Basic Imaging Functions page 27 Auto Exposure works in both Preview and Acquire Image modes.The AlphaView software calculates the Expose status ba for image acquisition appears.The status bar indicates the current status of the calculation.The status bar changes from red to yellow to blue to green to indicate progress.When it reaches green,the AlphaView soft. ware has calculated the correct exposure time.For exposure time calculations with saturated pixels in the image area,the status bar changes to pinkto indicate the issue. In Acquire Image mode,a green status bar indicates that the image is acquired.After achieving a green status the new image loads and the Camera Setup and Preview window closes.In Expose Preview mo e,the system continues repeated expose using the calculated exposure time until a different mode is selected. Switching from Expose Preview mode to Acquire Image mode does not reset the calculation.If the system changes from Expose Preview mode to Acquire Image mode after calculating the correct exposure time in Expose Preview mode,images are acquired using the last calculated exposure time.Similarly,if a different sensitivity(binning)mode is selected after finishing the exposure calculation.the e calculation is quickly con verted and no recalculation is required The Exposure Value(EV)unit is the same as used in film photography (also called a"stop).+I EV will double the exposure time while-I EV with halve the exposure time.The general exposure time using EV is found by the following formula: (EVCompensatedTime)=Time*2EV Where Time is the typical exposure time for the sample under test to reach full exposure as determined by the auto exposure tool. Setting Exposure Time Without Auto Expose Once the sample is positioned and the lens is properly focused.perform the following steps: 1.Close the Multilmage light cabinet door and check to ensure that the appropriate illumination source is turned on.In addition,check to ensure that the cabinet door indicator in the Cabinet Control software interface indicates Closed. 2.Uncheck the Auto Expose option. 3.Click the green Expose Preview buttn and select the desired expsure time the system for the desired image intensity quality Individual adjustments for mllseconds,seconds,minutes. and hours are available.The following list describes ideal exposure settings for different applications: For most white-light applications,using a 50 ms exposure is sufficient.Final aperture adjustments can be made to optimize the image quality. re range between 8 ms and 4 seconds is suffi- t The aperture s pen whe en performing any adjustments.Selection Show r these applications HluerChem HD2 and FC2 User Guide
Basic Imaging Functions page 27 FluorChem HD2 and FC2 User Guide Auto Exposure works in both Preview and Acquire Image modes. The AlphaView software calculates the correct exposure time for the current situation. In Expose Preview mode, a status bar appears above the Auto Expose interface. In Acquire Image mode, a status window featuring a colored Auto Expose status bar for image acquisition appears. The status bar indicates the current status of the calculation. The status bar changes from red to yellow to blue to green to indicate progress. When it reaches green, the AlphaView software has calculated the correct exposure time. For exposure time calculations with saturated pixels in the image area, the status bar changes to pink to indicate the issue. In Acquire Image mode, a green status bar indicates that the image is acquired. After achieving a green status, the new image loads and the Camera Setup and Preview window closes. In Expose Preview mode, the system continues repeated expose using the calculated exposure time until a different mode is selected. Switching from Expose Preview mode to Acquire Image mode does not reset the calculation. If the system changes from Expose Preview mode to Acquire Image mode after calculating the correct exposure time in Expose Preview mode, images are acquired using the last calculated exposure time. Similarly, if a different sensitivity (binning) mode is selected after finishing the exposure calculation, the calculation is quickly converted and no recalculation is required. The Exposure Value (EV) unit is the same as used in film photography (also called a “stop”). +1 EV will double the exposure time while –1 EV with halve the exposure time. The general exposure time using EV is found by the following formula: (EVCompensatedTime) = Time*2EV Where Time is the typical exposure time for the sample under test to reach full exposure as determined by the auto exposure tool. Setting Exposure Time Without Auto Expose Once the sample is positioned and the lens is properly focused, perform the following steps: 1. Close the MultiImage light cabinet door and check to ensure that the appropriate illumination source is turned on. In addition, check to ensure that the cabinet door indicator in the Cabinet Control software interface indicates Closed. 2. Uncheck the Auto Expose option. 3. Click on the green Expose Preview button and select the desired exposure time in to configure the system for the desired image intensity quality. Individual adjustments for milliseconds, seconds, minutes, and hours are available. The following list describes ideal exposure settings for different applications: • For most white-light applications, using a 50 ms exposure is sufficient. Final aperture adjustments can be made to optimize the image quality. • For UV fluorescence applications, using an exposure range between 8 ms and 4 seconds is sufficient. The aperture should be completely open when performing any adjustments. Selection Show Saturation is recommended for these applications
page 28 Chapter 2:Getting Started-Basic maging Functions .For low-light applications,such as chemiluminescence,a longer exposure time may be appropriate au Bumes uodn aBewl aul aunbop napw anbo pa au usnd 'saspo asau u ure time.If a good exposure time is un nown,an alternative method is to utilize the Auto Expose feature configured to super sensitivity.Auto Expose image generation-with signal intensity showing-generally completes in less than three minutes.After Auto Expose image generation,a different sensitivity(binning)mode can be selected to generate a better resolution image.For further images,the software automatically calculates the new exposure time. NOTE:When the system switches to Expose Preview mode,the image may flash or change brightness due to camera photon collection from the image over a longer period of time prior to sending the image to the computer's display readout Light Source and Filter Controls The mechanical controls on the cabinet and the AlphaView software's virtual cabinet control interface (labeled as Cabinet Controls)are linked and provide near-real time synchronization and updating of light source and filter selection.Selecting a desired option via the software interface produces the same result as using the physical controls on the cabinet NOTE:A slight delay occurs upon pressing the button until the light source is fully activated Standard lighting choices include: .Transillumination White:Protein gels,autorads,film,plates,and flasks .Transillumination UV:Fluorescent gels such EtBr,SYPRO Red.SYBR Safe.etc Reflective White:Colorimetric blots and membranes Reflective UV(optional):SYBR green,TLC plates,and Chemifluorescence ChromaLight (optional):GFP.Fluorescein,and SYBR green Filters selection is linked to appropriate sample visualization. Filter options include: Filter Position #Empty Position for Chemiluminescence detection .Filter Position #2:Orange filter(595/55 nm)for Ethidium Bromide,colorimetric stains,film,SYPRO Orange Filter Position #3:Optional green filter(537/40 nm)for SYBR Green,SYBR Safe and Fluorescein Filter Position #4:Optional red filter(620/40 nm)for SYPRO Ruby.SYPRO Red and Deep Purple Filter Position #5:Optional blue filter(460/40 nm)for Hoeschst Blue FluorChem HD2 and FC2User Guide
page 28 Chapter 2: Getting Started—Basic Imaging Functions FluorChem HD2 and FC2 User Guide • For low-light applications, such as chemiluminescence, a longer exposure time may be appropriate. In these cases, push the red Acquire Image button to directly acquire the image upon setting the desired exposure time. If a good exposure time is unknown, an alternative method is to utilize the Auto Expose feature configured to super sensitivity. Auto Expose image generation – with signal intensity showing—generally completes in less than three minutes. After Auto Expose image generation, a different sensitivity (binning) mode can be selected to generate a better resolution image. For further images, the software automatically calculates the new exposure time. NOTE: When the system switches to Expose Preview mode, the image may flash or change brightness due to camera photon collection from the image over a longer period of time prior to sending the image to the computer’s display readout. Light Source and Filter Controls The mechanical controls on the cabinet and the AlphaView software’s virtual cabinet control interface (labeled as Cabinet Controls) are linked and provide near-real time synchronization and updating of light source and filter selection. Selecting a desired option via the software interface produces the same result as using the physical controls on the cabinet. NOTE: A slight delay occurs upon pressing the button until the light source is fully activated. Standard lighting choices include: • Transillumination White: Protein gels, autorads, film, plates, and flasks • Transillumination UV: Fluorescent gels such EtBr, SYPRO Red, SYBR Safe, etc. • Reflective White: Colorimetric blots and membranes • Reflective UV (optional): SYBR green, TLC plates, and Chemifluorescence • ChromaLight (optional): GFP, Fluorescein, and SYBR green Filters selection is linked to appropriate sample visualization. Filter options include: • Filter Position #1: Empty Position for Chemiluminescence detection • Filter Position #2: Orange filter (595/55 nm) for Ethidium Bromide, colorimetric stains, film, SYPRO Orange • Filter Position #3: Optional green filter (537/40 nm) for SYBR Green, SYBR Safe and Fluorescein • Filter Position #4: Optional red filter (620/40 nm) for SYPRO Ruby, SYPRO Red and Deep Purple • Filter Position #5: Optional blue filter (460/40 nm) for Hoeschst Blue
Basic Imaging Functions NOTE:If exposure time is less than 100 ms,yet signal is strong dose the aperture by stops. Turbo Modes and Speed/Resolution Settings The AlphaView System offers different speed and resolution settings to allow image acquisition across a variety of needs.Tweaking resolution and speed settings allow image acquisition to occur as quickly as possible.with the highest resolution possible,or somewhere in between.Available acquisition modes include 5 binning modes and 2 Turbo modes. 20280四￥511日 网同.+可m AESp□0：0：石 setting Super Speed 的中 Closed Figure 2-14:Turbo Modes and Speed/Resolution Settings FluorChem HD2 and FC2 User Guide
Basic Imaging Functions page 29 FluorChem HD2 and FC2 User Guide NOTE: If exposure time is less than 100 ms, yet signal is strong, close the aperture by stops. Turbo Modes and Speed/Resolution Settings The AlphaView System offers different speed and resolution settings to allow image acquisition across a variety of needs. Tweaking resolution and speed settings allow image acquisition to occur as quickly as possible, with the highest resolution possible, or somewhere in between. Available acquisition modes include 5 binning modes and 2 Turbo modes. Figure 2-14: Turbo Modes and Speed/Resolution Settings
page 30 Chapter 2:Getting Started-Basic maging Functions The camera modes are selected by the Turbo Mode check box When the Turbo Mode checkbox is unchecked the camera operates in Normal mode.Normal mode is useful for those imaging applications that require wide dynamic and quantification of low signal intensities.When the Turbo mode checkbox is checked the camera operates in Turbo Mode.Turbo Mode is used for the applications that require fast image acquisition to quickly visualize samples even those with low signal intensities Both Normal and Turbo mode can be used with each of five binning modes and Auto Expose functions to optimize image acquisition speed and for fluorescence and chemiluminescence applications. Speed/Resolution Settings Normal/Ultra Mode Recommended for Fluorescence and Colorimetric Imaging,this mode captures the image using the CCD sensors full 2048x2048 pixel resolution to generate highest quality.vibrant images that are ready for quantitative analysis and publicatior Medium/High Mode with a2x pixel bin to decrease th exposure times from full resolution images by approximately a factor of four. High/Medium Mode Recommended for Chemiluminescence.this mode captures images with a 3x3 pixel bin(683x683 pixels) with decreased image acquisition time and medium resolution.This mode decreases exposure times from full resolution images by approximately a factor of nine Fast/Low Mode Recommended for Chemiluminescence,this mode captures images with a 4x4 pixel bin (512x 512 pixels) Similar to Super Speed this mode decrease exposure time but with a trade off in resolution.This mode decreases exposure times from full resolution n images by approximately a factor of sixteer Super Speed Mode Recommended for Chemiluminescence,this s mode captures the image performing an8x8 pixel bin(256x 256 pixels).This mode significantly decreases the exposure time required for acquiring a long chemiluminescent exposure.This mode's resolution is set to 256x256 pixels and is not recommended for precise quantitative analysis or publication.Super Speed mode is ideal for Expose Preview functionality when using Auto Expose to determine the chemiluminescent blot signal level It is also useful for rapid visualization of signals for a qualitative assessment of the sample. luorChem HD2 and FC2User Guid
page 30 Chapter 2: Getting Started—Basic Imaging Functions FluorChem HD2 and FC2 User Guide The camera modes are selected by the Turbo Mode check box. When the Turbo Mode checkbox is unchecked the camera operates in Normal mode. Normal mode is useful for those imaging applications that require wide dynamic and quantification of low signal intensities. When the Turbo mode checkbox is checked the camera operates in Turbo Mode. Turbo Mode is used for the applications that require fast image acquisition to quickly visualize samples even those with low signal intensities. Both Normal and Turbo mode can be used with each of five binning modes and Auto Expose functions to optimize image acquisition speed and for fluorescence and chemiluminescence applications. Speed/Resolution Settings Normal/Ultra Mode Recommended for Fluorescence and Colorimetric Imaging, this mode captures the image using the CCD sensors full 2048x2048 pixel resolution to generate highest quality, vibrant images that are ready for quantitative analysis and publication. Medium/High Mode Recommended for Chemiluminescence, this mode captures images with a 2x2 pixel bin to decrease the required exposure time while maintaining a high resolution (1024x1024) image. This mode decreases exposure times from full resolution images by approximately a factor of four. High/Medium Mode Recommended for Chemiluminescence, this mode captures images with a 3x3 pixel bin (683x683 pixels) with decreased image acquisition time and medium resolution. This mode decreases exposure times from full resolution images by approximately a factor of nine Fast/Low Mode Recommended for Chemiluminescence, this mode captures images with a 4x4 pixel bin (512x 512 pixels) Similar to Super Speed this mode decrease exposure time but with a trade off in resolution. This mode decreases exposure times from full resolution images by approximately a factor of sixteen Super Speed Mode Recommended for Chemiluminescence, this mode captures the image performing an 8x8 pixel bin (256 x 256 pixels). This mode significantly decreases the exposure time required for acquiring a long chemiluminescent exposure. This mode’s resolution is set to 256x256 pixels and is not recommended for precise quantitative analysis or publication. Super Speed mode is ideal for Expose Preview functionality when using Auto Expose to determine the chemiluminescent blot signal level. It is also useful for rapid visualization of signals for a qualitative assessment of the sample
Basic Imaging Functions pmge3引 Gray Scale Optimization for Saturation and Contrast Displays Figure 2-15 illustrates the check boxes available for modifying the displayed image ☐Chemi Display ☐Show Satuatio ☐Auto Contrast ☐Reverse Figure 2-15:Modifying the Displayed mag Any images set for analysis must not be over-exposed(too light)or under-exposed (too dark)for maximum clarity.Using the Show Saturation option(located below the camera control functions)displays the image areas assigned to each end of the gray scale spectrum.After inspecting the image's saturation,the imaging controls can be adjusted accordingly to optimize the image. The the the Saturation mage acquisition.The Saturation Palette is a modified gray scale palette in which green replaces black(gray level O).and red replaces white (gray level 255.4.095,or 65,535).With this palette,the image's over-and under-exposed areas appear as green or red,while areas within the linear range of the CCD chip appear in gray scale. During image acquisition,it is critical to note the image's regions that appear red or green.The exposure time and the camera aperture can be adjusted to minimize the amount of red and green in the image area. Eliminating red and green in the actual sample area is esp ecially critical for images that will be quantified.The saturation view can be turned off by unchecking Show Saturation once the red and green areas have been minimized and/or eliminated The other three selections(Chemi Display Auto Contrast,and Reverse)act as visualization tools designed to enhance contrast and provide flexibility image viewing.The Auto Contrast option displays the image with automatic black,white,and gamma adjustmer mation (black/white leve).Reverse The Chemi Display option utilizes both auto contrast and reverse functions,as well as providing gamma adjustments,and is intended for use with chemiluminescent samples. NOTE:These options are also available in the Movie Mode. NOTE:These Contrast Display options are only visualization tools and do not change any image data acquired by the CCD camera. FluorChem HD2 and FC2 User Guide
Basic Imaging Functions page 31 FluorChem HD2 and FC2 User Guide Gray Scale Optimization for Saturation and Contrast Displays Figure 2-15 illustrates the check boxes available for modifying the displayed image. Figure 2-15: Modifying the Displayed Image Any images set for analysis must not be over-exposed (too light) or under-exposed (too dark) for maximum clarity. Using the Show Saturation option (located below the camera control functions) displays the image areas assigned to each end of the gray scale spectrum. After inspecting the image’s saturation, the imaging controls can be adjusted accordingly to optimize the image. The Show Saturation option allows the user to access the Saturation Palette during image acquisition. The Saturation Palette is a modified gray scale palette in which green replaces black (gray level 0), and red replaces white (gray level 255, 4,095, or 65,535). With this palette, the image’s over- and under-exposed areas appear as green or red, while areas within the linear range of the CCD chip appear in gray scale. During image acquisition, it is critical to note the image’s regions that appear red or green. The exposure time and the camera aperture can be adjusted to minimize the amount of red and green in the image area. Eliminating red and green in the actual sample area is especially critical for images that will be quantified. The saturation view can be turned off by unchecking Show Saturation once the red and green areas have been minimized and/or eliminated The other three selections (Chemi Display, Auto Contrast, and Reverse) act as visualization tools designed to enhance contrast and provide flexibility image viewing. The Auto Contrast option displays the image with automatic black, white, and gamma adjustments according to the image histogram information (black/white levels). Reverse shows the sample as a negative by switching the black and white values. The Chemi Display option utilizes both auto contrast and reverse functions, as well as providing gamma adjustments, and is intended for use with chemiluminescent samples. NOTE: These options are also available in the Movie Mode. NOTE: These Contrast Display options are only visualization tools and do not change any image data acquired by the CCD camera