{"id":39673,"date":"2023-12-05T11:47:26","date_gmt":"2023-12-05T11:47:26","guid":{"rendered":"https:\/\/photonics.laser2000.co.uk\/?p=39673"},"modified":"2023-12-05T11:47:26","modified_gmt":"2023-12-05T11:47:26","slug":"image-stitching-for-microscope-applications-using-%c2%b5manager","status":"publish","type":"post","link":"https:\/\/stagingphotonics.laser2000.co.uk\/?p=39673","title":{"rendered":"Image Stitching for Microscope Applications using \u00b5Manager"},"content":{"rendered":"\n<h3 class=\"wp-block-heading\" id=\"h-introduction\">Introduction<\/h3>\n\n\n\n<p>Image stitching is the process of acquiring and merging a sequence of images into a single, large and detailed image. Microscope image stitching is widely used in biomedical research and material science to generate high-magnification images of sample areas which are much larger than the field of view (FOV). A wide FOV increases the probability of capturing rare and interesting events, while a high magnification supports building a detailed understanding of those events.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img decoding=\"async\" src=\"https:\/\/stagingphotonics.laser2000.co.uk\/wp-content\/uploads\/2023\/12\/500px-Image-stitching.png\" alt=\"\" class=\"wp-image-39676\"\/><\/figure>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>The microscopy image stitching process consists of two main steps: acquiring the images and combining them. During image acquisition, the camera is moved across the area to be captured and a sequence of images are captured in a pre-defined pattern. Combining these images is done in software.<\/p>\n\n\n\n<p>Zaber&#8217;s motorized XY microscope stages make image stitching easy by enabling rapid and accurate automated image capture. This article will walk through the image stitching process using a Zaber microscope stage and the popular third-party microscopy software tool \u00b5Manager.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"Equipment\">Equipment<\/h3>\n\n\n\n<p>In this example, we will use modules from the&nbsp;<strong><a href=\"https:\/\/stagingphotonics.laser2000.co.uk\/nucleus\" target=\"_blank\" rel=\"noreferrer noopener\">Nucleus\u2122 automated microscopy platform<\/a><\/strong>&nbsp;which provides a complete set of interchangeable hardware modules and software tools for building your bespoke inverted or upright standalone microscope or optical subsystem. Other microscope cameras have a similar performance. Try our online configuration tools guide you to choose microscope parts, automatically add any addition parts, and provide pricing at each step. You also contact Zaber if you are interested in configuring a system for your application.<\/p>\n\n\n\n<p>Hardware:<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li><a href=\"\/products\/manual-and-motorised-motion\/systems-controllers-and-accessories\/scanning-microscope\/motorized-inverted-microscope-platform\/\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>Zaber MVR Microscope<\/strong><\/a>&nbsp;&#8211; Automated inverted microscope with motorized XY movement, focus, and filter cube changes and integrated 3 channel LED illumination. Or<\/li>\n\n\n\n<li><strong><a href=\"https:\/\/stagingphotonics.laser2000.co.uk\/products\/manual-and-motorised-motion\/systems-controllers-and-accessories\/scanning-microscope\/motorized-xy-microscope-stages-with-built-in-controllers\/\" target=\"_blank\" rel=\"noopener\">X-ASR<\/a><\/strong>&nbsp;&#8211; Cost effective two-axis XY microscope stages with integrated controller and optional KX14B accessory kit with power and USB connection. Or<\/li>\n\n\n\n<li><strong><a href=\"https:\/\/stagingphotonics.laser2000.co.uk\/products\/manual-and-motorised-motion\/systems-controllers-and-accessories\/scanning-microscope\/linear-motor-xy-microscope-stage-integrated-controller\/\" target=\"_blank\" rel=\"noopener\">X-ADR<\/a><\/strong>&nbsp;&#8211; High-speed and ultra-high repeatability linear motor XY microscope stage with integrated controller and optional KX14B accessory kit with power and USB connection.<\/li>\n<\/ul>\n\n\n\n<p>Software:<\/p>\n\n\n\n<ul class=\"wp-block-list\">\n<li><a href=\"https:\/\/micro-manager.org\/Download_Micro-Manager_Latest_Release\" target=\"_blank\" rel=\"noopener\"><strong>\u00b5Manager<\/strong><\/a>&nbsp;&#8211; An open-source microscopy application developed and maintained by Open Imaging which provides a common control interface for a wide range of instruments.<\/li>\n\n\n\n<li><a href=\"https:\/\/imagej.net\/software\/fiji\/\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>Fiji<\/strong><\/a>&nbsp;&#8211; An open-source image analysis platform.<\/li>\n<\/ul>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"Set-up\">Set-up<\/h3>\n\n\n\n<p>To configure the stage and controller, you must first install \u00b5Manager, add the hardware, and follow the set-up instructions here. The X-ASR microscope stage should be set up as a two-axis&nbsp;<em>XYStage<\/em>&nbsp;device in \u00b5Manager.<\/p>\n\n\n\n<p>While adding the&nbsp;<em>XYStage<\/em>&nbsp;device in the Hardware Configuration Wizard, look for the OpenCVgrabber device in the Available Devices list and add it as well. OpenCVgrabber uses the open-source OpenCV library of computer vision drivers which supports most USB cameras, including the models from Dino-Lite. The list of installed devices should be similar to Figure 1.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img decoding=\"async\" src=\"https:\/\/stagingphotonics.laser2000.co.uk\/wp-content\/uploads\/2023\/12\/500px-Harware-configuration-wizard.png\" alt=\"\" class=\"wp-image-39682\"\/><figcaption class=\"wp-element-caption\">Figure 1: Hardware Configuration Wizard<\/figcaption><\/figure>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>After successfully configuring the hardware, an image from the microscope should be visible in the ImageJ window.<\/p>\n\n\n\n<p>Next, set the origin of the microscope stage by selecting&nbsp;<em>Tools &gt; Stage Position List &gt; Set Origin<\/em>. This moves the stage to the &#8216;home&#8217; and &#8216;away&#8217; limit sensors which establishes a reference for positioning, as well as defining the usable travel range. Figure 2 highlights the&nbsp;<em>Set Origin<\/em>&nbsp;button and the&nbsp;<em>Create Grid<\/em>&nbsp;button for the next step.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img decoding=\"async\" src=\"https:\/\/stagingphotonics.laser2000.co.uk\/wp-content\/uploads\/2023\/12\/300px-Stage-position-list.png\" alt=\"\" class=\"wp-image-39683\"\/><figcaption class=\"wp-element-caption\">Figure 2: Calibrating the Microscope Stage<\/figcaption><\/figure>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>Finally, calibrate the movement of the stage to the microscope field of view by setting the pixel size. Position the stage near the center, and focus on an area with sharp, clear details.<\/p>\n\n\n\n<p>Select&nbsp;<em>Devices &gt; Pixel Size Calibration<\/em>&nbsp;to add a new entry. Give the profile name which matches the objective you are currently using. Select the camera model name under the properties tab to associate it with the calibration data. Click&nbsp;<em>Measure<\/em>, then select&nbsp;<em>Automatic<\/em>&nbsp;and set the&nbsp;<em>Safe travel radius<\/em>&nbsp;to 10000 \u00b5m. Click Start. Save the calibration settings when the process is complete.<\/p>\n\n\n\n<p>Repeat this process by creating a new entry in&nbsp;<em>Pixel Size Calibration<\/em>&nbsp;whenever a different magnification of the microscope is needed.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"Capture_an_Image_Array\">Capture an Image Array<\/h3>\n\n\n\n<p>Now that the devices are configured and calibrated, \u00b5Manager can automatically capture the required images in sequential order. To create the grid of source images, select&nbsp;<em>Tools &gt; Multi-Dimensional Acquisition<\/em>. In the window, check the&nbsp;<em>Multiple Position (XY)<\/em>&nbsp;box and select&nbsp;<em>Edit position list\u2026<\/em>. Select&nbsp;<em>Create Grid<\/em>&nbsp;to open the Tile Creator.<\/p>\n\n\n\n<p>The \u00b5Manager Tile Creator tool will automatically determine the number and position of images required to cover the sample area.<\/p>\n\n\n\n<p>Select the Hand tool:<\/p>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img decoding=\"async\" src=\"https:\/\/stagingphotonics.laser2000.co.uk\/wp-content\/uploads\/2023\/12\/Hand-tool.png\" alt=\"\" class=\"wp-image-39684\"\/><\/figure>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>and align the top left corner of the Snap\/Live view with the top left corner of the region you want to capture. In the Tile Creator, there will be four Set buttons as shown in Figure 3. Select the top&nbsp;<em>Set<\/em>&nbsp;button and then the left&nbsp;<em>Set<\/em>&nbsp;button. Drag the Snap\/Live view to show bottom right corner to be captured and press the right and bottom&nbsp;<em>Set<\/em>&nbsp;buttons.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img decoding=\"async\" src=\"https:\/\/stagingphotonics.laser2000.co.uk\/wp-content\/uploads\/2023\/12\/Tile-creator.png\" alt=\"\" class=\"wp-image-39685\"\/><figcaption class=\"wp-element-caption\">Figure 3: Creating the Grid<\/figcaption><\/figure>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>The Tile Creator allows you to specify a desired overlap. 15% is a good starting value which ensures that no details on the edges of images are missed, and allows smooth blending between images. Increasing the overlap value can be useful if you are using a lens which has optical distortion or decreased brightness away from the center of the lens. Depending on the pixel size and overlap values that are set, the original positions (top, right, left, bottom) may be shifted slightly to fit the grid calculated within \u00b5Manager.<\/p>\n\n\n\n<p>When you are finished, choose&nbsp;<em>OK<\/em>&nbsp;in the Tile Creator window and Close in the Stage Position List window. If you&#8217;d like to save all of the images, check&nbsp;<em>Save Images<\/em>&nbsp;and choose a directory to save them in. Finally, when you are ready to capture the images, select&nbsp;<em>Acquire<\/em>&nbsp;and the stage will start moving to each position in the grid while capturing images.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"Stitch_Images\">Stitch Images<\/h3>\n\n\n\n<p>Download Fiji from the link in the Equipment section above, you can open it by running the included executable file. Go to&nbsp;<em>Plugins &gt; Stitching &gt; Grid\/Collection Stitching<\/em>.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img decoding=\"async\" src=\"https:\/\/stagingphotonics.laser2000.co.uk\/wp-content\/uploads\/2023\/12\/250px-Aquisition-order.png\" alt=\"\" class=\"wp-image-39686\"\/><figcaption class=\"wp-element-caption\">Figure 4: The default acquisition order for Grid\/Collection stitching using a Zaber ASR XY stage<\/figcaption><\/figure>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>Select&nbsp;<em>Type: Column-by-column, Order: Up &amp; Left<\/em>. Fill in the grid size, tile overlap, image directory, and file name information. The optimal settings for tile overlap can be determined by using the same pixel size and overlap settings that were used when creating the grid.<\/p>\n\n\n\n<p>The individual image files will be loaded by FIJI sequentially based on their file names. Images saved by \u00b5Manager use a different naming convention than is expected by FIJI. Rename the images using naming convention&nbsp;<em>tile_{ii}.tif<\/em>&nbsp;where&nbsp;<em>{ii}<\/em>&nbsp;is incremented, starting at&nbsp;<em>{01}<\/em>. Click&nbsp;<em>OK<\/em>&nbsp;to start stitching. The stitched image will be displayed once the process is complete.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\" id=\"Example_Output\">Example Output<\/h3>\n\n\n\n<p>As an example, we captured an array of high-resolution images of the Zaber logo on one of our business cards as shown in Figure 5.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img decoding=\"async\" src=\"https:\/\/stagingphotonics.laser2000.co.uk\/wp-content\/uploads\/2023\/12\/500px-High-res-image-result.jpg\" alt=\"\" class=\"wp-image-39687\"\/><figcaption class=\"wp-element-caption\">Figure 5: Original Card Being Magnified<\/figcaption><\/figure>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>When the grid was created, it required 2 rows of 13 images to cover the area. A sample of the acquired images are shown in Figure 6.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img decoding=\"async\" src=\"https:\/\/stagingphotonics.laser2000.co.uk\/wp-content\/uploads\/2023\/12\/450px-Original-picture.png\" alt=\"\" class=\"wp-image-39688\"\/><figcaption class=\"wp-element-caption\">Figure 6: Original Pictures<\/figcaption><\/figure>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>Finally, Figure 7 shows the high-resolution assembled image from the Fiji stitching plugin.<\/p>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<figure class=\"wp-block-image aligncenter size-full\"><img decoding=\"async\" src=\"https:\/\/stagingphotonics.laser2000.co.uk\/wp-content\/uploads\/2023\/12\/500px-Image-stitched.jpg\" alt=\"\" class=\"wp-image-39689\"\/><figcaption class=\"wp-element-caption\">Figure 7: Zaber Logo Image Stitched<\/figcaption><\/figure>\n\n\n\n<div style=\"height:44px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<p>Please email&nbsp;<a href=\"mailto:sales@laser2000.co.uk\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>sales@laser2000.co.uk<\/strong><\/a>&nbsp;or call&nbsp;+44 (0) 1933 461 666&nbsp;to speak with one of our experts if you are interested in a custom system.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Introduction Image stitching is the process of acquiring and merging a sequence of images into a single, large and detailed image. Microscope [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[17,21],"tags":[],"class_list":["post-39673","post","type-post","status-publish","format-standard","hentry","category-blogs","category-microscopy-blogs"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO Premium plugin v22.6 (Yoast SEO v27.7) - https:\/\/yoast.com\/product\/yoast-seo-premium-wordpress\/ -->\n<title>Image Stitching for Microscope Applications using \u00b5Manager - Laser 2000<\/title>\n<meta name=\"description\" content=\"Image Stitching for Microscope Applications. 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