The Angle Three™ Small Animal Stereotaxic Instrument connects to your computer via USB and passes position data from the 3 linear axes and the two rotational axes to your computer via a USB port.  The target is given in atlas coordinates.  Shown manipulator angles and skull landmarks, the software calculates and displays, given any tilt and/or rotation of the manipulator, and tilt and size of the animal’s head, how far you have to move on each of the 3 linear axes to reach your selected target.  No head orientation adjustment of the animal, or  adjustment of the manipulator(s) angles, is needed.  No pilot studies  for non-atlas age animals are needed.

Virtual Skull Flat™ for three planes> The Angle Three™ small animal stereotaxic instrument software corrects  for any head tilt in the AP plane. Shown the location of Bregma and of Lambda by touching down and clicking there, the software then calculates the angle of head tilt and the size of the head. Given the angle and size, it calculates the coordinates of the target in this animal. No need to physically level the head, the computer precisely calculates how to reach the target where it is now, given the animal tilt and size, and manipulator orientation.
Startup Alignment of the Manipulator> Upon startup, the software must acquire the location of straight vertical for tilt, and parallel to the earbars for rotate. When power is turned on, and the software initiated, 3 small yellow caution signs appear in the upper corner. Tool tips tell you that one is for tilt, the next for rotate, and the next for select target. The following easy steps must be done before proceeding. Unlock the tilt hinge and tilt outward away from the animal. That caution sign will turn into a blue information sign at a point. Lock the tilt at any selected angle.  Next, unlock the rotate and turn it outward out the open end of the “U” until the icon clears. Lock the rotate at any selected angle.  These simple movements tell the Angle Three™ Software precisely where straight vertical and parallel to earbars are located. It will not be necessary to ever move there!  The data will be used to calculate the revised coordinates of where the target is in this animal. Select your target by clicking on it in the atlas, manually typing it in, or clicking an icon with saved coordinates.
New! Automatic Bregma-Lambda Scaling!
Rodents, unlike humans, keep growing for their entire life. The head expands. The brain expands. Thus, distance from Bregma of a selected target increases with age (Paxinos, Watson, Pennisi, and Topple, 1985). If the rodents you select for your study are not within the size range published in the atlas, the atlas coordinates will not be accurate.  The usual solution would be pilot studies to home in on the target coordinates in your animals.  The Angle Three™ small animal stereotaxic instrument softtware calculates where the target is in each animal, given head and manipulator angles.
Several studies around the 1980’s sought to define a way to achieve reproducible stereotaxic accuracy in research animals over a range of sizes. The distance between the skull bone sutures Bregma and Lambda was found to have a high correlation with the distance between brain structures in mice, and use as a correction factor for stereotaxic coordinates was suggested (Slotnick and Leonard, 1975). Similar findings for the rat were also reported (Slotnick and Brown, 1980).
The anterior edge of the anterior commissure is directly below Bregma in both mice and rats (Paxinos, Watson, Pennisi, and Topple, 1985), and this relationship is maintained with ageing. Coordinates of other structures in brain move away from Bregma with ageing.
Schuller worked with wild caught bats of all ages and genders (Schuller, Radtke-Schuller & Betz, 1986). He worked on the auditory side of echolocation, and could not use earbars. An automated custom stereotaxic instrument, motorized, was developed for this project.  They touched down at multiple points on a head held by glue from an out of the way position, and a PDP-8 with custom software. From models built, he could scale the location of a structure in any given bat, and achieved considerable success with accuracy of electrode placement. He estimated 1 to 1.5 hours for every surgery using this equipment and procedure.
Possible reasons for the low rate of adoption of these methods included time for math on every coordinate for every surgical procedure, and cost at that time of the computer hardware. Every year, I meet a few scientists at SFN that are in fact regularly doing Bregma-Lambda scaling with a calculator on the operating table, or an Excel Spreadsheet open on screen, and report that  it improves their accuracy. Some of the rest of us are not that great at number memory and entry. Errors on the keyboard may cause errors of accuracy even with this technique. However, with the Angle Three™ small animal stereotaxic instrument, Bregma-Lambda scaling and virtual skull flat happen automatically in the background.
We must touch Bregma and Lambda anyway to test skull flat, clicking at each one gives also the distance between them. The B-L distance in this animal divided by B-L distance in the atlas animal can be used as a scaling factor to adjust for head size.
Other useful features:

• Single or Dual manipulator versions are available.
• Compatible with probe holders, earbars, and head holders from Kopf, Stoelting, MyNeurolab, & Leica Biosystems.
• “Export” button to compile a digital record of each surgery into a file.
• To try out an approach, and see what structures you will pass through on the descent, remove the probe holder after locating Bregma and Lambda, and advance to target coordinates as if really moving through brain. You will see on screen the plane you would be passing through, and your present position on it. If acceptable, reverse the DV drive, reinstall the probe holder, and proceed. If not, readjust Tilt or Rotation to a better position, and try again.
• USB> The Angle Three™ small animal stereotaxic instrument connects, for data and power, by one USB cable for each manipulator to any device that has USB and can run the included software.  A 27″ monitor is recommended.

Angled Approaches:
In the past, stereotaxic surgery was predominantly done straight down. This avoided intense math and pilot work to select one angle, and was more securely locked in position than angled approaches. This procedure has the drawback of always passing through the same structures above the target. This confounds the variables “action-at target” and “path to the target”.  With the Angle Three™ small animal stereotaxic instrument, we recommend doing every surgery from a different angle, which may happen anyway if the skull is not at Skull Flat. Reaching exactly the same target with high precision, but from different angles, is better evidence that the treatment at the target caused the observed results, rather than damage or leaking on the path down.
Accuracy of Stereotaxic Surgery
The limiting factor of accuracy of any stereotaxic surgery is the researcher’s ability to move the tip to the exact point of Bregma, and the exact point of Lambda. These are not necessarily the exact points of suture crossing (See: Paxinos & Watson, p.ix; Franklin and Paxinos, p. xi). Biological variability will make that somewhat of a ± value even with the most careful selection of the landmarks. This instrument is more precise in measurement than is biological variance of Bregma. Given that, biological variance of the zero setting is the primary error source. Higher precision (sub micron) could be provided by the instrument, but this would not improve probe placement accuracy. The automated features avoid trial and error settings, and reduce risk of human error, while allowing more rapid operation.

Options:

The 3600 Angle Three™ Small Animal Stereotaxic Instrument includes the “U” frame base, left side manipulator with movement sensors, a standard probe holder, and a base mounted electronics box under the “U” frame, and the Angle Three™ software. 

The customer selects 1) the mouse and/or the rat brain atlas software, installed with the Angle Three™.  The researcher may select on screen between installed atlases if both are included., 2) the mouse and/or the rat head holder hardware equipped with gas adaptors, and 3) whether to acquire a right side manipulator to make a Dual model.   Earbar style is optional, and available for both species.  Rat or mouse earbars with 45° taper to avoid tympanic membrane puncture are available. Mouse earbars with cupped ends or points are available.  Cupped ends are also preferrable for working with neonatal animals.  Accessories from other manufacturers (Kopf, Stoelting, MyNeurolab, Leica Biosystems) may be used on this instrument.  A bone drill adapted for mounting on a stereotaxic instrument, and the drill probe adaptor needed to mount it are available.  This works very efficiently on Angle Three™ dual maniplator stereotaxic models.

Citations:
Paxinos G, Watson C, Pennisi M, Topple A. Bregma, lambda and the interaural midpoint in stereotaxic surgery with rats of different sex, strain and weight. Journal of Neuroscience Methods: 1, pg 39-43, 1985
Slotnick, B.M. and Brown, D. L., Variability in the Stereotaxic Position of Cerebral Points in the Albino Rat. Brain Research Bulletin 5(2): 135-139, 1980
Slotnick, B.M, and Leonard, C.M. A Stereotaxic Atlas of Albino Mouse Forebrain. Rockville, MD: US Department of Health, Education and Welfare, 1975.
Schuller, G., Radtke-Schuller, S. Betz, M. , A stereotaxic method for small animals using experimentally determined reference profiles. Journal of Neuroscience Methods 18 339-350, 1986

Paxinos, G, Watson, C. The Rat Brain in Stereotaxic Coordinates, Elsevier Publishing, 7th Edition, 2013
Paxinos, G., Franklin, K., The Mouse Brain in Stereotaxic Coordinates, Elsevier Publishing, 5th Edition, 2019

Research and development partly supported by NIH SBIR R43 award NS055600 from NINDS.