally be used to minimize photobleaching. The Line Scan function is used to perform a series of scans along the x direction of the sample at a user selected Y position. The function allows the user to view a plot of the intensity as a function of the distance along the scan line and is used to focus the lens to optimize the sensitivity of the ScanArray. The focus for each fluorophore should be maximized each time a protocol is created. Routine scanning using an established protocol should not require regular adjustment to the focus. If the focus needs to be adjusted, it should be done before the sensitivity is adjusted (laser an/or PMT). The Line Scan Dialog Box The Line Scan dialog box is presented when the Focus/Line Scan button is clicked on the Adjust Hardware Settings dialog box or after the scan tool is used to define a line of interest on an image. The Line Scan dialog box contains the following information: The Fluorophores field. Focusing is done separately for each fluorophore. Select the fluorophore of interest, perform the focus operation and then go on to the next fluorophore. The Starting X Position and Size fields are used to indicate the region of the line to be monitored (e.g. each scan is from 4 to 14 mm from the left edge of the microarray). The edit boxes reflect the scan line selected on an image with the line scan tool or can be entered manually here. The Y-Stage field is used to select the position of the sample in the Y direction, and is used to move the microarray to view different regions. This reflects the position selected as an image with the Line Scan tool or can be adjusted here. Gross movements can be made by dragging the slider, fine movements can be made by clicking on the slider and using the up/down arrow keys. The Focus field is used to change the position of the lens to maximize the signal. Gross movements can be made by dragging the slider. Fine (5µm) movements can be made by clicking on the focus slider and using the left/right arrow keys to adjust the focus in increments. Adjust until the signal is maximized. Note: You must locate the line scan in an area on the sample that has a signal in order to determine focus. Nominal focus is at without a cover slip. With a cover slip that does not cover the sample to the edges of the slide, the nominal focus is and with a cover slip that covers the entire slide, it is approximately The scan region in the center of the position presents the data for the present scan. If data for the scan of two (or more) fluorophores are presented, they will appear in different colors. the curve), and is a numerical representation of the signal amplitude.

8 The Auto Focus radio buttons allow the user to select whether the auto-focus algorithm maximizes the signal level or the slope of edge transitions. Edge still works when the signal is saturated, but requires an edge to the scan line. Level works on an area of uniform signal, but requires a signal below saturation. The Current Average Signal field presents the averaged signal over the scanned range (the area under The Graticule field is used to select the appearance of the graticule, which is a pattern of horizontal lines in the display which users can select to suit their preferences. Determining the Focus Position for a Given Line The optimum focus position can be determined by the Auto-Focus command or by manually changing the Focus Position to maximize the intensity for each set of conditions. Note: If the signal approaches 100 %, reduce the Laser power or the PMT gain to avoid saturation of the signal. Automated Determination of the Focus To Manually Determine the Focus: Determining the Focus for the Entire Microarray Automated Determination of the Focus The Automated determination on the focus may be used when you have selected a scan line that includes features that have measurable signal from all of the fluorophores in the protocol. To use the Auto-Focus feature: Set the area parameters. Click Scan with each fluorophore in the protocol to ensure that there are spots at the indicated Y position. If spots are not observed with a reasonable intensity for each fluorophore, move to a new Y position and run another scan. The button will be recessed and the system will collect intensity data along the X position. When the signal is maximized, click the Scan button again to stop the operation. The scan data will be presented in the Scan region and the Current Average Signal field. When a line has been obtained that includes spots of reasonable intensity, press the Auto Focus button. The system will automatically scan the X-position line and move the lens as appropriate to determine the optimum lens position. The operator can stop the Auto-Focus process at any time by pressing the Auto Focus button again. When the optimum lens position is determined, it will be indicated on the Focus Position slide bar. Note: If the signal approaches 100 %, reduce the Laser power or the PMT gain to avoid saturation of the PMT or use AutoFocus in Edge mode.

9 To Manually Determine the Focus: Set the parameters. Click Scan to ensure that there are spots at the indicated Y position. If spots are not observed with a reasonable intensity, move to a new Y position and run another scan. When a line has been obtained that includes spots of reasonable intensity, record the Current Average Signal Change the focus position and continue to monitor the signal. Continue to change the focus position and observe the Current Average Signal until you have determined the position that produces maximum signal. Click the Scan button again to stop the line scan. e) Repeat for each fluorophore. For a good approximation, focus with one fluorophore and enter that focus position for the other fluorophores as well. Determining the Focus for the Entire Microarray The procedure to determines the focus for a given Y position which may be (or may not be representative) of the complete microarray. It is recommended that the above procedure be performed near the center of the array field so that the best focus is held throughout the field. The Auto-Balance, Auto-Range Function Auto-Balancing and Auto-Ranging are used to adjust the PMT gain and laser power to match the brightness of a sample. Role of the Auto-Balance, Auto-Range Function To Auto-Range and Auto-Balance the System Role of the Auto-Balance, Auto-Range Function Auto-ranging is the instrument function whereby the sensitivity is adjusted to match the brightness of a sample. Based on the size of the microarray spots, it selects a quick-scan resolution that acquires at least two scan lines across each spot. From this sample of the data, the part of the region where the 4 brightest adjacent pixels is identified. Lastly, the instrument adjusts either the PMT or the laser setting so that these 4 brightest pixels will produce data at a user-selected intensity level on the next scan. Generally, a user will select that target intensity level to be around 90% so that the dynamic range of the instrument is fully utilized. Auto-balancing is the same process, repeated for each fluorophore. This leaves the instrument with settings that will cause the next scan to produce data where the brightest features in each image will all be at the same user-selected brightness level. This is called balancing the channels. Auto-Balancing and Auto-Ranging (ABAR) would most often be performed on areas that contain microarray control spots, housekeeping or other genes that produce maximum signals.

10 Different ABAR areas can be chosen for different fluorophores. To Auto-Range and Auto-Balance the System Select the fluorophore of interest by clicking on the number in the left column of the Fluorophores box. In the ABAR section of the Adjust Hardware Settings dialog, the area on the sample in which the auto-ranging will be done can then be selected with the mouse on the cartoon of the slide area, or by entering position and size numbers into the edit boxes.