- ibidi heating system - stage top incubator for life cell imaging

Transcription

1 Instructions - ibidi heating system - stage top incubator for life cell imaging Please also see the Add-on: - gas incubation with active gas mixer - Instructions, - ibidi heating system - Page 1 of 15

2 Dear user, Live cell imaging enhances the power of cell microscopy immensely. Intracellular transport phenomena can be tracked with high resolution fluorescence microscopy. But also low magnification microscopy of living cells is of great interest like in migration or proliferation experiments. The examples show the wide variety of possible requirements in the optical setup of microscopes often being used in one lab. Designing our heating system, therefore, it was important to provide maximal flexibility. The goal was to make it compatible with more or less every type of microscope, regardless type and brand. The idea of the ibidi heating system is creating a micro-environment for cell culture and microscopy. The basic requirements of the system are: Stable conditions for temperature, humidity, O 2, and CO 2 Compatible to any inverted microscope Suitable for all ibidi µ-slides and µ-dishes Use of all objective lenses (up to 100x oil) Portable from one microscope to another Easy handling Reasonable pricing After we thought about the issue we came up with the idea of searching for a standard format used on inverted microscopes. The solution is simple: the multi-well format. There are large numbers of plates (, 12 well, 24 well, 96 well, ) and all of them have the same outer dimensions: mm x 85.5 mm. Consequently, for every inverted microscope there exists a stage or a holder in this format. This kind of holder is our basis for all microscope platforms. Based on this idea we designed a system which is capable of meeting all the requirements. The outstanding features are the universal usage and the heated lid, which prevents water condensation effects on top and provides stable temperature conditions. CO 2, O 2, and humidity controls complete the system. Thank you for purchasing the ibidi heating system. Best success with it and please give us feedback! Roman Zantl, Elias Horn, ibidi R&D Martinsried, Germany, July 17 th, 2007 Instructions, - ibidi heating system - Page 2 of 15

3 TABLE OF CONTENTS 1) PRINCIPLE MADE FOR LIVE CELL IMAGING ) SYSTEM OVERVIEW... 5 A) MULTI-WELL FORMAT SOLUTION... 5 B) SPECIFIC SOLUTIONS FOR NIKON AND OLYMPUS... 7 C) WEIGHT TABLES: ) TEMPERATURE CONTROLLERS HT 50 / HT A) APPLICATIONS... 9 B) SETTING INTO OPERATION... 9 C) INDICATION OF THE SET VALUE D) CHANGE OF THE SET VALUE ) TEMPERATURE CALIBRATION A) CALIBRATION ROUTINE B) INFLUENCE OF SURROUNDING TEMPERATURE AND AIR FLOW ) TROUBLESHOOTING A) FOCUS NOT STABLE B) EVAPORATION TOO HIGH ) CATALOG NUMBERS ) PERFORMANCE ) WEEE/ROHS COMPLIANCE STATEMENT Instructions, - ibidi heating system - Page 3 of 15

4 1) Principle Made for live cell imaging The stage top incubator consists of a heated plate and a heated lid designed for keeping cells at 37 C in microscopy slides and dishes. The heated lid prevents condensation effects inside the entire incubation system. Humidity, CO 2 enriched, and/or O 2 reduced/enriched air can be flushed in which makes the heating chamber a fully controlled incubator right on the microscope. This enables the user to perform live cell imaging. Schematic drawing of the ibidi heating system Heated lid and heated plate (without insert) The top of the lid and the bottom plate are actively heat-controlled. The heated plate has the outer dimensions of a 96-well plate (127.5 mm x 85.5 mm). Instructions, - ibidi heating system - Page 4 of 15

5 2) System overview a) Multi-well format solution The multi-well format solution provides a solid basis for heating and optional for gas incubation of any inverted microscope that provides a stage or frame for multi-well plates. Inserts are available for all µ-slides, any other regular slides (75.5 mm x 25.5 mm), or 35 mm dishes (ibidi µ-dish). Other inserts can be made on request. Outer dimensions of heated plate and heated lid: Length: Width: Height (with lid): mm 85.5 mm 25.5 mm Due to the height we recommend condensers with working distances of 26 mm. The height of the lid also can be lowered specifically for condensers with lower working distances. Information on gas incubation (CO 2, O 2, humidity) is provided in the Add-on instructions: - gas incubation with active gas mixer - Instructions, - ibidi heating system - Page 5 of 15

6 HEATED LID The heated lid (127.5 mm x 85.5 mm) provides excellent optical quality and allows all standard microscopy techniques, including differential interference contrast (DIC). The upper glass part is electrically heat controlled. The heated lid fits accurately to the heated plate. Heated lid For gas incubation the lid is equipped with an air flow inlet and a humidity sensor. HEATED PLATE AND INSERTS The heated plate provides the base for the heating system. Its format (127.5 mm x 85.5 mm) fits every multi-well stage or multi-well holder (e.g. for 96-well plates) on the microscope. It is completely electrically heated. The heated plate is the counterpart to the heated lid. The two-part inserts put the µ-slides or µ-dishes into position and press them down. They are passively heated by the heated plate and heated lid. Heated plate with inserts Heated plate, heated lid, and inserts are positioned by strong Neodym Magnets! Please contact us for a non-magnetic system in case magnetic fields disturb your experiment! Instructions, - ibidi heating system - Page 6 of 15

7 b) Specific solutions for Nikon and Olympus This solution provides heating / gas incubation for inverted Nikon and Olympus microscopes without multi-well plate holders. Dimensions of the round heated plates: Diameter (Nikon): Diameter (Olympus): Inner diameter (both): 108 mm 110 mm 20 mm Do not clean lid and plate with alcohol! Use ultrapure water (e.g. Milipore water) instead! 1) Nikon is a registered trademark of Nikon Corp. 2) Olympus is a registered trademark of Olympus Corp. Instructions, - ibidi heating system - Page 7 of 15

8 c) Weight tables: SINGLE PARTS: Weight [g] Nikon plate (108 mm) 70 Olympus plate (110 mm) 72 Heated plate (96-well format) 130 Heated lid 80 Inserts COMBINATIONS: Weight [g] Nikon plate (108 mm) with lid 150 Olympus plate (110 mm) with lid 152 Heated plate (96-well format) with lid 290 to 330 and Inserts Instructions, - ibidi heating system - Page 8 of 15

9 3) Temperature controllers HT 50 / HT 200 a) Applications The control units HT 50 / HT 200 were designed for temperature controlling of ibidi heating systems. HT 50 can control one unit, HT 200 can control two. Other applications must previously be consulted with the manufacturer. Please take into account that each controller is adjusted for one or two certain heating devices which are distinguished by their serial number. ibidi can recalibrate a control unit for other heating devices on request. b) Setting into operation The control unit should be used in dry rooms only. The control units must be adjusted to the corresponding voltage (230 V or 115 V). The actual voltage setting may be read and/or adjusted at the integrated fuse holder of the power cord connector at the rear side. In order to change these settings it is necessary to pull out the fuse holder from the casing. Then pull out the fuse socket from the fuse holder. Rotate end to end and replace it so that the proper voltage is displayed through the window. Please do not forget to insert the proper fuse, before putting back the fuse holder into the casing (please watch the value of the fuses when changing the input voltage!). See the heat controller table or inscription on the device. Connect the respective plug of the heating systems to the unit and switch on. The output jacks are performance-oriented assigned to the heating system. The "ON/OFF" switch is at the rear side of the unit. After switching on, the controller starts automatically a self-test, and then the actual value is indicated. HT 50 and HT 200 are labeled with a number code assigned to one single heating device. Do never mix up control units and heating systems! Instructions, - ibidi heating system - Page 9 of 15

10 Be aware when the system is switched on: 24V AC Voltage flow through the downside of the glass plate. It is highly recommended not to touch the downside or to contact it with any metal. This could cause a short circuit that may destroy the controller and/or the lid. c) Indication of the set value When pushing the "SET" button once quickly, the set value is indicated for about 5 seconds. d) Change of the set value Press the SET button for 2 seconds. After that, the set value is indicated, while a LED above the decimal point flashes. The temperature set value may now be adjusted with the buttons 5 or u. The set value is automatically stored after 15 seconds without pushing any button respective after having pushed the SET button once. After that, the actual value is indicated again. Control unit HT50 HT200 Picture Net dimensions (w x h x d): 185x80x180mm Net weight approx.: 1.7kg 1.8kg Environmental temperature: +5 C to +40 C Control range: Environmental temperature to + 55 C Deviation: +/- 0.2K Temperature sensor: PTC KTY 81/121 Input voltage: 230 V / 50 Hz V / 60 Hz Output jacks: 1 output jack 2 output jacks Input voltage: Power input: Power output: Output voltage: Temperature controller: Fuses 230 V / 50 Hz 315mA(T) 115 V / 60 Hz 630mA(T) 230 V / 50 Hz V / 60 Hz 50 VA 50 VA V AC (adjusted by the manufacturer) Electronic two point controller (number depends on the model) with digital LED-display. DIN (5 x 20 mm) Instructions, - ibidi heating system - Page 10 of 15

11 4) Temperature calibration Depending on the room temperature, air conditioning, heat loss via microscope, chamber type or objective lens, and humidification, the temperature has to be calibrated from time to time. Before the first experiment a calibration is required. The heat calibration should be done once for each used chamber type, in order to control temperatures with an accuracy of 1 C or better. For calibration our system comes with a digital thermometer with a heat sensor. a) Calibration routine The recommended calibration routine is fairly simple. Follow the described steps for initial temperature calibration: Connect the temperature sensor to the thermometer. Install the temperature sensor directly to the chamber and µ-slide as indicated in the figure. Fill the chamber with medium, and place the sensor as close as possible to the later position of the cells. Use the prepared µ-slide with a hole in its lid. Set all parameters and conditions as during the later experiments (room temperature, air conditioning, air flow, illumination, microscope settings ). Set the temperature of heating lid and heating frame to usual values (lid 42 C, plate 38 C). After 30 min read the temperature of the sensor. Change the temperature of the heated plate and the heated lid until you reach the required temperatures measured by the sensor. Wait 30 min after each temperature change for system equilibration. Check the temperature of the system from time to time again. The displayed temperatures are valid for the heated plate and heated lid only. The temperature near the cells might be different (lower in most cases)! Heating system with temperature sensor for calibration Instructions, - ibidi heating system - Page 11 of 15

12 b) Influence of surrounding temperature and air flow The surrounding temperature has an effect on the temperature in the microscopy chambers. Small rooms can be heated up significantly by devices like computers, camera controllers, etc. In this case we recommend equilibrating the room temperature to the typical experimental conditions at least 2-3 hours before calibrating. The heat sensor can also be used to measure room temperatures. Air flow can enhance the effect of changes in room temperature. In case air flow (air conditioning etc.) can not be stopped, we recommend decreasing possible effects by protecting the microscope from strong air flow. Please contact us for different possibilities. 5) Troubleshooting a) Focus not stable Especially during time lapse experiments focus drift is an annoying effect. Focus stability is mainly influenced by mechanical changes and temperature instabilities. Follow these recommendations to keep your cells in focus: Switch on all components (heating, gas incubation, computer, other equipment) at least 60 min before starting the experiment. After you put the µ-slide onto the microscope wait 20 min before starting a time lapse experiments to achieve temperature and immersion oil equilibration. (*) Keep the room temperature as stable as possible. Air conditioning should either be working continuously or should be switched off. Do not change temperature during experiments. Avoid door/window openings as this could change temperature rapidly. Eliminate all sources of mechanical vibrations. Use a damped table for your microscope. (*) In case the experiment needs to be started immediately after placing the slide on the microscope or after closing the lid we recommend controlling the focus for 20 min. In the first minutes after starting the experiment temperature equilibration might influence the focus/z-position of the cells. b) Evaporation too high Depending on the incubating conditions, small volumes might evaporate quickly, especially during long term experiments. We suggest using Parafilm (Pechiney Plastic Packaging Company) or silicon oil AR 200 (Fluka, Sigma-Aldrich) to decrease evaporation. If you have an actively controlled humidifying device increase the set value of relative humidity. PARAFILM PROCEDURE: Small stripes of Parafilm on the reservoirs of the slides prevent evaporation effects very well. Fill the slide as recommended. Afterwards, adapt the Parafilm until it fits tightly. Instructions, - ibidi heating system - Page 12 of 15

13 SILICON OIL PROCEDURE: Overlaying the medium with sterile silicon oil prevents all evaporation effects and is compatible with cell culture. Please don t use mineral oil as this would be harmful to the µ-slides. Equilibrate oil and medium inside the incubator overnight. This step avoids the formation of air bubbles and pre-warms the solutions to 37 C. Afterwards, fill your slide with cells and medium. Overlay the medium s surface with an appropriate amount of silicon oil. Don t drip the oil directly on the surface, but let it down run the edges by pressing the pipette tip directly on the upper side of the reservoir. E.g., for µ-slide VI, fill each reservoir with 30 µl cell-free medium and 30 µl silicone oil. Please contact us for further troubleshooting. (info@ibidi.com) 6) Catalog numbers The following catalog numbers correspond to the standard devices for heating system. For pricing please contact your local distributor or ibidi. Catalog number Details/Content Nikon plate with lid Nikon plate without lid Olympus plate with lid Olympus plate without lid well format with lid well format without lid Insert for 96-well format for µ-slide I and µ-slide I Luer Insert for 96-well format for µ-slide 8 well, µ-slide 2x9 well, µ-slide 18 well, µ-slide VI and µ-slide Angiogenesis Insert for 96-well format for µ-dish 35mm, low Insert for 96-well format for µ-dish 35mm, high Insert for 96-well format for µ-slide y-shaped and µ-slide Chemotaxis Information on gas incubation (CO 2, O 2, humidity) is provided in the Add-on instructions: - gas incubation with active gas mixer - Instructions, - ibidi heating system - Page 13 of 15

14 7) Performance The following time lapse movie was made using the ibidi heating system and CO 2 incubation. rat fibroblast cells, phase contrast, 10x objective lens, 60 hours 00 hours 12 hours 24 hours 36 hours 48 hours 60 hours For more videos please visit Heating system on Zeiss Axiovert 100 Instructions, - ibidi heating system - Page 14 of 15

15 8) WEEE/RoHS Compliance Statement EU Directives WEEE and RoHS To Our Valued Customers: We are committed to being a good corporate citizen. As part of that commitment, we strive to maintain an environmentally conscious manufacturing operation. The European Union (EU) has enacted two Directives, the first on product recycling (Waste Electrical and Electronic Equipment, WEEE) and the second limiting the use of certain substances (Restriction on the use of Hazardous Substances, RoHS). Two Categories of products covered by the WEEE Directive are currently exempt from the RoHS Directive Category 8, medical devices (with the exception of implanted or infected products) and Category 9, monitoring and control instruments. All of our products fall into either Category 8 or 9 and are currently exempt from the RoHS Directive. We will continue to monitor the application of the RoHS Directive to its products and will comply with any changes as they apply. Recycling is offered for our products which are within the scope of the WEEE Directive. Products falling under the scope of the WEEE Directive available for sale after August 13, 2005 will be identified with a wheelie bin symbol. Do Not Dispose Product with Municipal Waste. Special Collection/Disposal Required. Please contact us for feedback and further information. ibidi GmbH Am Klopferspitz Martinsried Germany info@ibidi.de Tel.: Fax: Instructions, - ibidi heating system - Page 15 of 15