The authors are thankful to the Coordination for the Improvement of Higher Education Personnel (CAPES) - Finance Code 001 and the Federal University of ABC (UFABC). Jo&#227;o R. Sato received financial support from the S&#227;o Paulo Research Foundation (FAPESP, Grants Nos. 2018/21934-5, 2018/04654-9, and 2023/02538-0).

Real-Time Recording and Mapping of Human Interaction with 3D Virtual Objects

The authors have no conflicts of interest to disclose.

As previously stated, this paper aims to present a detailed procedure of real-time mapping of fixation and saccade motion data on interactive 3D objects, which is easily customizable and only uses software available for free, providing step-by-step instructions to make everything work.
While this experimental setup involved a highly interactive task, such as moving a 3D object to match another object&#39;s orientation with PR in two of the three possible axes, we ensured thorough documentation...

Mental rotation (MR) is a cognitive ability that enables individuals to mentally manipulate and rotate objects, facilitating a better understanding of their features and spatial relationships. It is one of the visuospatial abilities, a fundamental cognitive group that was studied as early as 18901. Visuospatial abilities are an important component of an individual&#39;s cognitive repertoire that is influenced by both inherited and environmental factors2,3,4,5. Interest in visuospatial abilities has grown throughout the twentieth century due to mounting evidence of their importance in key subjects such as aging6 and development7, performance in science, technology, engineering, and mathematics (STEM)8,9, creativity10, and evolutionary traits11.
The contemporary idea of MR derives from the pioneering work published by Shepard and Metzler (SM) in 197112. They devised a chronometric method using a series of &#34;same or different&#34; tasks, presenting two projections of abstract 3D objects displayed side by side. Participants had to mentally rotate the objects on some axis and decide if those projections portrayed the same object rotated differently or distinct objects. The study revealed a positive linear correlation between response time (RT) and the angular disparity (AD) between representations of the same object. This correlation is known as the angle disparity effect (ADE). ADE is regarded as a behavioral manifestation of MR and became ubiquitous in several influential subsequent studies in the field13,14,15,16,17,18,19,20,21,22,23,24,25. The 3D objects employed in the SM study were composed of 10 contiguous cubes generated by the computer graph pioneer Michael Noll at Bell Laboratories26. They are referred to as SM figures and are widely employed in MR studies.
Two advancements were of great importance in Shepard and Metzler&#39;s seminal work; first, considering the contributions in the field of MR assessments. In 1978, Vanderberg and Kuze27 developed a psychometric 20-item pencil-and-paper test based on SM &#34;same or different&#34; figures that became known as the mental rotation test (VKMRT). Each test item presents a target stimulus. Participants must select among four stimuli, which ones represent the same object depicted in the target stimulus, and which do not. VKMRT has been used to investigate the correlation between MR ability and various other factors, such as sex-related differences6,21,24,28,29,30, aging and development6,31,32, academic performance8,33, and skills in music and sports34. In 1995, Peters et al. published a study with redrawn figures for the VKMRT35,36. Similarly, following the &#34;same or different&#34; task design, a variety of other libraries of computer-generated stimuli have been employed to investigate MR processes and to assess MR abilities (3D versions of the original SM stimuli19,22,23,37,38, human body mimicking SM figures25,39,40, flat polygons for 2D rotation41,42, anatomy, and organs43, organic shapes44, molecules45,46, among others21). The Purdue Spatial Visualization Test (PSVT) proposed by Guay in 197647 is also relevant. It entails a battery of tests, including MR (PSVT:R). Employing different stimuli from those in VKMRT, PSVT:R requires participants to identify a rotation operation in a model stimulus and mentally apply it to a different one. PSVT:R is also widely used, particularly in studies investigating the role of MR in STEM achievement48,49,50.
The second advancement of great importance in Shepard and Metzler&#39;s seminal work comprises the contributions to the understanding of the MR process, in particular, with the use of eye-tracking devices. In 1976, Just and Carpenter14 used analog video-based eye-tracking equipment to conduct a study based on Shepard and Metzler&#39;s ADE experiment. From their results on saccadic eye movements and RTs, they proposed a model of MR processes consisting of three phases: 1) the search phase, in which similar parts of the figures are recognized; 2) the transformation and comparison phase, in which one of the identified parts is mentally rotated; 3) the confirmation phase, in which it is decided whether the figures are the same or not. The phases are repeated recursively until a decision can be made. Each step corresponds to specific saccadic and fixational eye movement patterns in close relation to observed ADEs. Thus, by correlating eye activity to chronometric data, Just and Carpenter provided a cognitive signature for the study of MR processes. To date, this model, albeit with adaptations, has been adopted in several studies15,42,46,51,52,53.
Following this track, several ensuing studies monitoring behavioral18,19,22,23,25,34,40,54,55 and brain activity20,22,56,57 functions during stimuli rotation were conducted. Their findings point to a cooperative role between MR and motor processes. Moreover, there is a growing interest in investigating problem-solving strategies involving MR in relation to individual differences 15,41,46,51,58.
Overall, it can be considered that the design of studies aiming at understanding MR processes is based on presenting a task with visual stimuli that requests participants to perform an MR operation that, in turn, entails a motor reaction. If this reaction allows rotation of the stimuli, it is often called physical rotation (PR). Depending on the specific objectives of each study, different strategies and devices have been employed for data acquisition and analysis of MR and PR. In the task stimulus presentation step, it is possible to change the types of stimuli (i.e., previously cited examples); the projection (computer-generated images in traditional displays22,23,25,29,40,41,59, as well as in stereoscopes19 and virtual60 and mixed43 reality environments); and the interactivity of the stimuli (static images12,27,36, animations61, and interactive virtual objects19,22,23,43,53,59).
MR is usually inferred from measures of RTs (ADE), as well as ocular and brain activity25,46,62. Ocular activity is measured using eye-tracking data consisting of saccadic movements and fixations14,15,42,51,52,54,58,60, as well as pupillometry40. RT data typically arise from motor response data recorded while operating various devices such as levers13, buttons and switches14,53, pedals53, rotary knobs19, joysticks37, keyboard61 and mouse29,58,60, drive wheels53, inertial sensors22,23, touch screens52,59, and microphones22. To measure PR, in addition to the RTs, the study design will also include recording manual rotations of interactive stimuli while participants perform the MR task22,23,52,53.
In 1998, Wohlschl&#228;ger and Wohlschl&#228;ger19 used &#34;same or different&#34; tasks with interactive virtual SM stimuli manipulated with a knob, with rotations limited to one axis per task. They measured RT and the cumulative record of physical rotations performed during the tasks. Comparing situations with and without actual rotation of the interactive stimuli, they concluded that MR and PR share a common process for both imagined and actually performed rotations.
In 2014, two studies were conducted employing the same type of tasks with virtual interactive stimuli22,23. However, the objects were manipulated with inertial sensors that captured motion in 3D space. In both cases, in addition to RTs, rotation trajectories were recorded - the evolution of rotation differences between reference and interactive stimuli during the tasks. From these trajectories, it was possible to extract both cumulative information (i.e., the total number of rotations, in quaternionic units) and detailed information about solution strategies. Adams et al.23 studied the cooperative effect between MR and PR. In addition to RTs, they used the integral of the rotation trajectories as a parameter of accuracy and objectivity of resolution. Curve profiles were interpreted according to a three-step model63 (planning, major rotation, fine adjustment). The results indicate that MR and PR do not necessarily have a single, common factor. Gardony et al.22 collected data on RT, accuracy, and real-time rotation. In addition to confirming the relationship between MR and PR, the analysis of rotation trajectories revealed that participants manipulated the figures until they could identify whether they were different or not. If they were the same, participants would rotate them until they looked the same.
Continuing this strategy, in 2018, Wetzel and Bertel52 also used interactive SM figures in &#34;same or different&#34; tasks using touchscreen tablets as the interface. In addition, they used an eye-tracking device to obtain cumulative data on fixation time and saccadic amplitude as parameters of the cognitive load involved in solving MR tasks. The authors confirmed the previous studies discussed above regarding the relationships between MR and PR and the task-solving processes. However, in this study, they did not use fixation mapping and saccades data for the stimuli.
Methodological approaches for mapping eye-tracking data over virtual 3D objects have been proposed and constantly improved, commonly by researchers interested in studying the factors related to visual attention in virtual environments64. Although affordable and using similar eye-tracking devices, apparently, these methods have not been effectively integrated into the experimental repertoire employed in mental rotation studies with interactive 3D objects such as those previously mentioned. Conversely, we did not find any studies in the literature reporting real-time mapping of fixation and saccade motion data on interactive 3D objects. There seems to be no convenient method to integrate eye-activity data with rotation trajectories easily. In this research, we aim to contribute to filling this gap. The procedure is presented in detail, from data acquisition to graphical output generation.
In this paper, we describe in detail a method for studying mental rotation processes with virtual interactive 3D objects. The following advances are highlighted. First, it integrates quantitative behavioral motor (hand-driven object rotation via a computer interface) and ocular (eye-tracking) data collection during interaction sessions with 3D virtual models. Second, it requires only conventional computer equipment and eye-tracking devices for visual task design, data acquisition, recording, and processing. Third, it easily generates graphical output to facilitate data analysis - angular disparity, physical rotation, quaternionic rotation trajectories, and hit mapping of eye-tracking data over 3D virtual objects. Finally, the method requires only free software. All developed code and scripts are available free of charge (https://github.com/rodrigocnstest/rodrigocnstest.github.io).

<table><tbody><tr><td>Firefox</td><td>Mozilla Foundation (Open Source)</td><td></td><td>Any updated modern browser that is compatible with WebGL (https://caniuse.com/webgl), and in turn with Jmol, can be used</td></tr><tr><td>GNU Octave</td><td>Open Source</td><td></td><td>https://octave.org/</td></tr><tr><td>Google Apps Script</td><td>Google LLC</td><td></td><td>script.google.com</td></tr><tr><td>Google Sheets</td><td>Google LLC</td><td></td><td>https://www.google.com/sheets/about/</td></tr><tr><td>Laptop</td><td></td><td></td><td>Any computer that can run the eye tracking system software.</td></tr><tr><td>Mangold Software Suite</td><td>Mangold&amp;nbsp;</td><td></td><td>Software interface used for the Eye tracking device. Any Software that outputs the data with system time values can be used.</td></tr><tr><td>Mouse</td><td></td><td></td><td>Any mouse capable of clicking and dragging with simple movements should be compatible. Human interfaces analogous to a mouse with the same capabilities, such as a touchscreen or pointer, should be compatible, but may behave differently.</td></tr><tr><td>Vt3mini</td><td>EyeTech Digital Systems</td><td></td><td>60 Hz. Any working Eye Tracking device should be compatible.</td></tr></tbody></table>

three-dimensional mapping of the rotation of interactive virtual objects with eye-tracking data

We present a method for real-time recording of human interaction with three-dimensional (3D) virtual objects. The approach consists of associating rotation data of the manipulated object with behavioral measures, such as eye tracking, to make better inferences about the underlying cognitive processes.
The task consists of displaying two identical models of the same 3D object (a molecule), presented on a computer screen: a rotating, interactive object (iObj) and a static, target object (tObj). Participants must rotate iObj using the mouse until they consider its orientation to be identical to that of tObj. The computer tracks all interaction data in real time. The participant&#39;s gaze data are also recorded using an eye tracker. The measurement frequency is 10 Hz on the computer and 60 Hz on the eye tracker.
The orientation data of iObj with respect to tObj are recorded in rotation quaternions. The gaze data are synchronized to the orientation of iObj and referenced using this same system. This method enables us to obtain the following visualizations of the human interaction process with iObj and tObj: (1) angular disparity synchronized with other time-dependent data; (2) 3D rotation trajectory inside what we decided to call a &#34;ball of rotations&#34;; (3) 3D fixation heatmap. All steps of the protocol have used free software, such as GNU Octave and Jmol, and all scripts are available as supplementary material.
With this approach, we can conduct detailed quantitative studies of the task-solving process involving mental or physical rotations, rather than only the outcome reached. It is possible to measure precisely how important each part of the 3D models is for the participant in solving tasks, and thus relate the models to relevant variables such as the characteristics of the objects, cognitive abilities of the individuals, and the characteristics of human-machine interface.

Evolution of angular disparity and other variables 
As depicted in step 3.3.1 in Supplemental File 2, two canvases are presented to the participant on the video monitor screen, displaying copies of the same 3D virtual object in different orientations. On the left canvas, the target object (tObj) remains static and serves as the target position or tObj position. On the right canvas, the interactive object (iObj) is shown in a different position and allows the partici...

Author Spotlight: Insights into the Analysis of Human Interaction with 3D Virtual Objects

We have developed a simple, customizable, and efficient method for recording quantitative processual data from interactive spatial tasks and mapping these rotation data with eye-tracking data.

Three-Dimensional Mapping of the Rotation of Interactive Virtual Objects with Eye-Tracking Data

We present a method for real-time recording of human interaction with three-dimensional (3D) virtual objects. The approach consists of associating ...

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891 Views.  University of São Paulo, Institute of Chemistry. We have developed a simple, customizable, and efficient method for recording quantitative processual data from interactive spatial tasks and mapping these rotation data with eye-tracking data.

Video: Author Spotlight: Insights into the Analysis of Human Interaction with 3D Virtual Objects

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

Creating Virtual-hand and Virtual-face Illusions to Investigate Self-representation

Studies investigating how people represent themselves and their own body often use variants of &#34;ownership illusions&#34;, such as the traditional rubber-hand illusion or the more recently discovered enfacement illusion. However, these examples require rather artificial experimental setups, in which the artificial effector needs to be stroked in synchrony with the participants&#39; real hand or face&#8212;a situation in which participants have no control over the stroking or the movements of their real or artificial effector. Here, we describe a technique to establish ownership illusions in a setup that is more realistic, more intuitive, and of presumably higher ecological validity. It allows creating the virtual-hand illusion by having participants control the movements of a virtual hand presented on a screen or in virtual space in front of them. If the virtual hand moves in synchrony with the participants&#39; own real hand, they tend to perceive the virtual hand as part of their own body. The technique also creates the virtual-face illusion by having participants control the movements of a virtual face in front of them, again with the effect that they tend to perceive the face as their own if it moves in synchrony with their real face. Studying the circumstances that illusions of this sort can be created, increased, or reduced provides important information about how people create and maintain representations of themselves.

Here, we describe virtual-hand and virtual-face illusion paradigms that can be used to study body-related self-perception/-representation. They have already been used in various studies to demonstrate that, under specific conditions, a virtual hand or face can be incorporated into one&#39;s body representation, suggesting that body representations are rather flexible.

Studies investigating how people represent themselves and their own body often use variants of &#34;ownership illusions&#34;, such as the traditional ...

Behavior

Using Eye Movements Recorded in the Visual World Paradigm to Explore the Online Processing of Spoken Language

In a typical eye tracking study using the visual world paradigm, participants' eye movements to objects or pictures in the visual workspace are recorded via an eye tracker as the participant produces or comprehends a spoken language describing the concurrent visual world. This paradigm has high versatility, as it can be used in a wide range of populations, including those who cannot read and/or who cannot overtly give their behavioral responses, such as preliterate children, elderly adults, and patients. More importantly, the paradigm is extremely sensitive to fine grained manipulations of the speech signal, and it can be used to study the online processing of most topics in language comprehension at multiple levels, such as the fine grained acoustic phonetic features, the properties of words, and the linguistic structures. The protocol described in this article illustrates how a typical visual world eye tracking study is conducted, with an example showing how the online processing of some semantically complex statements can be explored with the visual world paradigm.

The visual world paradigm monitors participants' eye movements in the visual workspace as they are listening to or speaking a spoken language. This paradigm can be used to investigate the online processing of a wide range of psycholinguistic questions, including semantically complex statements, such as disjunctive statements.

In a typical eye tracking study using the visual world paradigm, participants' eye movements to objects or pictures in the visual workspace are recorded ...

Three Dimensional Vestibular Ocular Reflex Testing Using a Six Degrees of Freedom Motion Platform

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the vestibular organ results in mild to severe equilibrium problems, such as vertigo, dizziness, oscillopsia, gait unsteadiness nausea and/or vomiting. A good and frequently used measure to quantify gaze stabilization is the gain, which is defined as the magnitude of compensatory eye movements with respect to imposed head movements. To test vestibular function more fully one has to realize that 3D VOR ideally generates compensatory ocular rotations not only with a magnitude (gain) equal and opposite to the head rotation but also about an axis that is co-linear with the head rotation axis (alignment). Abnormal vestibular function thus results in changes in gain and changes in alignment of the 3D VOR response.
Here we describe a method to measure 3D VOR using whole body rotation on a 6DF motion platform. Although the method also allows testing translation VOR responses 1, we limit ourselves to a discussion of the method to measure 3D angular VOR. In addition, we restrict ourselves here to description of data collected in healthy subjects in response to angular sinusoidal and impulse stimulation.
Subjects are sitting upright and receive whole-body small amplitude sinusoidal and constant acceleration impulses. Sinusoidal stimuli (f = 1 Hz, A = 4&deg;) were delivered about the vertical axis and about axes in the horizontal plane varying between roll and pitch at increments of 22.5&deg; in azimuth. Impulses were delivered in yaw, roll and pitch and in the vertical canal planes. Eye movements were measured using the scleral search coil technique 2. Search coil signals were sampled at a frequency of 1 kHz.
The input-output ratio (gain) and misalignment (co-linearity) of the 3D VOR were calculated from the eye coil signals 3.
Gain and co-linearity of 3D VOR depended on the orientation of the stimulus axis. Systematic deviations were found in particular during horizontal axis stimulation. In the light the eye rotation axis was properly aligned with the stimulus axis at orientations 0&deg; and 90&deg; azimuth, but gradually deviated more and more towards 45&deg; azimuth.
The systematic deviations in misalignment for intermediate axes can be explained by a low gain for torsion (X-axis or roll-axis rotation) and a high gain for vertical eye movements (Y-axis or pitch-axis rotation (see Figure 2). Because intermediate axis stimulation leads a compensatory response based on vector summation of the individual eye rotation components, the net response axis will deviate because the gain for X- and Y-axis are different.
In darkness the gain of all eye rotation components had lower values. The result was that the misalignment in darkness and for impulses had different peaks and troughs than in the light: its minimum value was reached for pitch axis stimulation and its maximum for roll axis stimulation.
Case Presentation
Nine subjects participated in the experiment. All subjects gave their informed consent. The experimental procedure was approved by the Medical Ethics Committee of Erasmus University Medical Center and adhered to the Declaration of Helsinki for research involving human subjects.
Six subjects served as controls. Three subjects had a unilateral vestibular impairment due to a vestibular schwannoma. The age of control subjects (six males and three females) ranged from 22 to 55 years. None of the controls had visual or vestibular complaints due to neurological, cardio vascular and ophthalmic disorders.
The age of the patients with schwannoma varied between 44 and 64 years (two males and one female). All schwannoma subjects were under medical surveillance and/or had received treatment by a multidisciplinary team consisting of an othorhinolaryngologist and a neurosurgeon of the Erasmus University Medical Center. Tested patients all had a right side vestibular schwannoma and underwent a wait and watch policy (Table 1; subjects N1-N3) after being diagnosed with vestibular schwannoma. Their tumors had been stabile for over 8-10 years on magnetic resonance imaging.

A method is described to measure three-dimensional vestibulo ocular reflexes (3D VOR) in humans using a six degrees of freedom (6DF) motion simulator. The gain and misalignment of the 3D angular VOR provide a direct measure of the quality of vestibular function. Representative data on healthy subjects are provided

The vestibular organ is a sensor that measures angular and linear accelerations with six degrees of freedom (6DF). Complete or partial defects in the ...

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.	
	Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 &mu;m z-axis and a 0.23 &mu;m step size, corresponding to a 180 x 135 x 10 &mu;m volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

A Methodology for Capturing Joint Visual Attention Using Mobile Eye-Trackers

With the advent of new technological advances, it is possible to study social interactions at a microlevel with unprecedented accuracy. High frequency sensors, such as eye-trackers, electrodermal activity wristbands, EEG bands, and motion sensors provide observations at the millisecond level. This level of precision allows researchers to collect large datasets on social interactions. In this paper, I discuss how multiple eye-trackers can capture a fundamental construct in social interactions, joint visual attention (JVA). JVA has been studied by developmental psychologists to understand how children acquire language, learning scientists to understand how small groups of learners work together, and social scientists to understand interactions in small teams. This paper describes a methodology for capturing JVA in colocated settings using mobile eye-trackers. It presents some empirical results and discusses implications of capturing microobservations to understand social interactions.

Using multimodal sensors is a promising way to understand the role of social interactions in educational settings. This paper describes a methodology for capturing joint visual attention from colocated dyads using mobile eye-trackers.

With the advent of new technological advances, it is possible to study social interactions at a microlevel with unprecedented accuracy. High frequency ...

Controlled Rotation of Human Observers in a Virtual Reality Environment

The low cost and availability of Virtual Reality (VR) systems have supported a recent acceleration of research into perception and behavior under more naturalistic, multisensory, and immersive conditions. One area of research that has particularly benefited from the use of VR systems is multisensory integration, for example, the integration of visual and vestibular cues to give rise to a sense of self-motion. For this reason, an accessible method for the controlled physical rotation of an observer in a virtual environment represents a useful innovation. This paper presents a method for automating the rotation of an office swivel chair along with a method for integrating that motion into a VR experience. Using an example experiment, it is demonstrated that the physical motion, thus produced, is integrated with the visual experience of an observer in a way consistent with expectations; high integration when the motion is congruent with the visual stimulus and low integration when the motion is incongruent.

The controlled physical rotation of a human observer is desirable for certain experimental, recreational, and educational applications. This paper outlines a method for converting an office swivel chair into a medium for controlled physical rotation in a virtual reality environment.

The low cost and availability of Virtual Reality (VR) systems have supported a recent acceleration of research into perception and behavior under more ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex (pre-SMA and SMA proper) using a combination of fiber dissection techniques on cadaveric specimens and magnetic resonance (MR) tractography. The protocol will also describe the procedure for a white matter dissection of a human brain, diffusion tensor tractography imaging, and three-dimensional documentation. The fiber dissections on human brains and the 3D documentation were performed at the University of Minnesota, Microsurgery and Neuroanatomy Laboratory, Department of Neurosurgery. Five postmortem human brain specimens and two whole heads were prepared in accordance with Klingler's method. Brain hemispheres were dissected step by step from lateral to medial and medial to lateral under an operating microscope, and 3D images were captured at every stage. All dissection results were supported by diffusion tensor imaging. Investigations on the connections in line with Meynert's fiber tract classification, including association fibers (short, superior longitudinal fasciculus I and frontal aslant tracts), projection fibers (corticospinal, claustrocortical, cingulum, and frontostriatal tracts), and commissural fibers (callosal fibers) were also conducted.

The purpose of this study is to show each step of the fiber dissection technique on human cadaveric brains, the 3D documentation of these dissections, and the diffusion tensor imaging of the anatomically dissected fiber pathways.

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex ...