The Angle Three™ Small Animal Stereotaxic instrument and software has all the features of the  Angle Two™, with some important updates and improvements. It also has three new built-in features for greater precision.

Virtual Skull Flat™ for two planes> The Angle Two™ corrected automatically and precisely for any head tilt in the AP plane. Touch down at Bregma and click, touch down at Lambda and click, and the computer then calculated the head tilt, and calculated the change-from-atlas position of the selected target coordinates. No need to actually attempt to accurately level the head, the computer precisely calculates how to reach the target where it is now, given the tilt. Angle Three™ includes that, and a similar feature for correcting any lateral head tilt.
Startup Alignment of Tilt and Rotate> 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. These easy steps must be done before proceeding. Unlock and tilt outward away from the animal. That caution sign will turn into a blue information sign at a point. Next, rotate out until similar detection. These simple movements tell the stereotaxic instrument where vertical and parallel are located. It will not be necessary to ever move there! The linear coordinates of your target given tilt and rotation will be precisely calculated and displayed. Select your target by clicking on it in the atlas, manually typing it in, or clicking an icon with saved coordinates from last time. But consider this before selecting:
New! Automatic Bregma-Lambda Scaling!
Rodents, unlike humans, keep growing for their entire life. The head expands. The brain expands. Coordinates from Bregma of a selected structure increase with age and growth. (Paxinos, Watson, Pennisi, and Topple, 1985). Thus, if the rodents you select for your study are not within the size range published in the atlas, you may need to do pilot studies with similar sized animals to find the coordinates of that structure in that size brain. Repeated pilots may be required to home in on it.
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 and suggestions 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, or cost of the automated setups. 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, and feel 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™, 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 ratio of B-L distance in this animal/B-L distance in the atlas animal can be used as a scaling factor that will positively get results closer to the target than not using them. However, the software includes default but user adjustable scaling factor adjustments to very much home in on reliable targeting with only the atlas values entered.
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 keep a digital record of each surgery in 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™ connects, for data and power, by one USB cable for each manipulator to any device that has USB and can run the included software.

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 action-at-target with needle leakage or other along the path to the target, which may then cause or alter some of the dependent variable results. With the Angle Three™, 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. If somebody bumps the unit and the angle slips before entering brain, that is irrelevant, the software notices and corrects the to-target coordinates.
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 +or – value even with the most careful selection of the landmarks. This instrument is more precise in measurement than the zeroing error range. 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 5 encoders, a standard probe holder, 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™, 2) the mouse and/or the rat head holder hardware, and whether to have mounted a right side 5 encoder manipulator to make a Dual model.  The researcher may select on screen between installed atlases if both are included. Rat head holder earbars are 45° taper to avoid tympanic membrane puncture. Accessories from other manufacturers (Kopf, Stoelting, MyNeurolab, Leica Biosystems) may be used on this instrument.

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.