Culture Myograph System Model 202CM User Manual Version 1.
Culture Myograph 202CM
Culture Myograph System - Model 202CM User Manual Trademarks Pentium is a registered trademark of the Intel Corporation. Windows, Windows 95, Windows 98, Windows ME, Windows NT, Windows 2000 and Windows XP are registered trademarks of Microsoft Corporation. VediView software is a trademark of Photonics Engineering. Euresys Picolo Frame grabber. All other trademarks are the properties of their respective owners. DMT A/S reserves the right to alter specifications as required.
Safety The Culture Myograph System has been designed for use only in teaching and research applications. It is not intended for clinical or critical life-care use and should never be used for these purposes: nor for the prevention, diagnosis, curing, treatment, or alleviation of disease, injury or handicap. • • • • • • • • • • • Do not open the unit: the internal electronics pose a risk of electric shock. Do not use this apparatus near water.
EMC / EMI This equipment has been tested and found to comply with the limits for a Class B Digital device, pursuant to part 15 of the FCC rules. These limits are designed to provide reasonable protection against harmful interference in residential installations. This equipment generates, uses and can radiate radio frequency energy and, if not installed and used in accordance with the instructions, may cause harmful interference to radio communications.
Certificate of Conformity DMT A/S, Skejbyparken 152, 8200 Aarhus N., Denmark, hereby declares its responsibility that the following product: Culture Myograph System– Model 202CM Version 1.4 is covered by this certificate and marked with CE-label and conforms with the following standards: EN 60 065 (IEC 65) Safety requirements for mains operated Electronic and related apparatus for household and similar general use.
Contents Safety....................................................................................................................... 4 EMC / EMI................................................................................................................5 Approvals. .................................................................................................................5 Contents....................................................................................................................
3.6 FAQ................................................................................................................... 38 Chapter 4 - DMT 202CM Control Program.............................................39 4.1 Control of Temperature and Light Intensity.......................................... 39 4.1.1 Connecting the Heat Controller and the Microscope................................... 39 Chapter 5 - Culture Myograph Manual. ................................................... 41 5.
About this Manual This manual contains a complete list of procedures describing how to install, maintain and get started using the Culture Myograph System – Model 202CM – Version 1.4 Chapter 1 provides a comprehensive view of the construction and basic features of the complete Culture Myograph System. Chapter 2 describes step-by-step how to set-up a complete 202CM Culture Myograph System, including all various accessories. Chapter 3 is a complete manual to the VediView Culture Myograph software.
Unpacking the Myograph System Please take a few minutes to carefully inspect your new Culture Myograph System for any damage, which may have occurred during handling and shipping. If you suspect any kind of damage, please contact DMT immediately and the matter will be pursued as soon as possible. If the packing material appears damaged, please retain it until a possible claim has been settled. We recommend that you store the packing material for any possible future transport of the Culture Myograph System.
Chapter 1 - System Overview 1.
1.2 DMT Microscope Light intensity regulation plug Lever to fasten culture Myograph Unit (Fig. 1.1) Infrared light scource Plug for connection of temperature probe Zeiss Achromat 10X X Y Z level regulation / 0.25 objective of objective focus Figure 1.2 DMT Microscope 1.3 Culture Myograph Heat Controller Power ON Led Connection to the Culture Myograph Units USB-Port for Connection to Computer Figure 1.
Chapter 2 - Setting up 2.1 The Complete Culture Myograph System – 202CM Filter Peristaltic Pump (Optional) DMT Microscope DC Adapter (90240V) (Rear Panel) Waste Bottle Power Heat Controller Rear Panel PC Frame Grabber Connection PC Data Acquisition and Analysis Software (Optional) Heat Controller Front Panel Pressure Regulator Front Panel Outlet Superfusion buffer Inlet Superfusion buffer Inflow Perfusion buffer USB Connections Electrical connections Figure 2.
2.2.1 Setting up the Complete Culture Myograph System 202CM This section describes how to connect the cables in the culture myograph system as illustrated in Fig. 2.1. Before connecting any of the myograph equipment, ensure that the frame grabber card is installed into the PCI slot of the computer. Please see installation instructions in Section 2.2.2. 1. Ensure that the heat controller is switched off, on the rear panel before proceeding with the connection procedure. 2.
2.2.2.1 Software and Picolo Driver Installation Insert the Culture Myograph 202CM installation CD, to install the 202CM Control program for the Heat Controller and Microscope, the drivers for the Picolo Frame Grabber and the VediView software package. Normally the computer will automatically start software for installing the programs when you insert the CD. If this does not happen, run the program “setup.exe” from the CD.
Click “Install” The 202CM Control program is now installing from the CD.
Ignore the warning. Click “OK” It is recommended to install the software in the default folder. Click “Next”. Setup Type, choose Typical. Click “Next” Click ”Next” User Manual vers. 1.
Click “Yes” The Picolo Frame Grabber is now installing from the CD. The Picolo Frame Grabber is now installed.
It is recommended to install the software in the default folder. Click “Next” Click “Next” Click “Next” The VediView Software package is now installed. Click “Finish” User Manual vers. 1.
2.2.2.2 DMT 202CM VCP driver installation Connect the Heat Controller to the computer’s USB port, using a standard USB cable. The first time you do this, you must install the VCP drivers as described in Appendix 5. When the VCP driver for the Heat Controller is installed, connect the DMT microscope to the computer’s USB port, using a standard USB cable. The first time you do this, you must install the VCP drivers as described in Appendix 5. 2.3 Experiment Setup 2.3.
Note: A sterile micro filter (0.20µm) is connected between the pressure manometer reservoir and the two perfusion reservoirs to prevent contamination of the perfusion buffer. 2.2.2 Perfusion Flow Control Regulating the difference in height between the two flow reservoirs allows control of the perfusion flow velocity. The principle is illustrated in Fig. 2.3. In Fig. 2.3A the two reservoirs are equal in height and no flow will occur. In Fig. 2.
2.3.3 Setup and Control of Superfusion Flow Peristaltic Pump Micro Filters Culture Myograph Chamber Superfusion Buffer Oxygen Supply Waste Bottle Figure 2.4 Illustration of perfusion flow control The superfusion circuit consists of an inflow and an outflow from the culture myograph chamber. Both flows are driven by the same peristaltic pump, which leads the superfusion buffer from the reservoir to the culture myograph chamber and finally to a waste bottle.
Chapter 3 - VediView™ Software Manual 3.1 Introduction to VediView™ VediView™ provides an interface for image analysis of small blood vessels mounted in a culture myograph and includes data logging facilities for recording dynamic events. VediView™ uses data acquired by a frame grabber hardware, which receives video signals. These signals may come from a camera (with video output) or from a video recorder.
3.2 User Interface 3.2.1 Menus Menus are located across the top of the VediView™ interface. All the actions available using the control bars is also available from the Menus. This common Windows feature is very useful for example when the control bars are turned off. Fig. 3.2 shows how the data menu is used to open a file for data recording. Figure 3.2 Access to VediView™ functions via the menus 3.2.
3.2.3 Toolbar VediView™ contains a "dockable" tool bar containing buttons with specific icons used as short cuts to various VediView™operations (i.e. in the menus and control bars). The tool bar appears in the top of the interface underneath the menus. Some users find the iconic representation more convenient when they have become more familiar with the software. Figure 3.6 VediView™ Toolbar By passing the pointer over each icon and pausing, a “tool tip” will remind the user of the function of each icon.
3.2.4 Main Window The main window can show any of the graphical displays of the control bars, which are the frame grabber image, data control or analysis image. The main window display may be configured from the View menu or simply by a mouse click on the relevant control bar display. The relevant pop-up menu for each control bar can be revealed by a right mouse button click when the Windows cursor is anywhere in the main window. The three options for the main window are shown on below: Figure 3.
3.2.5 Modality Many aspects of the VediView™ software layout are similar to standard Windows applications (e.g. Microsoft Office). Main menus are available for all actions and dialogue boxes. A "dockable" toolbar containing icons is also provided for the most important actions. “Tool tips” are implemented throughout the software, reminding users of the function of active controls (icons, list boxes, radio buttons, etc.) when the mouse icon is allowed to dwell over each control.
All control bars can be “undocked” and re-sized for convenience in a given experiment. Once the control bar has been re-sized it may be “docked”, then careful sizing and positioning is required if the interface is to maintain a well-balanced appearance. Users are advised to experiment with these facilities to fully appreciate their potential. Once a user has designed and configured the appearance of the interface, the setting may be saved and subsequently loaded.
3.3.2.1 Displaying Images The real time frame-grabbing mode may be switched on or off by means of the “start/stop display” button or from the pop-up menu activated (click on ) from the control bar. The same action is also available by selecting the “start/stop image display” icon on the tool bar. The rate at which frames are displayed in the interface depends upon the size of the control bar and whether frames are also being displayed in the main window. 3.3.2.
3.3.2.6 Distance Measurement If the user wishes to measure a distance on the image, VediView™ can supply the dimensions of any rectangle drawn in the main window. Drag and click the mouse to select a rectangle in the main window and then select the "measure rectangle" icon on the tool bar. A dialogue box will appear, giving the rectangle dimensions in selectable units. The default units are image pixels.
3.3.3.1 Selecting Data for Display The data control bar can be used to display up to six parameters in the interface. These can be selected from the list box menus. Control parameters of the myograph (e.g. pressures) or derived parameters (e.g. lumen diameter) can be displayed. The display has an internal memory of several hours where parameters are stored even if they are not displayed.
Data logging is not automatic and must be activated by selecting (or creating) a file. Files are chosen by clicking the “start data logging” button in the data control bar or by selecting the “start logging data” menu item in the pop-up menu (click on ) on the data control bar. When data logging is active the “cross” indicator becomes a “tick” indicator .
3.3.4.2 Calibration of Image Analysis Calibration of image dimensional analysis can be done in sensible units (e.g. microns) defined by the user. To perform such a calibration the user is referred to How To... Calibrate the Image Analysis. The default calibration is performed in units defined by the image data resolution (i.e. pixels). VediView™ can calculate both flow rate and vascular resistance but it requires knowledge about the fluid viscosity and vessel length.
Figure 3.21 No image analysis possible Figure 3.20 Invalid vessel If the analysis rectangle contains a “valid” vessel but only has the outer edges determined by edge detection, VediView will indicate that an estimation algorithm is being used to determine the inner (or lumen) edges (Fig. 3.22 and 3.23). This condition will be clear from the analysis results window showing the green "model" icon . Figure 3.22 One edge detection Figure 3.
VediView can analyse vessels positioned in either the vertical or horizontal planes. The configure analysis dialog box can be used to select the appropriate orientation of vessel (default vertical). This feature is useful in some microscope set-ups where the vessel appears horizontal in the captured image. 3.3.4.5 Logging and Reviewing Results of Analysis The configuration and control of data logging is performed from the data control bar.
5. Once the dimensions and units of the calibrated rectangle are correctly entered, click the ”calibrate” button on the dialog box. The VediView™ image analysis is now calibrated. All parameters passed to other control bars or logged will be calibrated. 6. To save the calibration settings for future experiments, a calibration file (*.cal) can be saved and subsequently loaded from the Calibration dialog box.
interface is done through reconfiguration of the control bars and main window contents. In the example shown in Fig. 3.28 the data control bar has been removed. The easiest way to do this is from the icon on the tool bar. The graphical contents of the analysis control bar has been selected to the main window and subsequently the analysis control bar itself has been removed (using the tool bar icon ).
3.6 FAQ There are only six parameters in the graphical display, how do I display other data acquired or calculated by VediView™? All the data acquired or calculated within VediView™ can be displayed but the maximum number appearing in any given display is six. The data control bar is used for selecting the appearance of the display. Whatever the status of the data display is, all parameters are calculated and stored internally.
Chapter 4 - DMT 202CM Control Program 4.1 Control of Temperature and Light Intensity In addition to the VediView™data acquisition and analysis software a program is installed on the computer allowing temperature and light intensity control of the Culture Myograph 202CM system. To run the DMT 202CM control program, please select the “202CM controller” which was copied to your desktop during installation.
Set-temperature: The temperature controller tab is used to adjust the set temperature for each chamber (range: 10 - 450C with 0.10C accuracy). If a temperature is changed, click the button Change and the change is up loaded to the heat controller. The setting is permanently stored in the heat controller. Status: “Status” indicates status for each chamber: : The Myograph is not connected to the heat controller. : The Myograph is connected. The heat has not reached set temperature.
Chapter 5 - Culture Myograph Manual 5.1 The Culture Myograph Unit The culture myograph unit is placed on the DMT microscope as illustrated in Fig. 5.1. Press the black lever to lock the culture myograph unit onto the DMT microscope. 5.1.1 Glass Cannula Adjustment The glass cannulas are adjustable in all X Y Z directions. The adjustments are illustrated in Fig. 5.1 Horizontal adjustment, please see fig. 5.2 Vertical adjustment Longitudinal adjustment Myograph unit lock Figure 5.
Horizontal Adjustment: Horizontal adjustment is performed as illustrated in Fig. 5.1 and 5.2. Gently loosen the screw fixing the chamber and carefully move the myograph chamber in either clockwise or counter clockwise direction to adjust the horizontal alignment of the glass cannulas. Horizontal Adjustment Screw. Loosen this screw and carefully turn CW or CCW. Observe how the pippettes move in horisontal plane. Figure 5.
5.1.2 Removal and Mounting of Glass Cannulas and Chamber Replacement of glass cannulas and demounting of the culture myograph chamber for sterilisation are common routines in culture myograph experiments. The procedure is illustrated in Fig. 5.3. Fixation screw O-rings Lock-screw Figure 5.3 How to free the glass cannulas from the culture myograph unit Removal of Left Glass Cannula: Carefully remove the two O-rings (marked by the two arrows) to free the glass cannula from the culture myograph unit.
5.1.3 Changing O-rings Each locknut on the culture myograph chamber is equipped with rubber O-rings (ø1.07 x 1.27mm) to keep the chamber tight. These O-rings will from time to time need to be replaced. The procedure is illustrated in Fig. 5.4. Figure 5.4 Changing O-rings. • Unscrew the locknut to reveal the O-ring and remove it carefully using small forceps or a similar tool. • Insert the new O-ring into the chamber hole and push it back until it makes contact with the bottom of the hole.
5.2 The DMT Microscope The DMT microscope is an invert microscope equipped with a Zeiss Achromat 10X / 0.25 objective and build-in CCD camera. The objective is adjustable in all X Y Z directions using the three micro positioners on the front as illustrated in Fig. 5.5. Figure 5.5 How to adjust the position and focus of the microscope in the X Y Z directions.
5.3 Pressure Regulator The Pressure Regulator is connected to the culture myograph system to generate a pressure on the mounted vessel from the perfusion buffer. The pressure is adjusted using the knob and air release valve shown in Fig. 5.6. Figure 5.6 Pressure Regulator 5.4 Culture Myograph Maintenance The Culture Myograph System – Model 202CM is a very delicate and sophisticated piece of research equipment.
In cases of red or brown discolorations appearing on the chamber sides, the following cleaning procedure will work in most cases: 1. Incubate the myograph chamber for 30 minutes with 20µl of a 2mM T-1210 Tetrakis-(2-pyridylmethyl)-ethylenediamine solution dissolved in double distilled water. 2. Use a swab-stick to mechanically clean all the affected surfaces during the last 15 minutes of the incubation period. 3. Wash the myograph chamber several times with double distilled water. 4.
Chapter 6 - Myograph Accessories and Spare Parts This chapter contains a complete list of equipment needed to set-up a basic culture myograph system. In addition the chapter contains a list of special culture myograph accessories and spare parts. In addition to the main focus on development and manufacturing of myograph systems, DMT has specialised in offering our customers first class laboratory equipment needed for a basic myograph setup and at very competitive prices. Please see: www.dmt.dk or www.
6.2 Culture Myograph 202CM System Accessories This section contains a list of special accessories available for the Culture Myograph System - Model 202CM: • DMT FlowMeter – Model 161FM 6.3 Culture Myograph 202CM System Spare Parts This section contains a complete list of standard spare parts available for the Culture Myograph 202CM. For parts not listed in this section or for special parts, which need to be custom made, please contact DMT for further information.
Chapter 7 - Getting Started 7.1 Dissection Protocol for Small Mesenteric Arteries The culture myograph technique is versatile in that a large variety of physiological and pharmacological studies of ring preparations from different species can be performed. Mostly, the culture myograph is used for investigation of small blood vessels and as an example this chapter describes the dissection of rat mesenteric arteries. 50 1.
Second Branch First Branch Figure 7.1 Branch of the mesenteric arteries 6. Use high quality forceps and ocular dissection scissors (see Chapter 6.1) to dissect the vessel segment of interest. Start cutting through the mesenteric membrane along both sides of the vessel, about 1-2mm from the vessel. To avoid accidentally cutting the artery always cut along the length of the vessels and never perpendicular to them (Fig. 7.2A-B). B A Figure 7.
• The branch points of arteries are V-shaped whereas those of veins are more U-shaped. • The arterial wall contains a thick layer of smooth muscle cells compared to the vein wall, which only contains a single or a few layers of smooth muscle cells. The histological difference is clearly visible in the stereo microscope. • If you still have difficulty and the vein and artery still contain some blood then try to move the blood forward by very gently squeezing the vessels with a forceps.
9. Clean the artery by removing any remaining adipose or connective tissue. Gently pull away adipose or connective tissue to make the connective tissue membrane become visible. Cut the membrane to remove the tissue. 10. Cut the distal end of the artery section to be investigated. Afterwards cut the proximal end while ensuring that the vessel segment has the correct length (Fig. 7.5A-C). Figure 7.5 A, B and C Cutting free the artery to be studied A B C User Manual vers. 1.
7.2 Mounting Protocol for Small Arteries This section shortly describes the basic technique of mounting small vessels in the culture myograph: • One end of the vessel is care-fully mounted and secured (with two fine nylon sutures) to one of the hollow glass micro-cannulas. • The lumen is flushed gently with PSS to remove any blood or debris. • The second end of the vessel is then mounted and secured to the second micro-cannula.
7.3 Buffer Recipes 7.3.1 Physiological Saline Solution (PSS) 1x PSS (1000ml): Solution 1. Chemical NaCl KCl MgSO4 - 7H2O KH2PO4 MW (g/mol) 58.44 74.56 246.48 136.09 Conc. (mmol/l) 118.99 4.69 1.17 1.18 Conc. (g/l) 6.954 0.350 0.289 0.161 Solution 2. Chemical CaCl2 - 2H2O MW (g/mol) 147.02 Conc. (mmol/l) 2.50 Conc. (g/l) 0.368 Solution 3. Chemical NaHCO3 EDTA Glucose MW (g/mol) 84.01 372.24 198.77 Conc. (mmol/l) 25.00 0.
25x Concentrated PSS (1000ml) Solution 1 Chemical MW (g/mol) NaCl 58.44 KCl 74.56 CaCl2 - 2H2O 147.02 Conc. (mmol/l) 118.99 4.69 2.50 Conc. (g/l) 173.850 8.750 9.200 Solution 2 Chemical MgSO4 - 7H2O KH2PO4 MW (g/mol) 246.48 136.09 Conc. (mmol/l) 1.17 1.18 Conc. (g/l) 7.225 4.025 Solution 3 Chemical EDTA MW (g/mol) 372.24 Conc. (mmol/l) 0.03 Conc. (g/l) 0.
Appendix 1 - Terms of Warranty Warranty DMT A/S warrants to the original user that myograph systems manufactured by DMT A/S will be free from defects in materials and workmanship for a period of three years after the date of delivery. DMT A/S will repair or replace any defective part, subject to the conditions, limitations and exclusions. Exclusions Spare parts of myograph systems manufactured by DMT A/S, are disclaimed from any warranty.
Appendix 2 - Service Check A myograph working at optimal performance is extremely important for success when studying small blood vessels or other small tubular tissues. To make sure that our customers always are dealing with first class myographs, DMT offers a Myograph Service Check at a very favourable price. The Myograph Service Check includes a complete separation and inspection of all mechanical and electronic parts.
Appendix 3 - Shipping Instructions If the myograph system needs to be sending back for service or repair, please read the following shipping instructions very carefully. Before you start packing the myograph system, please remember that you are dealing with very delicate equipment and therefore care must be taken. DMT recommends that each part of the myograph system be wrapped individually (i.e.
Appendix 4 - System Specifications Chamber Unit: Vessel size: Chamber: Chamber volume: Capillary ports: Superfusion ports: Base window: Chamber material: Chamber cover: > 50µm Single Max. 2ml OD 1.2mm Two built-in 16 x 0.1mm Acid resistant stainless steel Yes Culture Myograph: Alignment: Adjustment: Flow: Temperature range: Temperature resolution: Temperature control: Temperature probe: Two capillary holders X, Y & Z directions Two 25 ml bottles for flow adjustment Ambient temp - 50 �� °� C 0.
Appendix 5 - Fuse Changing The main fuse of the myograph system is placed inside the power inlet on the Heat controller. If the fuse blows it is easily changed using the following procedure. Important: If the fuse needs to be changed, make sure that the replacement fuse is equal to the one blown. Specifications: T2A / 250V, 6.3 x 32mm Use a small screwdriver to open the voltage selector block Red Fuse Block Fuse Fuse User Manual vers. 1.
Appendix 6 - VCP – Driver Installation The first time the 202CM Heat Controller and the DMT microscope is connected to the PC the VCP drivers must be installed. They are on the installation CD delivered with 202CM. To install VCP drivers for 202CM under Windows XP please follow the instructions below. Be sure the Installation CD is inserted in the PC.
If Windows XP is configured to warn when unsigned (non-WHQL certified) drivers are about to be installed, the following screen will be displayed. Click “Continue Anyway” to continue with the installation. If Windows XP is configured to ignore file signature warnings, no message will appear. The following screen will be displayed as Windows XP copies the required driver files: Windows should then display a message indicating that the installation was successful. Click “Finish” to complete the installation.
When the drivers for the Heat controller and the microscope are installed the 202CM Controller Software can be opened and connected as described in paragraph 4.1.1. Please note that if the Heat Controller or the DMT Microscope is connected to another USB port on the PC, the procedure above must be repeated for the specific USB port.
DMT A/S Skejbyparken 152 DK-8200 Aarhus N Denmark Tel.: +45 87 41 11 00 Fax: +45 87 41 11 01 www.dmt.dk support@dmt.dk or sales@dmt.dk DMT - Asia Everwin Gardens Rm 502, Block B 521 Wanping Nan Lu Shanghai 200030 China Tel: + 86 (0) 21 64869685 Fax: + 86 (0) 21 64280591 sales@dmt-asia.com support@dmt-asia.com DMT-USA, Inc. P.O. Box 681373 Marietta, GA 30068-0023 USA Tel.: +1 770 612 8014 Fax: +1 678 302 7013 www.dmt-usa.com support@dmt-usa.com or sales@dmt-usa.
