Leica Angle Two™ Computer-Assisted Stereotaxic System User Manual Living up to Life
Intended Use Statement The Angle Two™ system is designed for stereotaxic surgery on small animals only and is not intended for use on humans. Trademarks Leica, Leica Microsystems and logo are trademarks of Leica Microsystems GmbH HRB 5187 and used under license. Angle Two is a trademark of Leica Biosystems Richmond Inc. 36-2899171. Other trademarks are the property of their owners. Copyright Leica Biosystems Richmond Inc. owns the copyright on this document and any associated software.
Contents Leica Angle Two™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Intended Use Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trademarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Copyright. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 Introduction The Leica Angle Two™ stereotaxic system is designed to perform surgery at any angle and position. Reaching the target site is easier and more accurate than manual systems as the Angle Two is computer assisted – all calculations are done for you – meaning no user errors and no pilot studies are needed. Probe position is set with three linear movements (in anterior/posterior, medial/lateral and dorsal/ventral axes) and two rotary movements (tilt and rotation).
2 Instrument Installation The Leica Angle Two™ system needs to be assembled before use. Use the information below to check you have received all parts and to assemble the system. 2.1 Checklist The list below has the standard items supplied in the Angle Two system. Additional or alternative items may be included depending on the configuration ordered.
Instrument Installation 2.2 Assembly When unpacked the Angle Two instrument has either one or two manipulators folded over the U frame. 1. Unlock the tilt clamp and gently lift each manipulator upright into position. The rotation encoder will point down and the tilt encoder to the left, away from the U frame. Figure 1. Unlock the tilt clamp (highlighted) and gently lift the manipulator upright into position Leica Angle Two™ User Manual Rev A02 © Leica Biosystems Richmond Inc.
Instrument Installation 2. Connect the five instrument cables into the ports at the rear of the computer. Each cable and its corresponding port has the same label. If you have a second manipulator, insert the cables into the second card provided. Figure 2. Angle Two cables and computer ports 3. Connect the mouse and keyboard into the computer’s USB ports. 4. Connect the monitor and computer power cables into an outlet. 5. Turn on the computer. The Angle Two icon is displayed on the desktop.
3 Components This chapter describes the parts of the Leica Angle Two™ system and their functions. The components are divided into the following sections: “U Frame” on page 10 – includes the ear bars, nose clamp and incisor bar. “Manipulator: Linear Movements” on page 13 – the three coarse drives that move the manipulator in the AP, ML and DV directions and the fine drive for small movements in the DV direction.
Components 3.1 U Frame Figure 4 shows the Angle Two U frame with a rat adapter and 45° ear bars attached. Some of the part names in the figures below link to sections of the manual where the part is discussed in more detail. Nose Clamp Incisor Bar Ear Bars U Frame Figure 4. The Angle Two U frame The animal is held in the instrument U frame with the head at the closed end of the ‘U’ and the tail at the open end.
Components Caution Do not use the rat incisor bar on a mouse. A mouse adaptor is available from Leica Microsystems Note: Hamsters and gerbils can use the rat ear bars and incisor bar. Nose Clamp The nose clamp is placed over the animal’s nose and gently lowered using the thumbscrew indicated. The nose clamp helps to keep the animal in position when injectable anesthesia is used. Nose clamp Vertical control Removes the nose clamp Figure 6.
Components The ear bars are locked into position using the locking levers. The right lever has a right-hand thread, and the left lever a left-hand thread. The levers only need a quarter turn to lock the ear bars. Figure 7. Turn the lever to lock the ear bar into place Caution Use 45° ear bars if post-surgical testing is planned. Do not use 18° ear bars as they will break the tympanic membrane of an animal. Caution Do not use the 45° or 18° ear bars on a mouse.
Components 3.2 Manipulator: Linear Movements The manipulator is attached to the U frame and is used to move the probe. The manipulator has three linear movements: AP (Anterior/Posterior): moves horizontally along the length of the instrument ML (Medial/Lateral): moves horizontally across the width of the instrument DV (Dorsal/Ventral): moves vertically towards the animal. AP ML DV Figure 8.
Components Each linear movement is controlled by a coarse drive and, in the DV direction, by an additional fine drive. When locating the target site, first use the ML and AP coarse drives to move the probe into place above the target. Then lower the probe until it reaches the brain using the coarse DV drive. Finally, engage the fine drive to continue DV movements to the target within the brain. DV drive ML drive Fine drive AP drive Figure 9.
Components Fine Drive The fine drive (see Figure 9) provides greater control and smoother movement in the DV direction when placing or removing the probe from the brain. The fine drive helps prevent inserting the probe too deep. Using the Fine Drive 1. Push the fine drive towards the cog to engage it. Figure 10. Engaging the fine drive Figure 11. Disengaging the fine drive 2. Turn the fine drive to move in the DV direction. 3. When finished, pull the drive away from the cog to disengage.
Components 3.3 Manipulator: Tilt, Rotation and Swing-Out Tilt, rotation and swing-out allow surgery to be performed at any angle. To change the tilt or rotation, or use the swing-out, unlock the clamps indicated. Swing-Out Tilt Rotation Figure 12. The swing-out, rotation and tilt clamps Tilt changes the manipulator angle and in turn the probe’s angle of approach. The primary tilt can be in either the AP or ML planes. Rotation rotates the plane of tilt between AP and ML. Figure 13.
Components Tilt The primary tilt plane can be in either the AP or ML directions (see Figure 15). Always remember to update the software with the primary tilt direction (“Tilt Direction” on page 29). To adjust the tilt loosen the tilt clamp and move the manipulator to the desired angle, then retighten the clamp. A scale below the clamp shows the amount of tilt. Figure 15. Tilt movement in the AP (left) and ML (right) directions Changing From AP to ML Tilt 1.
Components 2. Unlock the rotation clamp and rotate the base of the manipulator 90° in the opposite direction to the swing-out in the preceding step, so the tilt encoder is facing the front of the instrument and the probe is back over the U frame. The tilt is now in the ML direction (see Figure 15). 3. Retighten the clamps. Changing From ML to AP Tilt 1.
Components Swing-Out Use swing-out to change the primary tilt plane by 90°, between the ML and AP directions (see Figure 13) or to remove the probe from the area within the U frame and return it later to the same position. To change the primary tilt plane see “Tilt” on page 17. To swing out the manipulator: 1. Unlock the swing-out clamp and push the manipulator up with your thumb just enough to clear the raised edge on the base of the manipulator. Swing-out clamp Figure 18.
Components 3.4 Probe Shaft A clamp attaches the probe shaft to the manipulator. The clamp has two holes for positioning the clamp onto the manipulator, and you can rotate the clamp to the left or right of the manipulator arm to give a total of four possible probe positions in the AP direction. Figure 19. Four positions for attaching the probe shaft to the manipulator The Angle Two comes with a standard probe holder.
Components Probe clamp screw Figure 20. The probe shaft screw Attaching a Probe At the bottom of the standard probe shaft is a plastic block with three grooved faces and one flat face. Insert the probe into one of the grooves (grooves are 1 mm apart) and tighten the metal clamp to secure. Probe shaft Clamp screw Grooves on the plastic block Metal clamp Figure 21. The probe holder at the bottom of the probe shaft Leica Angle Two™ User Manual Rev A02 © Leica Biosystems Richmond Inc.
Components 3.5 Vernier Scales All Leica stereotaxic instruments have vernier scales for manual measurements. While the instrument software makes all calculations for you, some manual measurements might be needed, for example to center the ear bars side to side. To use the scales follow the example below. The main scale, graduated in millimeters, is on the ear bar, and the vernier is on the U frame. To measure the position of the ear bar: 1.
Components 3.6 Computer The computer consists of a desktop case, monitor, mouse and keyboard. The computer comes with the Angle Two software that includes an integrated rat or mouse brain atlas. The computer should only be used to operate the Angle Two instrument. If your computer is to be networked we recommend installing antivirus or firewall software only. If you require additional species atlases contact Leica Microsystems (see “Contacting Leica Microsystems” on page 2).
4 Software Overview This chapter describes the Angle Two™ software and its functions. The topics covered are: “Atlas” on page 25 – how to select an atlas and view the coronal plates. “Modes” on page 26 – explains what Set Target, To Target, Atlas and Vertical are used for. “Initial Settings” on page 29 – describes how to set the tilt direction, zero the rotation and tilt angles, enter offsets and indicate bregma and lambda to the software.
Software Overview 4.1 Atlas The instrument comes with either one mouse or one rat brain atlas installed. Contact Leica Microsystems to see what other animal atlases are available. Click Select Atlas to choose an atlas. The atlas consists of a series of coronal plates displayed in the center of the screen. The position of the current plate relative to the rest of the brain is shown in the top left corner. Click Rostral and Caudal to move through the coronal plates.
Software Overview 4.2 Modes Four modes are used during surgery: Set Target, To Target, Atlas and Vertical. Each mode is used for a specific function and has its own set of coordinate boxes that appear beneath the mode selection area. Click the appropriate mode to activate it for use. Modes Coordinate boxes for the selected mode Figure 25. The four modes and coordinate boxes Set Target Select Set Target mode to enter the position of the target site – where the probe will go.
Software Overview 3. Manually enter the coordinates into the Target (Red) boxes. Target coordinates Target position Saved coordinates Figure 26. Target coordinates can be entered manually into the coordinate boxes, loaded from saved coordinates or by positioning the mouse on the screen and clicking at the position If performing bilateral surgery, after the first target site has been located and probe placed, click +/- beside the ML coordinates box.
Software Overview Atlas The Atlas mode shows the exact position of the probe including the rotation, tilt and the deviation of the animal’s skull from 0° (skull flat). The Skull value is only determined once the bregma and lambda points have been located. To save the atlas coordinates click Save Named Point (see “Exporting” on page 32). Vertical Vertical mode is available for users wanting to measure the vertical position from the top of the brain rather than the top of the skull.
Software Overview 4.3 Initial Settings Before locating the target site, you must do the following on the Angle Two software: Select the tilt direction Zero the tilt and rotation angles Enter any probe offsets Enter the locations of the reference points bregma and lambda Zero tilt and rotation and set offsets Bregma button Lambda button Tilt direction Figure 28.
Software Overview Enter Offsets Offsets are used when performing surgery on animals that do not fall within the weight range of the atlas being used. Offsets are a distance in millimeters that is added or subtracted from the probe’s path to the target site. The offsets are determined in pre-experiments by locating the target site using the atlas coordinates. The amount that the atlas coordinates miss the actual target site is determined and used as the offset values.
Software Overview 4.4 Coordinates Coordinates can be saved or exported at any time while the instrument is being used. Double click on saved coordinates to load the target coordinates from a previous study, rather than relocating the target position. Saving coordinates Exporting coordinates Saved Coordinates Figure 30. Saving coordinates, exporting coordinates and saved coordinates Saving The target site, probe position or any other coordinates can be saved by clicking Save Named Point (see Figure 30).
Software Overview 1. Enter a name and description. 2. In Choose Source Point select which coordinates you want to save: 3. Target Position – saves the current target site Device Position – saves the current position of the probe Input – saves a new target position, which you must provide coordinates for. Click Save. Once the coordinates are saved they will appear in the area beneath the atlas with the entered save name.
5 Alignment Align the instrument whenever the software is started. Alignment is necessary to check the horizontal (ML) and vertical (DV) planes are perpendicular. To align the instrument the rotation and tilt must be set so the scales read zero. The alignment kit is installed onto the instrument, with the dial indicator positioned where the probe would normally be. Then the dial indicator is moved across and up the T bar, mounted in the ear bar slots.
Alignment 5.2 Procedure The primary tilt of the instrument can be in either the ML or AP direction so there are two procedures for the vertical alignment. Select the correct instruction set for your instrument set up. Set the Rotation and Tilt The instrument’s rotation and tilt must be moved so that the scales read zero. 1. Unlock the tilt and rotation clamps, and move the instrument so the scales read zero, as shown in Figure 35. 2. Relock the clamps when finished.
Alignment 5. Attach the clamp adaptor to the dial indicator by sliding the raised area of the dial indicator into the groove of the adaptor. Clamp adaptor Dial indicator Figure 36. The raised area of the dial indicator slides into the groove of the clamp adaptor 6. Slide the clamp covering towards the middle of the clamp to reveal the clamp hole. Push the top of the clamp adaptor into the clamp hole and release the covering to lock the adaptor in position. Clamp screw Clamp covering Figure 37.
Alignment 8. Attach the other end of the alignment shaft into the manipulator, and tighten the thumbscrew. Figure 38. The dial indicator attached to the Angle Two 9. Remove the ear bars and install the T-bar. Lock it into position. Figure 39. The T-bar locked into position Leica Angle Two™ User Manual Rev A02 © Leica Biosystems Richmond Inc.
Alignment Horizontal Alignment The ML horizontal path of the probe is controlled by the rotation of the instrument. This part of the alignment procedure checks the ML horizontal path is perpendicular. To test this the dial indicator is moved horizontally along the length of the T-bar. 10. Use the AP drive to position the dial indicator spring against the T-bar. AP drive Dial indicator spring T-bar Figure 40. The dial indicator spring against the T-bar 11.
Alignment 12. Use the ML drive to move the spring horizontally along the T-bar. Move the indicator to one side and then across the total length of the bar to the other side. Figure 42. Move the dial across the T-bar to check the horizontal alignment The instrument is horizontally aligned if the dial arm only moves several divisions back and forth, but stays roughly in the same position. The instrument is not horizontally aligned if the dial arm continuously moves in one direction.
Alignment AP Tilt 13. If the tilt is in the AP direction use the ML drive to position the dial indicator spring in the middle of the T-bar. Do not move the AP drive. Figure 43. Tilt (1) is in the AP direction (2) 14. Move the spring to the bottom of the bar and then all the way up using the DV drive. Figure 44.
Alignment 15. When the AP tilt is aligned, select ll To AP on the computer screen (see Figure 28). 16. Click Setup and click Zero Angles to zero the Tilt and Rotation. Figure 45. The set up dialog and Zero Angles button NOTE: if you cannot vertically align the instrument contact Leica Microsystems. ML Tilt 13. If the tilt is in the ML direction position the dial indicator spring against the side face of the Tbar (see Figure 47). Figure 46.
Alignment 14. Move the spring to the bottom and then up using the DV drive. Figure 47. Vertical alignment when the tilt is in the ML direction The instrument is aligned in the vertical axis if the dial arm only moves several divisions back and forth, but stays roughly in the same position. The instrument is not vertically aligned if the dial arm continuously moves in one direction.
6 Using the Instrument This chapter describes the whole process of using the Angle Two™ system to perform surgery; from starting the software to successfully placing the probe.
Using the Instrument Preparation 1. Ensure that the instrument is clean (see “Cleaning and Maintenance” on page 47). 2. Turn the computer on and start the Angle Two software (see Figure 3). 3. Align the instrument using the alignment kit (see “Alignment” on page 33). Selecting the Target Site The software opens in Set Target mode. 4.
Using the Instrument 11. Support the head and insert the right ear bar into the right ear. 12. Lower the right ear bar into the instrument and lock it in place. 13. Check that both ear bar scales have the same readings. If not, loosen both ear bar locks and move the animal until both scales agree, then relock. 14. Check that the ear bars are fully inserted in the ear canals. There are two tests for this: (i) gently push the nose side to side; (ii) swivel the nose up or down about the earbars.
Using the Instrument Locating the Target Site 22. Click To Target (see “To Target” on page 27) to view the distances to the target site. 23. Use the ML and AP drives to position the probe directly ‘above’ the target. As the target site is approached the ML and AP distances count down. 24. Continue to move the probe until the AP and ML axis displays read zero. A line appears on screen showing the path of descent (DV) towards the target.
Using the Instrument Probe Placement 30. Unlock the swing-out clamp and reposition the manipulator and probe over the animal. 31. Relock the swing-out clamp. 32. Use the coarse DV drive to approach the target site. 33. Engage the fine drive when about to enter the brain. A single blue circle appears around the blue crosshairs when the probe is 0.05 mm from the target site (see Figure 50). 34. Stop moving the probe once a second blue circle appears around the blue crosshairs.
7 Cleaning and Maintenance Caution Do not autoclave. The bushings and probe holder block can be damaged. Caution Do not lubricate the axis rod slides or threaded drive. The plastic bushings can absorb oil and cause the instrument to seize. Caution Do not use alcohol to clean the surface of the probe holder block, as it can cause cracking. Wash the instrument with a cloth or paper towel, dampened with water or dishwasher detergent.
8 Specifications Instrument Dimensions Base width 253 mm Base length 355 mm Base height 20 mm U frame width 165 mm U frame length 200 mm U frame height 55 mm Base and manipulator height 381 mm Base weight (no manipulator) 6.1 kg Resolution, Linear Scales Vernier 0.1 mm Digital display 0.01 mm Internal scale 0.005/rev Drive Advancement per Revolution Linear axis drive (screw pitch) 5 mm/rev Fine drive 0.
Specifications Tooth Bar Movement Ranges DV 20 mm AP 44.5 mm Probe Holder Diameter 7.88 mm Shaft length 200 mm Probe holder block 11 mm square (10 grooves spaced at 1 mm on 3 sides) Standard clamp holds 0.2 to 2 mm shaft Side clamp holds 0.2 to 4.5 mm shaft “C” clamp holds 0.2 to 4.5 mm shaft Set screw on shaft holder: 3/32″ Computer Voltage 115 V or 230 V, switch settable Current 3.6 A or 1.
Software Licence Terms Software Licence Terms The following License Terms govern your use of the accompanying software unless you have a separate signed agreement with Leica Microsystems. License Grant Leica Microsystems grants you a license to Use one copy of the Software. "Use" means storing, loading, installing, executing or displaying the Software. You may not modify the Software or disable any licensing or control features of the Software.
