Confocal Laser Scanning Microscopy

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1 Name of the Core Facility: Confocal Laser Scanning Microscopy CORE Forschungszentrum Immunologie Mainz Welcome to the CSLM Core Facility: The CLSM Core Facility enables working groups to incorporate high resolution confocal laser scanning techniques into their research programs. The CLSM core offers state of the art equipment, expertise, hands- on training and support in microscopic- based image collection and analysis. Advances in the molecular imaging field have led to more refined acquisition methods and to a dramatic increase in the number of useful reagents. For most working groups the time invested to learn these techniques may be overwhelming. Thus, and perhaps most importantly, the key benefit offered by CLSM Core Facility is the possibility for groups to efficiently achieve in the shortest possible time the highest quality imaging results using state of the art instrumentation. What is CLSM? Confocal Laser Scanning Microscopy (CLSM) is a fluorescence based imaging technique that provides greater resolution than conventional fluorescence microscopy. The optics are so arranged that information is collected from a very narrowly defined focal plane and out of focus blurring information is eliminated. Using this technique, it is possible to image thin x- y sections along the z- axis, which produces a stack of high resolution images (optical sections) devoid of out of focus light. This stack of images represents the thickness of the specimen. For example, imaging through one cell can routinely yield a stack of optical sections. The stack of x- y sections can then be projected together to provide an extended range of focus image and rendered into 3D reconstructions. Advantages of CLSM: 1. Clearer images Point illumination is achieved using lasers of defined wavelengths rather than UV lamps as a light source. The point detection is achieved by the addition of a pinhole in front of the fluorescence detector. Due to its point illumination/detection properties, out of focus light is excluded from reaching the detector. Because of this, only in- focus light is detected, hence sharp images are collected. 2. Increased sensitivity In confocal microscopic systems fluorescence from the sample is detected using highly sensitive photomultiplier tubes (PMTs). This is advantageous in that samples of low intensity

2 can be imaged. Furthermore, for samples that are sensitive to photobleaching, lower excitation intensity can be used hence allowing for longer imaging times. 3. Reduced photobleaching Because of the use of point illumination light sources (lasers) to excite the sample, only small areas of the samples are photobleached. 4. Three- dimensional reconstruction Due to its ability to perform optical sectioning, thicker samples can be imaged and reconstructed in an in- focus three- dimensional manner using image analysis software. These images can be displayed as rotating movies as well as stereo- images. 5. More accurate quantitation The low incidence of out of focus light in the digital images generated by CLSM enables more accurate quantification of images. Quantification of relative fluorescence levels within an image can be achieved using commercial or freeware image analysis software. What we offer: 1. Multiple simultaneous analyses Multiple fluorescence detectors allow simultaneous analysis of multiple cellular properties and/or markers. The range of useful fluorochromes is further expanded in instruments utilizing multiple lasers. 2. Live imaging A temperature controlled chamber with CO 2 input for long term live imaging of specimens is available. Autofocusing during imaging insures stable focus parameters. A motorized stage provides parallel image collection within a sample at multiple locations and/or another sample/treatment during a time series. 3. Guidance in experiment design Choice of mounting media, coverslips, immersion media and lenses, is probably one of the most important aspects of confocal microscopy. Also the potentials and possibilities should be clear from the beginning. 4. Advice for sample preparation An image is only as good as the sample. Talk to us. We produce thousands of images each year from a wide variety of samples. Use our experience. 5. Imaging as service To make it possible for all users to get the best results out of their samples, we offer the imaging service by the Facility Manager. Thus it is not necessary for the users to learn the CLSM or deal with problems. 6. User training For users that have on- going imaging projects and are interested in confocal laser scanning microcopy, it is possible to receive hands- on training. Once trained it is possible to make appointments and image alone.

3 System and requirements: Lasers and examples of compatible of dyes: Laser Wave length (ex) Example of Detectable Dyes UV-Diode 405 nm Hoechst, DAPI Argon Laser 458 nm, 488 nm, 514 nm CYP, GFP, FITC, Alexa 488 HeliumNeon 543 nm TexasRed, Cy3, Alexa546 HeliumNeon 591 nm HeliumNeon 633 nm Cy5, Alexa633, Toto3 NLO Photon excitable dyes The system includes a fully integrated workstation that incorporates image acquisition and image analysis software. A metamorph workstation is available for semi- automated quantitative analysis, for example motion of cells. Nomarski optics allows superimposition of signals from three fluorophores onto a DIC image. Zeiss software for reconstruction of 3- D images, presentation of image "galleries", Z- sectioning, time series, etc. The Zeiss Axiovert Inverted Scope used by the system has 10x, 25x and 63x oil, 10x and 40x water objectives and a Z- axis motor. All functions are computer controlled. A temperature controlled tissue culture stage, which holds chambers with cover slip bottoms and applies CO 2. Also important is the holder for your sample. Very convenient is to have a chamber for cultivating cells that has a bottom the thickness of a coverslip. Otherwise high resolution confocal imaging is not possible. There are different products on the market. Please contact us for further information.

4 Zeiss LSM 710 NLO

5 Image gallery Upper left: Differential interference contrast (DIC) image overlayed with confocal images. Other panels are examples of images from 2 and 3 channel image collections.

6 This image shows the liver of a (ROSA)26 ACTB- tdtomato- EGFP x LYS- M cre transgenic mouse. The liver tissue is red by the membrane targeted tdtomato- rot (mt) cassette. Removing the tdtomato cassette by homologuous cre recombination with a macrophage specific promoter, activating the silent green fluorescent protein, gives macrophages green fluorescence. Picture is a two photon Image of a 3x3 raster of 15 optical sections (135 pictures). User obligations: Users may reserve, in advance, only 1 session at a time. Additional sessions may be reserved after that session is completed. If you need to reserve multiple sessions in advance, please contact the facility manager to reserving time. For reservation of an appointment, please contact the facility manager (at least 1 week ahead of an appointment). If you are unable to use the microscope during the time that you have reserved, please let the facility staff know 24 hours in advance. Repeat "no- shows" will lose their sign- up privileges. Users who do not arrive within 15 minutes after the beginning of the time block that they have reserved will forfeit their time on the microscope and have to pay for one hour use. Before the first imaging session, the group leader/head of department must sign a form that costs will be covered.

7 Users are required to enter (IN LEGIBLE NOTATION) all necessary information in the log- book before and after they use the microscope. Users must CLEAN the microscope and dispose of garbage when finished. Duration: Sessions are counted hourly. One session should not exceed 3 hours in duration. Charges: FZI- members und cooperation partners: 25 per hour or part thereof External users: 50 per hour or part thereof Location: Verfügungsgebäude für Forschung & Entwicklung (#911) Obere Zahlbacher Straße Mainz Germany Contacts: Director: Dr. Dennis Strand Verfügungsgebäude für Forschung und Entwicklung Obere Zahlbacher Straße Mainz Germany Tel Fax dstrand@uni- mainz.de Manager: Dr. Steffen Lorenz Verfügungsgebäude für Forschung und Entwicklung (Office ) Obere Zahlbacher Straße Mainz Germany Tel Fax lorenzst@uni- mainz.de