1. Editorial. N 9 June Content

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1 N 9 June 2010 Content 1. Editorial 2. Timelapse: news and updates 3. n vivo rodent imaging setup available in Epalinges Workshops 5. Spotlight on mage Stitching 1. Editorial We welcome new and existing readers to this issue of the CF Newsletter. The aim of the newsletter has been to keep users of the facility and all those interested in the field of microscopy and imaging informed of the activities of our core facility. The Cellular maging Facility has experienced many exciting developments since its creation in 2003, that our research community directly benefits and are made possible by the determined and continuing support of the Faculty of biology and medicine of UNL and of CHUV. The latest major development has been the opening of a third branch of the facility on the Epalinges campus. At this stage, all three branches of the CF are operational and function as a single entity. With the increased panel of imaging resources offered and the increasing number of users, the core facility will do its best to maintain a high level of service over the next years. A significant challenge for the CF will be also to provide adequate answers to increasingly complex technological demands, for instance in the direction of live cell imaging. As you will see from this issue, significant efforts are currently deployed towards higher throughput imaging both for morphological and live cell imaging. hope that you will enjoy reading this 9 th issue of the CF Newsletter and welcome your feedbacks! Jean-Yves Chatton CF Coordinator 1

2 2. Timelapse: news and updates The CF has now three complete timelapse image acquisition stations. On the three locations of the CF (Bugnon, Dorigny and Epalinges) you will find brand new and updated setups representing state-of-the-art machines in this domain. The setups offer you many new possibilities and a great versatility for recording living cell samples over time and other dimensions. Our multi-dimensional acquisition systems on the three locations in Lausanne will allow you to follow your living samples over time and with a controlled temperature, humidity and CO 2 environment using stage-top incubators. Each incubator can be fitted with dedicated inserts that can accept multiwell plates (Falcon M12), glass bottom chambered slides (LabTek 2,4,8... chambers) or standard 35 or 60 mm glass bottom Petri dishes. On the systems with motorized xy stage controllers, multiple acquisition positions can be defined in terms of xy coordinates as well as of z series (stacks) in depth. n addition to all these spatial dimensions it is possible to configure your acquisition sessions for multiple channel and multiple wavelength recordings (fluorescence and brightfield). The different magnifications available cover the range from 10X up to 100X objectives. The best to do if you have questions is simply to contact the CF Technical Manager of your current location in Lausanne for more information about the videomicroscopytimelapse offer and capabilities. 2

3 3. n vivo rodent imaging setup available in Epalinges A whole animal in vivo imaging setup called Xenogen VS Lumina is available in Epalinges in the conventional animal facility. This setup allows imaging of both luminescent and fluorescent signals. t is only slightly invasive, as it only requires anesthesia during image acquisition (that can be performed using isoflurane) and luciferin injection in case of luminescent signal detection. t has therefore the advantage of potentially collecting a lot of information from single mice over time. Moreover, the system allows simultaneous imaging of up to five mice. The system consists of a dark imaging chamber equipped with a heating stage to maintain optimum body temperature, gas anesthesia, and with a very sensitive supercooled camera. Motorized filter wheels are used for fluorescence excitation and emission. The acquisition and processing of the data are done using the Living mage 3.1 software on the imaging setup. n addition, off-line versions of this software are also installed on the image processing workstations of CF Epalinges and CF Bugnon to facilitate data processing. Luminescence can be detected from cells or micro-organisms genetically modified to express luciferase, when they are in presence of the substrate luciferin. Nowadays, a lot of cells expressing luciferase are available commercially as models of cancer development or inflammation, for example. Moreover, light producing animal models using human or mouse inducible promoters to drive luciferase expression are also available commercially. They are designed for a variety of therapeutic areas including: Drug metabolism and toxicology nflammation Angiogenesis and Oncology Metabolic diseases Endocrine Signaling Organ Transplant Chemical Toxicity 3

As our system is equipped with a variety of fluorescence filters, multiple reporters can be simultaneously detected in vivo (e.g. Cy5.5, Alexa Fluor, DsRed, GFP etc ).

4 As our system is equipped with a variety of fluorescence filters, multiple reporters can be simultaneously detected in vivo (e.g. Cy5.5, Alexa Fluor, DsRed, GFP etc ). This enables the use of cells previously selected by FACS. However, despite a rather strong fluorescent signal, it is more complicated to perform in vivo fluorescent measurement (animal autofluorescence, light absorption by the tissue) as compared to luminescent imaging. Therefore, for suggestions regarding the fluorochromes or mice strains easier to use, it is highly recommended to contact the CF staff as soon as possible during the research protocol elaboration Workshops Like the previous years, imaging workshops are proposed to the CF users. Starting in June, this year s program tries to cover all the main topics we consider as a real plus for any scientist interested in imaging. Module Description Part Title Date Location Digital mage Handling ntroduction to commonly used functions and operations on digital images Compression, formats, resolution, useful tools for basic operations (Photoshop, Powerpoint, mage J, etc.) Basic transformations and operations 1 June 2010 CF-E 8 June 2010 CF-E mage J The use of the opensource image analysis and processing software magej magej basics, macros, description of plugins developed at CF 7 September 2010 CF-B Macroscopy / Xenogen maging on large samples, methods, instruments and softwares (macroscope, xenogen, image stitching) Macroscopic imaging 5 October 2010 CF-E Metamorph The use of the commercial image analysis and processing software Journals, automation, multidimensional acquisition, advanced functions. 2 November 2010 CF-D Colocalization Colocalization studies with multichannel images Setting up image acquisition parameters, cross-talk, 2D vs. 3D, non-standard techniques, quantitative estimates 30 November 2010 CF-B Workshops are held on Tuesdays at 12:15 to 14:00. Admission is free and open to anyone interested. These workshops are usually intended for max 15 pers. Therefore, we kindly ask those who wish to attend to register to jean-yves.chatton@unil.ch. 4

5 5. Spotlight on mage Stitching As white light and fluorescence microscopy becomes more and more friendly to use, users tend to ask more. One recurring demand is to acquire high resolution images together with large fields of view. This has always been quite difficult to obtain, because to image large fields of view you had to use small magnification objectives, sacrificing resolution to dimension. These last years we have seen the emergence of a new technique coming directly from photographers who wanted to produce panorama pictures: mage Stitching. The principle: The idea is quite straightforward: take highly detailed images (close-ups) and merge them together using a program like Photoshop to obtain a high resolution composite image. f this is made easy for a photographer with his camera, doing such a work with a microscope was quite more demanding for the scientists, until microscopy companies developed automated processes and software integration for this new approach. The CF offer: Currently several systems at the CF have the necessary components to do image stitching: The confocal microscopes Leica SP5 and the Zeiss LSM 710 (up to 100x100 tiles together with multichannel and Z stacks) at the Bugnon, the LSM 510 at the Dorigny campus, and the SP5 and the LSM510 at the Epalinges campus can more or less provide support for image stitching, as a built-in feature or using macros. 5

6 Regarding conventional microscopes, the Axiovision setup at the Bugnon is now equipped with the MosaiX module, dedicated to image stitching. MosaiX Features Generate precise, high-resolution images and virtual maps of large surfaces Use an overview image to interactively define multiple scan areas Use motorized stage to automatically scan entire samples Sort single images in a tile view Combine overlapping single images to a pixel-precise overview image Extract interesting image regions from the overview image and display them as high resolution images Extract selected image objects for further analysis Multiple extractions possible from a single overview image Reposition captured objects using the stage navigator MosaiX images are compatible with all image processing and measurement functions of AxioVision Beside these direct integrations of the image stitching feature, there are some thirdparty software that can work on images acquired almost anywhere. mage J with plugins, Metamorph, and Autostitch which is dedicated to this particular task, or even Photoshop, can do the job. Pretty heavy computer power might be needed, and specific acquisition protocols (like being sure to have some overlapping) are mandatory for successful stitching. f you feel the need of using such a technology, please contact Yannick KREMPP or your respective CF manager for further details on how this works using the system of your choice. 6

Multifluorescence The Crosstalk Problem and Its Solution

Multifluorescence The Crosstalk Problem and Its Solution If a specimen is labeled with more than one fluorochrome, each image channel should only show the emission signal of one of them. If, in a specimen