The Print Shop at Sheffield Hallam University is thanked for providing high quality reproductions of the portraits used in this study. Michael Pickard and Jonathan Dean are thanked for their valuable insights.

The protocol was approved by the Faculty Research Ethics Committee at Sheffield Hallam University for studies on human subjects.
1. Inter-observation Stage
<ol>
	<li>Identify how and why La Bella Principessa&#39;s expression appears ambiguous.
	<ol>
		<li>Display a good quality, frameless and exact sized foam backed digital copy of La Bella Principessa (33.2 cm in height x 23.8 cm in width) on an easel in diffuse lighting.</li>
		<li>Have four participants discuss the ambiguous expression in La Bella Principessa&#39;s portrait. Record the results of the discussion on a piece of paper. 
		Note: One participant must be the researcher and the others must be recruited from the academic community. 
		Note: Ensure that the participants can freely approach the portrait from any distance or angle.</li>
	</ol>
	</li>
</ol>
2. Structured Interview Stage
<ol>
	<li>Interview four different participants one at a time and have them answer the questions contained within the structured interview schedule: &#34;Please can you describe the Princess&#39;s expression? Do you think there are any differences in the Princess&#39;s expression when you view her from close or further away? If so, please explain your answer&#34;.
	<ol>
		<li>Display La Bella Principessa&#39;s portrait (33.2 cm H x 23.8 cm W) in the center of an A1 paper pad resting on an easel in a well illuminated room.
		<ol>
			<li>Instruct the participants to describe the princess&#39;s expression (e.g., happy or melancholic) and note any differences in the expression when viewed from close-up (to within a few centimeters of the portrait) or further away (more than three meters away) from the portrait. Have the participants write down their observations on paper.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Conduct a Grounded Theory analysis15,16,17,18 of participants&#39; descriptions to identify the most common words used to describe La Bella Principessa&#39;s expression (e.g., smiling, happiness, contentment) and changes to her expression with respect to distance (e.g., less content when closer, etc.).</li>
</ol>
3. Psychophysical Experiments Stage
<ol>
	<li>Conduct five experiments to test the five hypotheses formulated on the basis of findings from steps 1 and 2, as outlined below.
	<ol>
		<li>On Viewing Distance (experiment 1)
		<ol>
			<li>Conduct a between-subjects experiment with two independent variables (IVs): Portrait (with three levels: La Bella Principessa, Mona Lisa, and Portrait of a Girl); and Viewing Distance (with two levels: Close vs. Far). 
			Note: The dependent variable must be perceived contentment (i.e., how much on the scale of 1 to 7 the women in the portraits appear content).
			<ol>
				<li>Recruit 60 participants, 10 for each of the six conditions resulting from the combination of the factor levels indicated in 3.1.1.1. Ensure that the participants have normal or corrected-to-normal acuity (determined by self-reporting) and are naive with regard to the experimental design. Ensure that the participants are not familiar with either La Bella Principessa or the Portrait of a Girl and have not seen the Mona Lisa for at least one year.</li>
				<li>Display good quality and exact sized foam backed digital copies of La Bella Principessa (33.2 cm H x 23.8 cm W), Mona Lisa (77 x 53 cm), and Portrait of a Girl (45.5 x 32.7 cm) in the center of an A1 paper pad resting on an easel 1.80 m from the floor.</li>
				<li>Mount the portraits indicated in 3.1.1.1.2 in diffused lighting at the end of a long corridor with two doors 0.5 and 8 m from the portraits, respectively, and cover them with a sheet. Place two markers on the floor, one 0.5 m from the portrait for the Close condition, and the other 8 meters from the portrait for the Far condition.</li>
				<li>Randomly assign participants to the Close or Far condition and to one Portrait condition. Have the participants in the Close condition enter the corridor from the closest door, and the participants assigned to the Far condition enter the corridor from the furthest door.</li>
				<li>Have the participants stop at a location indicated by the marker on the floor and then unveil the portrait. Ensure that the participants are able to see the portrait from the assigned marker only and not experience any change in the viewing distance.</li>
				<li>Have the participants verbally rate the perceived contentment in the expressions of the woman viewed in the assigned portrait on a scale of 1 (not content at all) to 7 (extremely content). Record participants&#39; responses on a piece of paper (e.g., Participant 1; Close Condition; Mona Lisa; Contentment = 5).</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>On Blur Level (Experiment 2)
		<ol>
			<li>Conduct a within subjects experiment (i.e., each subject is presented with all the experimental conditions) with two IVs: Portrait (with three levels: La Bella Principessa, the Mona Lisa, and Portrait of a Girl); and Level of Blur (with eight levels of increasing blur). 
			Note: The dependent variable must be perceived contentment.
			<ol>
				<li>Recruit 10 participants who have not taken part in any previous stages of the study.</li>
				<li>Create a set of eight digital images for each of the three portraits with increasing levels of Gaussian Blur 12. Ensure that the Blur level increases in steps of 1 pixel over the range 0-7. 
				Note: In this blurring process pixels take contributions from their surrounding pixels and contributions from the center are de-emphasized.</li>
				<li>Set up a 22&#34; (20&#34; viewable) CRT monitor set at a resolution of 1,280 x 1,024 connected to a computer.</li>
				<li>Write software19 (see Supplemental file) that displays the portraits in a random order on the monitor; presents a random noise mask (i.e., a figure made by black and white 1 pixel large dots randomly distributed across the screen) for one second before the presentation of each picture; allows participants to enter their ratings of contentment from 1 to 7 using a keyboard; and records the ratings when the spacebar is pressed. 
				Note: See supplemental &#34;Code for exp 2.bak&#34; file for an example on how this could be programmed in True Basic19, although other programs can be used.</li>
				<li>Seat participants one at a time in a well-illuminated room 50 cm from the monitor.</li>
				<li>Have the participants rate the perceived contentment in the expression of La Bella Principessa, Mona Lisa and Portrait of a Girl from 1 to 7 and enter their ratings using a keyboard. Record the responses when the spacebar is pressed as per step 3.1.2.1.4 above.</li>
				<li>To eliminate any influences from the previous images affecting participants&#39; ratings of the new ones, present a random noise mask full screen that disappears automatically after one second for one second prior to the next image being shown. Allow the image to remain on the screen for as long as the participant desires.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>On Establishing the Source of the Illusory Effect (Experiment 3)
		<ol>
			<li>Conduct a repeated-measures experiment with two IVs: Level of Blur (with eight levels); and Mask Position (with four levels: No mask, Mouth mask, Eye mask, or both Mouth and Eye masks). 
			Note: The dependent variable must be perceived contentment.
			<ol>
				<li>Recruit 10 participants who have not taken part in any previous stages of the study.</li>
				<li>Use the same digital images of La Bella Principessa with different levels of Gaussian blur used in experiment 2. Using graphical software (e.g., Photoshop), create a solid black rectangle of 0.3 x 0.6 cm and place it over the mouth, or the eye or over both the mouth and the eye.</li>
				<li>Create three more sets of masked images to result in a total of four sets (each comprising eight images): For the first set create eight images for the No Mask condition; for the second set create eight images for the Mouth Mask condition; for the third set create eight images for the Eye Mask condition (of La Bella Principessa&#39;s visible eye); and for the fourth create eight images for the Eye and Mouth condition. 
				Note: The mask itself must not be blurred. See supplemental &#34;Code for exp 3.bak&#34; file for an example on how this could be programmed in True Basic, although other programs can be used.</li>
				<li>Use a CRT monitor and the same conditions as for experiment 2. Have the participants rate the perceived contentment of La Bella Principessa on a scale of 1 (not content at all) to 7 (extremely content) by pressing the correspondent key on the numeric keypad.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Distance on Perceived Mouth Slant (Experiment 4)
		<ol>
			<li>Conduct a between-subjects experiment (i.e., different subjects for the different conditions) with one IV (Viewing Distance with two levels: Close vs. Far). 
			Note: The dependent variable must be the perceived slant of the mouth.
			<ol>
				<li>Recruit 20 participants (10 in the Close Condition and 10 in the Far condition) who have not taken part in any previous stages of the study.</li>
				<li>Project an exact sized (33.2 cm H X 23.9 cm W) digital version of La Bella Principessa using a projector onto a screen connected to a computer at 1.80 m from the floor in a large room with diffused lighting.</li>
				<li>Place a wireless keyboard on top of a table at 0.5 m from the screen for the Close Condition. Place the wireless keyboard on top of a table 8 m from the screen for the Far Condition. 
				Note: Ensure that the sizes of the retinal images of the portrait in degrees of visual angle are the same as for experiment 1. Ensure that the participants view La Bella Principessa from the assigned location only and do not experience any change in the viewing distance.</li>
				<li>Using the programming software described above (3.1.3.1.3), create a 0.9 cm long black hairline measurement &#39;handle&#39; on the screen (the same length as La Bella Principessa&#39;s mouth). Place the hairline at the same level and 4 cm to the left of the mouth.</li>
				<li>Make the right side of the handle adjustable by pressing a button of a wireless mouse and record the perceived mouth slant angle when the spacebar key is pressed (see supplemental &#34;Code for exp 4.bak&#34; file).</li>
				<li>Have the participants to adjust the &#39;handle&#39; using the mouse. Instruct participants to press the mouse button to adjust the slant of the handle until it aligns with the perceived slant of the mouth of the woman in the portrait. Leave the images on the screen for as long as the participant desires.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Blur on Perceived Mouth Slant (Experiment 5)
		<ol>
			<li>Conduct a repeated-measures experiment with one IV (Blur with eight levels). 
			Note: The dependent variable must be the Perceived slant of the mouth.
			<ol>
				<li>Recruit 10 participants who have not taken part in any previous stages of the study.</li>
				<li>Using the same apparatus used for experiment 2 and the same on-screen facility used in experiment 4, present the eight versions of La Bella Principessa with different levels of blur in a random order on the screen (see supplemental &#34;Code for exp 5.bak&#34; file).</li>
				<li>As for experiment 4, instruct participants to adjust the slant of the &#39;handle&#39; until it aligns with the perceived slant of the mouth of the woman in the portrait. As in experiments 2 and 3 present a random noise mask on the screen one second prior to the next image being shown. Leave the images on the screen for as long as the participant desires. 
				Note: The software records the perceived mouth slant angle when the spacebar key is pressed.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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E., Arscott, J., Taggart, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gold Edition Reference Guide for the True BASIC Language System. , (2010).</li><li>Z&#246;llner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ueber+eine+neue+Art+von+Pseudoskopie+und+ihre+Beziehungen+zu+den+von+Plateau+und+Oppel+beschrieben+Bewegungs+phaenomenen.+[On+a+New+Kind+of+Pseudoskopics+and+its+Relationship+to+the+Motion+Phenomena+Described+by+Plateau+and+Oppel].">Ueber eine neue Art von Pseudoskopie und ihre Beziehungen zu den von Plateau und Oppel beschrieben Bewegungs phaenomenen. 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The authors have no competing financial interests.

The findings of the study conducted by Soranzo &#38; Newberry3 indicate that the ambiguity of the expression in Leonardo da Vinci&#39;s La Bella Principessa portrait is attributable to a visual illusion. A new mixed-methodology which combined three methods: Inter-observation, structured interviews, and psychophysical experiments was adopted. Firstly, the inter-observation method was used to shed some light on the &#39;ambiguity&#39; of the Princess&#39;s facial expression. This method which involves l...

This paper discusses the methodology used by Soranzo and Newberry3 to investigate the &#39;Uncatchable Smile&#39; illusion in Leonardo da Vinci&#39;s La Bella Principessa portrait. The overall goal of the methodology employed was to identify what makes the expression of the Princess ambiguous. The portrait shows the profile of a young woman identified as Bianca, the illegitimate daughter of Ludovico, who at around 13 years of age was to be married to a commander of the Duke&#39;s Milanese forces4. According to Kemp5, the portrait was commissioned by the Duke in honor of the celebration of her marriage in 1496. Kemp and Cotte6 noted that the expression of La Bella Principessa is ambiguous and &#34;subtle to an inexpressible degree&#34; (p.26), although the question remains as to what it is that triggers this ambiguity and whether this is similar to that perceived in Leonardo&#39;s Mona Lisa.
Soranzo and Newberry&#39;s3 study involved three stages which combined three different research methods. The rationale for using these different research methods was to conduct a research investigation that developed without prior beliefs, thus minimizing potential researcher bias. The first stage used the inter-observation method to investigate why the expression of the Princess is ambiguous; in the second, a structured interview was used to identify the psychological dimension underpinning the &#39;Uncatchable Smile&#39; illusion; and in the third stage experiments were conducted to test hypotheses formulated on the basis of the findings of the first two stages. Independent groups of participants took part in these three stages and the findings of each stage influenced the following stages. This is likely to have reduced researcher bias compared to if the researchers had determined the research questions themselves.
The advantages of combining different research methods over singular methods are as follows. First, the inter-observation stage enabled discussion to take place about where the ambiguity may have originated from (rather than the researchers imposing their own pre-determined view). Second, the interview stage enabled the identification of the psychological dimension to be investigated without imposing pre-defined categories. Third, the experimental stage enabled the perceptual change identified in the previous stages to be quantified in a controlled manner.
Inter-observation
As noted above, La Bella Principessa&#39;s expression is &#34;subtle to an inexpressible degree&#34;6. The first step of the research was to try to identify what it is about the portrait that makes the expression ambiguous. To do this, the inter-observation method posited by Paolo Bozzi and his colleagues7, 8 was conducted with three participants and the senior author. This method enables maximum information to be extracted from direct experience9 and involves looking together, discussing and mutually exchanging views. The first step of the research adhered to the guidelines provided by Bozzi who argued that observers should conjointly partake in the observational process in a natural environment (not in a laboratory); observers should be aware of the researcher&#39;s hypothesis (no hidden hypothesis); the instructions to participants should be broad and discussed together rather than providing strict instructions; the discussion should go beyond the first impression (for a discussion of the un-permeability of this method in relation to the so called &#34;conformity effect&#34; found by Asch11 see Bozzi7). According to Bozzi, the discussion should go beyond the first impression in order to explore all possible solutions. The conclusion is therefore reached through a process of &#34;negotiation&#34; between the participants and the researcher, and negotiation is the process through which a common account is determined10. Participants were presented with a copy of La Bella Principessa&#39;s portrait resting on an easel in diffuse lightning. General instructions were given; participants were requested to discuss the ambiguous expression of La Bella Principessa. In the negotiation stage, it became clear early on that the ambiguity in the Princess&#39;s expression, or the allure of the portrait, is triggered by a perceptual change. The Princess&#39;s expression is ambiguous because it is unstable; sometimes she looks happy and cheerful whilst at other times she looks melancholic and hostile. However, the conditions under which this change occurs were still not known. Further inspection and negotiation led to the unanimous conclusion that the change relates to the way the portrait is viewed: the Princess&#39;s expression appears to change when she is viewed from different distances. When the Princess is viewed from far away her mouth appears to take an upward direction, making her appear friendly and positive, but as soon as the portrait is approached, the Princess&#39;s mouth appears to take a downwards slant, making her look sad and unapproachable. In other words, it seems that the ambiguity in the Princess&#39;s expression is due to a visual illusion of direction, which depends on the viewing distance. Since spatial frequency changes with distance it was concluded that this change relates to spatial frequency.
The existence of a spatial frequency-dependent illusion of direction was also explored by using a blurred digital version of the portrait. A digital version of La Bella Principessa was presented on a computer screen and the level of blur was manipulated. The blurring process was essentially a convolution kernel of the pixel spreading type in which pixels take contributions from their surrounding pixels, while de-emphasizing contributions from the center12. In addition to appearing more cheerful and happy when viewed from a distance, La Bella Principessa also appeared more cheerful and happy when viewed as a blurred image. This supports the idea of a spatial frequency-dependent illusion. The concept of spatial frequency-dependent facial expression is supported by a wide understanding of how the human visual system is particularly sensitive to seeing and interpreting the subtleties of facial expression13, 14.
Structured Interviews
The results of the inter-observation stage led us to hypothesize that the Princess&#39;s expression is ambiguous because it is unstable and that this instability may be attributable to a spatial frequency-dependent perceptual change relating to the direction of the mouth. The assumption was that if there is a &#39;perceptual change&#39; which relates to the way the portrait is viewed, then there must be a controllable way to elicit one impression in the viewer over another (for example, the melancholic one rather than the cheerful one). The challenge was to find out how to control the viewer&#39;s impression of La Bella Principessa in order to generate one impression over the other. Before addressing this problem in a quantitative manner, structured interviews were conducted to determine the psychological dimension that best captures this perceptual change.
Four participants who had not taken part in the inter-observational stage of the study took part in a structured interview. Participants were shown an identical copy of La Bella Principessa&#39;s portrait resting on an easel in diffuse lighting. Participants were asked to describe the Princess&#39;s expression and to note any differences in the expression when viewed from close or further away. Interestingly, participants&#39; descriptions were not framed in terms of outward expressions such as &#39;smiling&#39; but more with the internal emotional state of the Princess. A Grounded Theory analysis15,16,17,18 of the descriptions was conducted and it was found that the most common words used to describe the Princess&#39;s expression were &#34;happiness&#34; and &#34;contentment&#34;. These findings suggested that the change in the Princess&#39;s expression might be better captured with the definition of a change in perceived &#34;contentment&#34;. Together, the inter-observational and structured interview stages informed the formulation of five hypotheses as follows: First, the viewer&#39;s distance from La Bella Principessa&#39;s portrait will influence their perception of her facial expression. Specifically, the ratings of contentment will be higher when the portraits are viewed from further away. Second, the portrait&#39;s level of blur will influence the perceived contentment of La Bella Principessa. Specifically, presenting digital versions of the portrait with an increased level of blur will lead to an increase in the viewer&#39;s perception of contentment. Third, the uncatchable smile illusion is mainly attributable to the mouth area of La Bella Principessa&#39;s portrait. Fourth, the perceived slant of La Bella Principessa&#39;s mouth will change when the distance of the portrait is manipulated. Specifically, the corner of the mouth will appear to take an upward turn when distance is increased. Fifth, the perceived slant of La Bella Principessa&#39;s mouth will change when a digital version of the portrait is presented with different levels of blur. Specifically, the corner of the mouth will appear to take an upward turn when the level of blur is increased.
Psychophysical Experiments
In the quantitative stage of the study, five psychophysical experiments were conducted to test the aforementioned hypotheses. In addition, since it was hypothesized that a similar effect would be observed for the Mona Lisa&#39;s expression, this portrait was also included in the investigation. Furthermore, to test whether the effect under investigation is specific to La Bella Principessa and the Mona Lisa or whether it may be general to all portraits of Leonardo&#39;s era, a control portrait was also used. This was the Portrait of a Girl painted around 1470 by Piero del Pollaiuolo. This was chosen as a suitable control for La Bella Principessa as the portrait is from the same period and there are similarities of size, presentation and appearance in the subject matter.

<table><tbody><tr><td>High quality same size reproductions of the following three portraits: The Mona Lisa and La Bella Principessa (Leonardo da Vinci) and Portrait of a Girl (Piero del Pollaiuolo)</td><td>The Print Shop - Sheffield Hallam University - Sheffield Hallam University, Howard Street, Sheffield, South Yorkshire S1 1WB, United Kingdom</td><td></td><td></td></tr><tr><td>Easel for resting portraits on</td><td>No particular brand needed</td><td></td><td></td></tr><tr><td>Laptop computer with wireless mouse for experiments</td><td>Lenovo</td><td></td><td></td></tr><tr><td>Programming software to write syntax to present the pictures in random order, record participant responses from the keyboard, and enable the measurement &#039;handle&#039; to be adjusted by participants</td><td>True Basic software</td><td></td><td></td></tr><tr><td>Image editing software</td><td>Adobe Photoshop CC 2015</td><td></td><td></td></tr></tbody></table>

investigating the 'uncatchable smile' in leonardo da vinci's la bella principessa: a comparison with the mona lisa and pollaiuolo's portrait of a girl

This paper discusses how the &#39;Uncatchable Smile&#39; illusion in Leonardo da Vinci&#39;s La Bella Principessa portrait was discovered. Kemp and Cotte1 described the expression of the Princess as ambiguous and &#34;subtle to an inexpressible degree&#34;. A combination of three methods was used (inter-observation, structured interviews, and psychophysical experiments) to identify what may underlie this &#39;ambiguity&#39;. The inter-observation and the structured interview methods were firstly applied to generate experimental hypotheses that were successively tested by a series of psychophysical experiments. The combination of these research methods minimizes the impact of the researcher&#39;s beliefs and biases in the development of the research design. It emerged that the ambiguity in La Bella Principessa is triggered by a change in the perceived level of contentment in her facial expression and that this perceptual change is attributable to a visual illusion relating to her mouth. Moreover, it was found that a similar effect can be observed in the Mona Lisa. As the smile in La Bella Principessa disappears as soon as the viewer tries to &#39;catch it&#39;, we named this visual illusion the &#39;Uncatchable Smile&#39;. The elusive quality of the Mona Lisa&#39;s smile2 is probably why the portrait is so famous, and so the existence of a similar ambiguity in a portrait painted by Leonardo prior to the Mona Lisa is even more interesting.

From the negotiation process of the Inter-observation stage it was ascertained that the ambiguity in the Princess&#39;s expression might be due to a visual illusion of direction which depends on the viewing distance. Since spatial frequency changes with distance it was concluded that this change relates to spatial frequency. A Grounded Theory analysis of the descriptions obtained in the Structured Interview stage was performed and it was found that observers tended to describe her change ...

Watch this Scientific Journal Video about Investigating the 'Uncatchable Smile' in Leonardo da Vinci's La Bella Principessa: A Comparison with the Mona Lisa and Pollaiuolo's Portrait of a Girl at JoVE

Investigating the 'Uncatchable Smile' in Leonardo da Vinci's La Bella Principessa: A Comparison with the Mona Lisa and Pollaiuolo's Portrait of a Girl

This paper discusses the methodology used to reveal the &#39;Uncatchable Smile&#39; illusion in Leonardo da Vinci&#39;s La Bella Principessa portrait. A combination of three methods was used (inter-observation, structured interviews, and psychophysical experiments), which led to an investigation that shaped itself without prior beliefs, thus reducing potential researcher bias.

This paper discusses how the &#39;Uncatchable Smile&#39; illusion in Leonardo da Vinci&#39;s La Bella Principessa portrait was discovered. Kemp and Cotte1 ...

investigating-uncatchable-smile-leonardo-da-vinci-s-la-bella

Research

JoVE Journal

Behavior

6.9K Views.  Sheffield Hallam University. This paper discusses the methodology used to reveal the 'Uncatchable Smile' illusion in Leonardo da Vinci's La Bella Principessa portrait. A combination of three methods was used (inter-observation, structured interviews, and psychophysical experiments), which led to an investigation that shaped itself without prior beliefs, thus reducing potential researcher bias.

Video: Investigating the 'Uncatchable Smile' in Leonardo da Vinci's La Bella Principessa: A Comparison with the Mona Lisa and Pollaiuolo's Portrait of a Girl

Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues

Mori's Uncanny Valley Hypothesis1,2 proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness (DHL) (Figure 1). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.

Investigation of the Uncanny Valley Hypothesis and affective experience requires an understanding of the hypothesis' dimension of human likeness (DHL). This protocol allows representation of the DHL and examination of categorical perception. Use of the same stimuli and fMRI to distinguish brain regions responsive to physical and category change is illustrated.

Mori's Uncanny Valley Hypothesis1,2 proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated ...

Brain Imaging Investigation of the Neural Correlates of Observing Virtual Social Interactions

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language affect our decisions in personal and professional life alike 1. These "friend or foe" judgements are often based on first impressions, which in turn may affect our decisions to "approach or avoid". Previous studies investigating the neural correlates of social cognition tended to use static facial stimuli 2. Here, we illustrate an experimental design in which whole-body animated characters were used in conjunction with functional magnetic resonance imaging (fMRI) recordings. Fifteen participants were presented with short movie-clips of guest-host interactions in a business setting, while fMRI data were recorded; at the end of each movie, participants also provided ratings of the host behaviour. This design mimics more closely real-life situations, and hence may contribute to better understanding of the neural mechanisms of social interactions in healthy behaviour, and to gaining insight into possible causes of deficits in social behaviour in such clinical conditions as social anxiety and autism 3.

This article demonstrates an experimental design in which whole-body animated characters are used in conjunction with functional magnetic resonance imaging (fMRI) to investigate the neural correlates of observing virtual social interactions.

The ability to gauge social interactions is crucial in the assessment of others&#x2019; intentions. Factors such as facial expressions and body language ...

Neuroscience

Holistic Facial Composite Creation and Subsequent Video Line-up Eyewitness Identification Paradigm

The paradigm detailed in this manuscript describes an applied experimental method based on real police investigations during which an eyewitness or victim to a crime may create from memory a holistic facial composite of the culprit with the assistance of a police operator. The aim is that the composite is recognized by someone who believes that they know the culprit. For this paradigm, participants view a culprit actor on video and following a delay, participant-witnesses construct a holistic system facial composite. Controls do not construct a composite. From a series of arrays of computer-generated, but realistic faces, the holistic system construction method primarily requires participant-witnesses to select the facial images most closely meeting their memory of the culprit. Variation between faces in successive arrays is reduced until ideally the final image possesses a close likeness to the culprit. Participant-witness directed tools can also alter facial features, configurations between features and holistic properties (e.g., age, distinctiveness, skin tone), all within a whole face context. The procedure is designed to closely match the holistic manner by which humans&#8217; process faces. On completion, based on their memory of the culprit, ratings of composite-culprit similarity are collected from the participant-witnesses. Similar ratings are collected from culprit-acquaintance assessors, as a marker of composite recognition likelihood. Following a further delay, all participants &#8212; including the controls&#160;&#8212; attempt to identify the culprit in either a culprit-present or culprit-absent video line-up, to replicate circumstances in which the police have located the correct culprit, or an innocent suspect. Data of control and participant-witness line-up outcomes are presented, demonstrating the positive influence of holistic composite construction on identification accuracy. Correlational analyses are conducted to measure the relationship between assessor and participant-witness composite-culprit similarity ratings, delay, identification accuracy, and confidence to examine which factors influence video line-up outcomes.

This applied experimental paradigm replicates circumstances by which an eyewitness to a real crime may create a holistic facial composite of the culprit from memory, and then attempt to identify the culprit from a video line-up containing the culprit or one in which he or she is not present.

The paradigm detailed in this manuscript describes an applied experimental method based on real police investigations during which an eyewitness or victim ...

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis (AFEA) software. Additionally, we demonstrate a method for extracting relevant emotional data output and plotting the emotional response of a population over a specified time frame. By time pairing each participant's treatment response to a control stimulus (baseline), the overall emotional response over time and across multiple participants can be quantified. AFEA is a prospective analytical tool for assessing unbiased response to food and beverages. At present, most research has mainly focused on beverages. Methodologies and analyses have not yet been standardized for the application of AFEA to beverages and foods; however, a consistent standard methodology is needed. Optimizing video capture procedures and resulting video quality aids in a successful collection of emotional response to foods. Furthermore, the methodology of data analysis is novel for extracting the pertinent data relevant to the emotional response. The combinations of video capture optimization and data analysis will aid in standardizing the protocol for automated facial expression analysis and interpretation of emotional response data.

A protocol for capturing and statistically analyzing emotional response of a population to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis software is described.

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial ...

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 ...

Eye Tracking During Visually Situated Language Comprehension: Flexibility and Limitations in Uncovering Visual Context Effects

The present work is a description and an assessment of a methodology designed to quantify different aspects of the interaction between language processing and the perception of the visual world. The recording of eye-gaze patterns has provided good evidence for the contribution of both the visual context and linguistic/world knowledge to language comprehension. Initial research assessed object-context effects to test&#160;theories of modularity in language processing. In the introduction, we describe how subsequent investigations have taken the role of the wider visual context in language processing as a research topic in its own right, asking questions such as how our visual perception of events and of speakers contributes to&#160;comprehension informed by&#160;comprehenders&#39; experience. Among the examined aspects of the visual context are actions, events, a speaker&#39;s gaze, and emotional facial expressions, as well as spatial object configurations. Following an overview of the eye-tracking method and its different applications, we list the key steps of the methodology in the protocol, illustrating how to successfully use it to study visually-situated language comprehension. A final section presents three sets of representative results and illustrates the benefits and limitations of eye tracking for investigating the interplay between the perception of the visual world and language comprehension.

The present article reviews an eye-tracking methodology for studies on language comprehension. To obtain reliable data, key steps of the protocol must be followed. Among these are the correct set-up of the eye tracker (e.g., ensuring good quality of the eye and head images) and accurate calibration.

The present work is a description and an assessment of a methodology designed to quantify different aspects of the interaction between language processing ...

Investigating the Effect of Visual Imagery and Learning Shape-Audio Regularities on Bouba and Kiki

This article presents a protocol for investigating the role of visual imagery in the bouba/kiki-effect, whether training in noticing the bouba/kiki shape-audio regularities affects the bouba/kiki-effect and the recognition of individual bouba and kiki shapes, and finally what mental images these regularities produce. To generate bouba/kiki shape-audio regularities, there were two types of shapes (filled; outlined) and two types of audio (word; non-word sound). Three groups of individuals participated in three experiments: Blind, blindfold, and vision. The experiments were conducted in fixed order across participants, with no break between them. In Experiment 1 (pre-test-post-test design with three repeated within-group measures) the participants were asked to pick out the shape they associated with the auditory bouba/kiki; in Experiment 2 (within-subject design), to name one shape and some audio (sometimes congruous; sometimes incongruous) as &#8216;bouba&#8217; or &#8216;kiki;&#8217; and in Experiment 3 (post-test only design), to draw the shape they associated with the auditory bouba/kiki. The results suggest that the blindfold-group draw upon visual imagery to solve new problems, but not long term; that training in noticing bouba/kiki shape-audio regularities affects the bouba/kiki-effect and the recognition of individual bouba and kiki shapes, but differently in each experimental group; and that all experimental groups create mental images of the most characteristic shape feature of bouba (curve) and kiki (angle). In fact, the effect of visual imagery is robust across tasks, but not long term; the effect of learning shape-audio regularities is robust long term, but not across tasks. The presented protocol is appropriate for investigating the effect of visual imagery and learning shape-audio regularities, when they occur and how robust they are; in specific individuals and groups of individuals. This protocol is unique in that it keeps under control both the visual imagery and the sensory information during training and testing.

The aim of the presented protocol is to investigate the role of visual imagery in the bouba/kiki-effect, whether training in noticing the bouba/kiki shape-audio regularities affects the bouba/kiki-effect and the recognition of individual bouba and kiki shapes, and finally what mental images these regularities produce.

This article presents a protocol for investigating the role of visual imagery in the bouba/kiki-effect, whether training in noticing the bouba/kiki ...

Applying Hyperspectral Reflectance Imaging to Investigate the Palettes and the Techniques of Painters

Reflectance Spectroscopy (RS) and Fiber Optics Reflectance Spectroscopy (FORS) are well-established techniques for the investigation of works of art with particular attention to paintings. Most modern museums put at the disposal of their research groups portable equipment that, together with the intrinsic non-invasiveness of RS and FORS, makes possible the in situ collection of reflectance spectra from the surface of artefacts. The comparison, performed by experts in pigments and painting materials, of the experimental data with databases of reference spectra drives the characterization of the palettes and of the techniques used by the artists. However, this approach requires specific skills and it is time consuming especially if the number of the spectra to be investigated becomes large as is the case of Hyperspectral Reflectance Imaging (HRI) datasets. The HRI experimental setups are multi-dimensional cameras that associate the spectral information, given by the reflectance spectra, with the spatial localization of the spectra over the painted surface. The resulting datasets are 3D-cubes (called hypercubes or data-cubes) where the first two dimensions locate the spectrum over the painting and the third is the spectrum itself (i.e., the reflectance of that point of the painted surface versus the wavelength in the operative range of the detector). The capability of the detector to simultaneously collect a great number of spectra (typically much more than 10,000 for each hypercube) makes the HRI datasets large reservoirs of information and justifies the need for the development of robust and, possibly, automated protocols to analyze the data. After the description of the procedure designed for the data acquisition, we present an analysis method that systematically exploits the potential of the hypercubes. Based on Spectral Angle Mapper (SAM) and on the manipulation of the collected spectra, the algorithm handles and analyzes thousands of spectra while at the same time it supports&#160;the user to unveil the features of the samples under investigation. The power of the approach is illustrated by applying it to Quarto Stato, the iconic masterpiece by Giuseppe Pellizza da Volpedo, held in the Museo del Novecento in Milan (Italy).

Hyperspectral Reflectance Imaging hypercubes include remarkable information into a large amount of data. Therefore, the request for automated protocols to manage and study the datasets is widely justified. The combination of Spectral Angle Mapper, data manipulation, and a user-adjustable analysis method constitutes a key-turn for exploring the experimental results.

Reflectance Spectroscopy (RS) and Fiber Optics Reflectance Spectroscopy (FORS) are well-established techniques for the investigation of works of art with ...

Chemistry

Pipeline for Analyzing Behavioral Responses to External Cues in MRI Environments

Profiling Maternal Behavior Responses During Whole-Brain Imaging

Recent advancements in whole-brain imaging tools have enabled neuroscientists to investigate how coordinated brain activity processes external cues, influencing internal state changes and eliciting behavioral responses. For example, functional magnetic resonance imaging (fMRI) is a noninvasive technique that allows for the measurement of whole-brain activity in awake, behaving mice using the blood oxygenation-level-dependent (BOLD) response. However, to fully understand BOLD responses evoked by external stimuli, it is crucial that experimenters also assess behavioral responses during scans. The MRI environment poses challenges to this goal, rendering commonly employed methods of behavioral monitoring incompatible. These challenges include (1) a restricted field of view and (2) the limited availability of equipment without ferromagnetic components. Presented here is a behavioral video analysis pipeline that overcomes these limitations by extracting valuable information from videos acquired within these environmental constraints, enabling the evaluation of behavior during the acquisition of whole-brain neural data. Employing methods such as optical flow estimation and dimensionality reduction, robust differences can be detected in behavioral responses to stimuli presented during fMRI scans. For example, representative results suggest that mouse pup vocalizations, but not pure tones, evoke significantly different behavioral responses in maternal versus virgin female mice. Moving forward, this behavioral analysis pipeline, initially tailored to overcome challenges in fMRI experiments, can be extended to various neural recording methods, providing versatile behavioral monitoring in constrained environments. The coordinated evaluation of behavioral and neural responses will offer a more comprehensive understanding of how the perception of stimuli leads to the coordination of complex behavioral outputs.

Presented here is a video analysis pipeline that overcomes challenges in behavioral monitoring within MRI environments, allowing for the detection of uninstructed behavioral responses to external cues. This analysis will facilitate a more comprehensive understanding of evoked internal state changes and brain-wide activity.

Recent advancements in whole-brain imaging tools have enabled neuroscientists to investigate how coordinated brain activity processes external cues, ...

Assessing Pupil-linked Changes in Locus Coeruleus-mediated Arousal Elicited by Trigeminal Stimulation

Current scientific literature provides evidence that trigeminal sensorimotor activity associated with chewing may affect arousal, attention, and cognitive performance. These effects may be due to widespread connections of the trigeminal system to the ascending reticular activating system (ARAS), to which noradrenergic neurons of the locus coeruleus (LC) belongs. LC neurons contain projections to the whole brain, and it is known that their discharge co-varies with pupil size. LC activation is necessary for eliciting task-related mydriasis. If chewing effects on cognitive performance are mediated by the LC, it is reasonable to expect that changes in cognitive performance are correlated to changes in task-related mydriasis. Two novel protocols are presented here to verify this hypothesis and document that chewing effects are not attributable to aspecific motor activation. In both protocols, performance and pupil size changes observed during specific tasks are recorded before, soon after, and half an hour following a 2 min period of either: a) no activity, b) rhythmic, bilateral handgrip, c) bilateral chewing of soft pellet, and d) bilateral chewing of hard pellet. The first protocol measures level of performance in spotting target numbers displayed within numeric matrices. Since pupil size recordings are recorded by an appropriate pupillometer that impedes vision to ensure constant illumination levels, task-related mydriasis is evaluated during a haptic task. Results from this protocol reveal that 1) chewing-induced changes in performance and task-related mydriasis are correlated and 2) neither performance nor mydriasis are enhanced by handgrip. In the second protocol, use of a wearable pupillometer allows measurement of pupil size changes and performance during the same task, thus allowing even stronger evidence to be obtained regarding LC involvement in the trigeminal effects on cognitive activity. Both protocols have been run in the historical office of Prof. Giuseppe Moruzzi, the discoverer of ARAS, at the University of Pisa.

To verify whether trigeminal effects on cognitive performance involve locus coeruleus activity, two protocols are presented that aim to evaluate possible correlations between the performance and task-related pupil size changes induced by chewing. These protocols may be applied to conditions in which locus coeruleus contribution is suspected.

Current scientific literature provides evidence that trigeminal sensorimotor activity associated with chewing may affect arousal, attention, and cognitive ...