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lunes, 30 de septiembre de 2019

En deuda con Turing

En el logo de Apple hay una manzana mordida....Steve Jobs, lo hizo en recuerdo de Alain Touring

Infancia y juventud
Alan Mathison Turing nació en Paddington el 23 de junio de 1912. Sus padres Julius y Ethel residían en la India debido a que Julius trabajaba de funcionario en la India, pero decidieron volver al Reino Unido para que su hijo naciera allí. Esto hizo que Alan tuviera una infancia peculiar debido a los constantes viajes de sus padres entre Inglaterra e India durante los cuales dejaban a sus hijos al cargo de amigos.
A los 12 años entra en Sherborne School. Su jefe de estudios dijo de él «si lo único que quiere ser es un especialista científico, está perdiendo el tiempo en una escuela pública». Durante su estancia en dicha escuela Turing perdió a su amigo Christopher Morcom por una tuberculosis bovina contraída tras beber leche de vaca infectada. Esto le hizo perder su fe religiosa y convertirse en ateo.
Tras Sherborne School, Turing fue a King’s College en Cambridge. A pesar de que destacó en el campo de las matemáticas y la computabilidad, en un artículo suyo de 1950 mostrará un toque filosófico/moralista ya que relacionó el concepto matemático de la computabilidad con problemas tradicionales como la separación de la mente y cuerpo, el libre albedrío y el determinismo.
En 1931 formaliza el concepto de máquina de Turing sustituyendo así el lenguaje formal que Kurt Gödel utilizaba sobre los límites de la computación y la demostrabilidad. En 1935 es nombrado profesor del King’s College, a la temprana edad de 22 años.
La Máquina de Turing
Desarrolló el concepto de la máquina de Turing. Una máquina de Turing es un dispositivo teórico que manipula símbolos de una cinta de entrada en función de unas reglas. Se define como un autómata, que mediante un cabezal lector que lee de una cinta de entrada símbolos de un alfabeto, cambiando entre estados en función de la entrada pudiendo rechazar o aceptar la cadena de entrada dependiendo del lenguaje que acepte. Dicha máquina era capaz de implementar cualquier problema matemático que pudiera representarse mediante un algoritmo. Formalmente se define en función de los estados que tiene dicho autómata el alfabeto de entrada y las transiciones que soportal. Es una herramienta básica para el campo de los autómatas y lenguajes formales.
Demostró el problema de la parada de una manera muy intuitiva, aunque dicha demostración la había publicado previamente Alonzo Church (cálculo lambda) con el que trabajaría en Princeton donde obtuvo en 1938 el Doctorado debido a sus estudios sobre la hipercomputación.
La siguiente etapa de su vida da un cambio radical ya que el objeto de sus estudios no es investigación sin rumbo sino un fin específico: El criptoanálisis.
La Segunda Guerra Mundial
Decide empezar a trabajar para el Ejército porque “Nada estaba haciendo nada al respecto”. En 1939 empieza a trabajar en Bletchley Park (estación secreta del Ejército) liderando el Hut- 8 que era una de las secciones de la estación Británica de “codebreaking” durante la 2ªWW. Fue uno de los principales protagonistas en el desmantelamiento y ruptura de la máquina Enigma, mediante la que el Eje ocultaba sus transmisiones. Tras la declaración de guerra del 3 de Septiembre , Turing se volcó en el criptoanálisis en Bletchley Park. Con el trabajo que habían realizado los criptoanalistas polacos, Turing desarrolló la “Bombe” que era una máquina capaz de romper el código de la Enigma. Pero no bastaba, el ejército polaco había interceptado una máquina enigma parecida a la que utilizaba el ejército alemán y sabían que aun así no les daría tiempo a descifrar mensajes, ya que cada día cambiaban la forma de cifrarlos.
En Diciembre de 1939 resolvió gran parte del indicador que era una parte de la configuración que se cargaba en la máquina cada día, en la misma noche concibió la idea de Banburismus (conocido como análisis secuencial). Desde 1940 en adelante el Hut-8 utilizó la bomba criptográfica para leer mensajes de la Lufftwaffe, en cambio el método utilizado por la Kriegsmarine era mucho más complejo y se tomaba por irrompible. Sin embargo Turing aprovechando el conocimiento que tenían de la máquina proporcionada por el ejército polaco desarrolló, el solo, un sistema para atacar el cifrado: El banburismo.
El banburismo es un proceso de criptoanálisis que desarrolló Alan Turing en Bletcheley Park (instalación militar orientada a romper la máquina enigma). El proceso se basaba en la probabilidad condicional secuencial para deducir información acerca de las configuraciones de la máquina Enigma. El objetivo del banburismo era reducir el tiempo necesario para que la «Bomba» identificara los patrones de los rotores ya que reducía mucho las posibilidades. En 1939 Turing consiguió romper el código ahora solo quedaba capturar mensajes trabajo que hizo la marina. El procedimiento aprovechaba la debilidad del indicador (configuración inicial de la Enigma) comparaban mensajes criptados con distintas configuraciones, si el desplazamiento sólo se diferenciaba de un carácter (CFE-CFT cada letra corresponde a un rotor), podían obtener dicho desplazamiento. En 1941 se empezó a descifrar formalmente mensajes, en particular del submarino U-boat. El Hut 8 aplicó dicho procedimiento durante 2 años, hasta 1943 cuando empezó a ser factible el plan de la bomba criptográfica. Esto dio una gran ventaja al bando aliado en la batalla del atlántico cuando EEUU entró en la guerra. En ese momento Turing tuvo un rol de ingeniero electrónico, ideó el concepto de la mecanización de la ruptura del material FISH (teletipo), aunque fue MHA Newman quien desempeñó el papel organizativo. La mezcla de las ideas sobre estadística de Turing junto con la electrónica a gran escala tuvo resultados trascendentales.
Durante los últimos años de la guerra, Turing colaboró en la creación del Colossus, una máquina totalmente electrónica, que tuvo gran importancia para la invasión de Europa por parte del bando aliado al descifrar un mensaje en el que los alemanes decían que el desembarco se iba a dar lugar en en Calais, al conocer esta creencia del enemigo, los americanos decidieron encauzar el desembarco a las playas de Normandía. Turing consideró dicha máquina como un cerebro primitivo.
Cabe destacar, que debido a ser secreto de estado, la implicación de Alan Turing en la desencriptación de códigos nazis no fue revelada al público hasta 1970. Por lo que murió sin que la gente supiera de su contribución a la victoria de la guerra.
Pasada la segunda guerra mundial
En 1944 ya mencionó al ingeniero civil Donald Bailey sobre la «construcción de un cerebro», entre 1945, año en el que comenzó a trabajar en el National Physical Laboratory, y 1947, un año antes de abandonar el NPL, se dedico a dicha empresa. Existía un proyecto similar llamado EDVAC desarrollado paralelamente por los americanos pero el Automatic Computing Engine (ACE) de Turing se diferenciaba del proyecto estadounidense en que incluía la implementación de funciones aritméticas en circuitos electrónicos. Turing tenía en mente crear una máquina que pudiera ser configurada para hacer cálculos algebraicos, desencriptar códigos, manipular archivos e incluso jugar al ajedrez.
En 1947 creó el Abbreviated Code Instruction, origen de los lenguajes de programación.
A diferencia de la época de la segunda guerra mundial, en aquella ocasión no tuvo el apoyo de ingenieros y científicos, por lo que el ACE no se llegó a construir.
Gracias a la colaboración del ingeniero Frederic Calland Williams en 1948 se da por primera vez una demostración del principio de la máquina de Turing. Mientras tanto, estuvo entrenando como corredor de fondo y estuvo cerca de participar en los Juegos Olímpicos de ese año en representación de Inglaterra.
Tras la salida del NPL, se puso al cargo del laboratorio de computación de la Universidad de Mánchester, donde realizó parte del software del Mark I y escribió el artículo «Computing Machinery and Intelligence». En este artículo Turing desarrolló la idea de la inteligencia artificial y propuso el test de Turing que es capaz de discernir una máquina de un ser humano.
En 1950, sus antiguos colegas del NPL hicieron una versión reducida de la máquina inicialmente ideada por Turing, la Pilot Model ACE.
Los dos últimos años de su vida Turing dedicó sus esfuerzos a la formación de patrones y la biología mecánica, más concretamente estudió el proceso que controla la distribución organizada de las células de los organismos (morfogénesis) y si éste seguía la secuencia de Fibonacci. Dichos estudios dieron como resultado su escrito «The Chemical Basis of Morphogenesis».
Juicio y muerte
Desde el final de la guerra la inteligencia británica había decidido vigilar a Turing, pues sabían de su homosexualidad y no querían que alguien que sabía tantos secretos de la seguridad británica estuviera expuesto al chantaje. Finalmente, en Marzo de 1952 Turing fue detenido con motivo de su homosexualidad, la cual fue descubierta a raíz de las relaciones que mantuvo con un joven mancuniano.
Tras el juicio, en el que no se quiso defender al no considerar que estuviera cometiendo ningún delito, se le dio a elegir entre la cárcel o un tratamiento hormonal a base de estrógenos para neutralizar su libido. Alan eligió lo segundo que, si bien le libró del presidio, lo llevó a unos cambios físicos y anímicos que desencadenarían en el final de su vida.
Alan Turing fue encontrado por su asistenta el 8 de Junio de 1954. Murió el día anterior por ingestión de cianuro. Oficialmente la muerte fue considerada suicidio, se dice que mordiendo una manzana a la que había inyectado el veneno, pero su madre defendió que murió por una ingestión accidental de cianuro tras un experimento químico.
Post mortem
En 1966 se creó el Premio Turing, organizado por la Association for Computing Machinery, está reconocido como el Nobel de la computación.
Numerosas películas y novelas han tratado el tema de la máquina enigma y el test de Turing. Un conocido ejemplo de este último, es el film futurista Blade Runner de 1982, en el que se realizan entrevistas a algunos sujetos con el objetivo de determinar si son humanos o máquinas.
En 1990 Hugh Loebner con la aprobación del «Centro para Estudios del Comportamiento de Cambridge» prometió un premio de 100000 dólares y una medalla de oro para la primera máquina cuyas respuestas fueran indistinguibles de las humanas. Cada año se da un premio de 3000$ y una medalla de bronce a aquella que se acerque más a este objetivo.
Más información del premio Loebner:
http://loebner.net/Prizef/2010_Contest/loebner-prize.html
Premio Loebner del 2010, un chat bot de nombre Suzette creado por Bruce Wilcox engañó a un juez tras 25 minutos de conversación:
http://www.newscientist.com/article/dn19643-prizewinning-chatbot-steers-the-conversation.html?DCMP=OTC-rss&nsref=online-news
Para hablar con el bot:
http://www.chatbots.org/chatbot/suzette/
En 2009, el primer ministro británico Gordon Brown escribió una carta de disculpa en la que reconocía la horrible forma en la que fue tratado.
Ejemplo de CAPTCHA
Ejemplo de CAPTCHA
Los captchas que normalmente nos encontramos por internet, son un tipo de test de Turing, de hecho, la palabra CAPTCHA es un acrónimo de «Completely Automated Public Turing test to tell Computers and Humans Apart».

https://histinf.blogs.upv.es/2010/11/01/breve-biografia-de-alan-turing/?fbclid=IwAR1kcbskmROaqDAs1oeQdhvpRoNAwMOcf7ygBKZEZHAfXn6XUdOvCgEGCVc





2.Que mide el test de Touring?
Una de las preguntas que se vienen haciendo hace mucho tiempo los hombres de ciencia –y otras personas comunes como los escritores– es si pueden existir máquinas inteligentes. Alan Turing, fundador de la era tecnológica, consideraba que para saber si un dispositivo poseía inteligencia había que confirmar que este podía comunicarse como lo hacemos los seres humanos. Para ello propuso lo que se ha dado en llamar el test de Turing. Veamos en qué consiste el test de Turing y cuáles han sido los resultados obtenidos desde su creación.

Test de Turing

El test de Turing fue presentado por su creador en 1950. Supuestamente, se trata de una prueba para medir la inteligencia, y su punto de partida es que si un dispositivo se comporta sistémicamente con inteligencia, entonces es una entidad inteligente.
Para llevar a cabo esta prueba, se coloca un juez en una habitación y, en otra contigua, una máquina y una persona. El primero debe juzgar, mediante el lenguaje, cuál es el dispositivo y cuál el ser humano.
Comenzará entonces a hacerles preguntas hasta descubrir las verdaderas identidades. Ambos, máquina y persona, pueden dar respuestas falsas a cada interrogante. La tesis central es que, si la máquina es hábil al responder, podrá confundir al juez.

Resultados de la prueba de Turing

A lo largo del tiempo se han realizado diversos test de Turing para medir inteligencia artificial. Los resultados no habían sido exitosos, esto es, ningún dispositivo había pasado favorablemente dicha prueba hasta la actualidad.
En 1990, se dio inicio a un concurso anual llamado Premio Loebner, cuyo objetivo es el mismo que el del Test de Turing. Hay un juez que tiene frente a sí dos computadoras, una dirigida por un ser humano y otra automática. Se establece un diálogo con cada una, mediante preguntas y respuestas, para discernir cuál es cuál. Si la computadora automática logra confundir al juez, el programa gana una gran cantidad de dinero. El concurso no ha tenido aún un ganador.

Éxitos en el Test de Turing

A pesar de que hasta ahora no se habían tenido éxitos en el test de Turing, he aquí que de pronto algo ha logrado pasar dicha prueba. Ocurrió en Londres, hace poco tiempo, como homenaje a los 60 años de la muerte de Alan Turing. Se trata de un chatbot, es decir, un robot para tener charlas por la web, de nombre Eugene Goostman, que simula ser un adolescente ucraniano. Al parecer, ha logrado convencer a más de 30 personas de que es un ser humano.
Sin embargo, los científicos se cuestionan qué mide el Test de Turing, pues la inteligencia a través del lenguaje tiene que apelar a distintos contextos para cada estructura, lo cual es muy complejo, pero si una máquina accede a grandes cantidades de información, es posible que pueda dar respuestas sensatas a las preguntas sin ser inteligente, solo por trucos y artimañas.
Un cuestionamiento central sigue en pie: ¿mide el Test de Turing realmente inteligencia? Tendremos que esperar a ver el curso que siguen las investigaciones en los años venideros. Quizás después de todo no sea tan descabellado imaginar un futuro donde haya robots que sueñen con ovejas.
vacacionesporeuropa.com/

3.La pelicula ENIGMA
(no seguir leyendo si hay intención de ver la pelicula)

Ante la crisis creativa de Hollywood, las biopics parecen ser una alternativa confiable para buscar cierta originalidad. La industria toma vidas extraordinarias para crear películas de fórmula con diferentes calidades. Para El código enigma, el director noruego, Morten Tyldum, y el guionista, Graham Moore, supieron elegir el momento perfecto en la vida de un genio para dar a conocer de manera emocionante a una de las mentes que cimentaron nuestros tiempos modernos, Alan Turing, cuyo mote al final del filme expira contundencia y claridad: “el inventor de la computadora”. El resultado es una película con tintes de thriller, en un contexto de guerra, con mensaje y denuncia, cuya variedad de ingredientes están empleados con puntualidad para no saturar el resultado, sino para sumar a favor de la película misma, sin que haya demasiada profundidad en alguno.
El código enigma siguió por lo menos dos de las reglas básicas para lograr una buena película biográfica. Primero: tiene como protagonista a alguien ya muerto, por lo que no pesa ni pesó su opinión ni su existencia sobre lo que vemos sobre él en pantalla. Sus realizadores pudieron actuar con libertad, honrando a su manera, aunque maniobrando con la realidad según su conveniencia y prioridades. Segundo: la película no intenta abarcar toda la vida de Turing; a través de un periodo muy concreto, da cuenta de su ímpetu, de su carácter, de las dificultades a las que se enfrentó y de sus inigualables logros. Y lo adereza con flashbacks, sin olvidar cuál es el platillo principal. Lo hace con soltura y el típico humor inglés sarcástico y autodenigrante, ideal para exacerbar personalidades fuertes, antisociales y seguras de sus objetivos.
Spoiler alert
El filme se enfoca en el periodo durante la Segunda Guerra Mundial en el que el matemático fue contratado para descifrar, junto con un grupo de especialistas, el Código Enigma, creado por una computadora, que usaban los Nazis para comunicarse estrategias de guerra. Los mensajes se transmitían por señales de radio, flotaban en el aire al alcance de cualquiera, pero eran intraducibles. Y Turing fue el líder del equipo que logró descodificarlo, como una especie de hacker, y lo hizo rompiendo paradigmas: inventando una máquina que, mediante un algoritmo, pudiera leer en minutos cualquier mensaje que hubiera sido creado con el mismo código de programación que otro y que otro y que otro, y que todos los que se enviaban entre sí los alemanes. Se trató de una guerra entre computadoras, en la que la británica venció a la alemana. Aunque el trasfondo de la historia son campos de batalla, las armas y los muertos, los familiares devastados, las ciudades bombardeadas, en El código enigma no vemos la catástrofe, pero sí una verdad aún más injusta y cruel, que ya habíamos visto, aunque en su vertiente burocrática, en Zero Dark Dirty (2012): mientras que en las guerras hay hombres dando su vida en los campos de batalla, son solo carne de cañón; las grandes decisiones se trazan con el cuerpo caliente, seguro, bien comido y bien vestido, y con la mente fría y calculadora, entre caras paredes. Es ahí donde se decide el destino de miles de vidas, donde se opta por el mal menor. El código enigma nos permite ver, aunque de forma apresurada, el dilema trágico en el que estos hombres de mentes y condiciones privilegiadas vivían, sabiendo que mientras ellos estaban relativamente seguros, de su trabajo dependían las vidas de millones. La película, creada para dar zancadas de emoción en emoción, no permite que nos detengamos en estos sufrimientos y les dedica apenas un puñado de diálogos. La mayor atención que recibe el tema es cuando uno de los colegas de Turing pide que eviten que se bombardee un barco de guerra británico (que, han descubierto, es blanco alemán), porque su hermano soldado estará ahí, y todos en el equipo saben que el precio de salvar a ese hombre podría ser perder la guerra misma, pues los alemanes se percatarían del conocimiento de los británicos y cambiarían inmediatamente el código ya descifrado, lo que retrasaría indefinidamente el fin de la guerra. Lo único que volvemos a saber de ese personaje es que, tiempo después, mientras trabajan, le da un empujón a Turing con el hombro, sin voltear a verlo, porque obviamente no ha superado el rencor. Pero ese choque más bien es un pretexto para seguir avanzando la historia hacia otros asuntos.
Como suelen resolverse las tramas demasiado especializadas en algún campo científico, la película simplifica la ciencia, sobreexplica los descubrimientos esenciales, exagera y/o trastoca rasgos de la personalidad y anécdotas de la vida íntima de los implicados, a favor de un guión y edición con ritmo, y de la accesibilidad y de la empatía que puedan provocar las situaciones más coloquiales en las que puedan verse envueltos los personajes. Todo le funciona a El código enigma. Aunque jamás intenta acercarse a la complejidad de los descubrimientos del matemático, muestra y dice lo suficiente para atrapar, envolviendo con las bien equilibradas actuaciones, sobre todo de Cumberbatch, con esa personalidad de genio, encantadoramente odioso, y de su contraparte en el equipo, Matthew Goode, que se muestra lo suficientemente seguro de sí mismo y perspicaz, para soportar, sin conceder demasiado fácilmente, sus desplantes a Turing. Lo mismo hace la música de Alexandre Desplat, que balancea suaves armonías con melodías aceleradas, o viceversa, acentuando la lejanía de la época que vemos y lo apremiante de la situación.
Los pequeños nudos en la historia que crean expectativa se destensan con humor, haciéndolo todo sumamente entretenido; por ejemplo, sucede así con la entrevista de trabajo entre Turing y un general de alto mando del ejército, en la que Turing no se pliega a la jerarquía marcial y desde su presentación en pantalla desafía a la autoridad con conocimiento y determinación, lo que será su modus operandi hasta el cumplimiento de la misión. Está también la subtrama de la única mujer matemática que colaboró en el proyecto, Joan Clarke (Keira Knightley), que aunque en la realidad ya estaba ahí cuando Turing se incorporó, en la película es reclutada por él. La relación entre ambos sirve para exacerbar el tema de las presiones sociales y legales que vulneraban a homosexuales y mujeres en ese entonces. En el caso de los primeros, eran ilegales; en el caso de las segundas, como siempre, no debían destacar como profesionistas, sino como amas de casa.
Por el tiempo que se le dedica, podría parecer que la homosexualidad de Turing es tratada como secundaria, pero en realidad la denuncia que se hace contrastando sus logros con el trato legal que recibió y las consecuencias que tuvo, termina siendo lo más importante en la película. Es claro que evitaron darle demasiado peso al tema para que no fuera etiquetada como una película progay. Toda la historia del desciframiento del código está impregnada de regresiones a su niñez, que ayudan a comprender su dificultad para relacionarse y comunicarse, su pasión por los códigos y las matemáticas, pero que sobre todo dan cuenta de su primer amor hacia un niño del internado en el que creció que lo ayudó a descubrir su vocación, a creer en él mismo y por quien eventualmente bautizó a su máquina como Christopher (en la película, no en la realidad). Las tribulaciones matemáticas están enmarcadas por el robo que sufrió Turing en su casa después de que terminara la guerra; así inicia el filme. Intermitentemente seguimos la subsecuente investigación que iniciaron las autoridades, de la que se desprendió su encarcelamiento por haber tenido una relación con otro hombre; tras ser enjuiciado, se le dio la opción de mantenerse en la cárcel o someterse a un tratamiento hormonal. La última vez que vemos a Turing en pantalla, Joan lo va a visitar. Él se ve como un remedo de sí mismo, ansioso, con manos temblorosas, incapaz de resolver un vil crucigrama de periódico. En realidad, las consecuencias del uso de las hormonas fueron mucho más drásticas. Prácticamente, las reacciones a las sustancias lo desaparecieron en vida, después de torturarlo. El verdadero drama que padeció este hombre, después de que sus descubrimientos, su tenacidad y su temperamento lograran acortar la guerra salvando así millones de vidas, se muestran en la película con subtítulos: su suicidio por las insoportables inyecciones. El tardío perdón de la Reina otorgado en 2013, 61 años después de su muerte.
Como al terminar la guerra, el expediente de Turing y de todas las investigaciones que se realizaron, se clasificaron durante décadas, ni su nombre, ni sus logros, ni su tragedia, son tan conocidos como debieran. El código enigma saca ventaja de esa ignorancia generalizada para llevarnos a trote de suspenso. Sus realizadores privilegian la imagen de un genio y los logros de un país (una vez más), frente a la de un reprimido y sacrificado por el sistema. El regusto es un tanto esquizofrénico: el fundador de uno de los pilares de nuestra época fue héroe y mártir casi en simultáneo. Es un acertijo que la película no intentó resolver.

https://enfilme.com/resenas/en-pantalla/el-codigo-enigma?fbclid=IwAR0GmadvEQpqdqJqSKn3g0u18Pp-pHfucy68DsH_59CJgrce8xE5cpYyLO4
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viernes, 27 de septiembre de 2019

Tatuajes ¿seguros?

Synchrotron-based ν-XRF mapping and μ-FTIR microscopy enable to look into the fate and effects of tattoo pigments in human skin

Scientific Reportsvolume 7, Article number: 11395 (2017) | Download Citation

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Abstract

The increasing prevalence of tattoos provoked safety concerns with respect to particle distribution and effects inside the human body. We used skin and lymphatic tissues from human corpses to address local biokinetics by means of synchrotron X-ray fluorescence (XRF) techniques at both the micro (μ) and nano (ν) scale. Additional advanced mass spectrometry-based methodology enabled to demonstrate simultaneous transport of organic pigments, heavy metals and titanium dioxide from skin to regional lymph nodes. Among these compounds, organic pigments displayed the broadest size range with smallest species preferentially reaching the lymph nodes. Using synchrotron μ-FTIR analysis we were also able to detect ultrastructural changes of the tissue adjacent to tattoo particles through altered amide I α-helix to β-sheet protein ratios and elevated lipid contents. Altogether we report strong evidence for both migration and long-term deposition of toxic elements and tattoo pigments as well as for conformational alterations of biomolecules that likely contribute to cutaneous inflammation and other adversities upon tattooing.

Introduction

In recent years, the seemingly unstoppable trend for tattoos has brought safety concerns into the spotlight1. Currently, basic toxicological aspects, from biokinetics to possible alterations of the pigments, are largely uncertain. The animal experiments which would be necessary to address these toxicological issues were rated unethical because tattoos are applied as a matter of choice and lack medical necessity, similar to cosmetics2. Consequently, the hazards that potentially derive from tattoos were as yet only investigated by chemical analysis of the inks and their degradation products in vitro 3,4,5,6. Even though toxicological data might be available for some ink ingredients individually, information on in vivo interactions of the ink’s components and their fate within the body is rare.
Tattoos and permanent make-up work by depositing insoluble pigments into the dermal skin layer (Fig. 1). In conjunction with tattoos, pigmented and enlarged lymph nodes have been noticed in tattooed individuals for decades7. After the traumatic insertion of inks during the tattooing procedure, the body will excrete as many components as possible via the damaged epidermis. Other ways to clean the site of entrance are through active transport to lymph nodes by phagocytizing cells, or passively along the lymphatic vessels8,9,10,11. In addition to observations in humans, an in vivo study in mice revealed colored lymph nodes after tattooing with an azo pigment12. Even though this leaves little doubt that the pigment originates from corresponding tattoos, the origin and fate of pigments in human lymph nodes have never been analytically investigated so far. Lately, the only study analyzing human lymph nodes in tattooed individuals assessed its contents on carcinogenic polycyclic aromatic hydrocarbons deriving from carbon black particles13.
Figure 1
figure1
Translocation of tattoo particles from skin to lymph nodes. Upon injection of tattoo inks, particles can be either passively transported via blood and lymph fluids or phagocytized by immune cells and subsequently deposited in regional lymph nodes. After healing, particles are present in the dermis and in the sinusoids of the draining lymph nodes. The picture was drawn by the authors (i.e., C.S.).
Full size image
Tattoo pigments consist of either inorganic colorful metals and its oxides, or of polyaromatic compounds, all of which are thought to be biologically inert. It can thus be expected to find a broad range of elements in tattooed human tissue—among them the sensitizers nickel (Ni), chromium (Cr), manganese (Mn), and cobalt (Co)—as parts of color-giving pigments or element contamination14,15,16,17. Besides carbon black, the second most common used ingredient of tattoo inks is titanium dioxide (TiO2), a white pigment usually applied to create certain shades when mixed with colorants. The toxicity of TiO2 depends on its speciation (crystal structure) which can be either rutile or the more harmful photocatalytically active anatase18. The latter can lead to the formation of reactive oxygen species when exposed to sunlight. Delayed healing is thus often associated with white tattoos, along with skin elevation and itching19.
The contribution of tattoo inks to the overall body load on toxic elements, the speciation of TiO2, and the identities and size ranges of pigment particles migrating from subepidermal skin layers towards lymph nodes have never been analytically investigated in humans before. The average particle size in tattoo inks may vary from <100 nm="" to="">1 µm20. Therefore most tattoo inks contain at least a small fraction of particles in the nano range.
Here, we analyzed tattooed human skin and regional lymph nodes originating from four donors (corpses). Inductively coupled plasma mass spectrometry (ICP-MS) was used to assess the general contents of elements in both tissues and tattoo inks after microwave digestion. Laser-desorption/ionization time-of-flight (LDI-ToF) MS facilitated the identification of organic pigments in enzyme-lysed samples. To precisely locate the different elements in the cutaneous and lymphatic microenvironments, to provide information on TiO2 speciation and to assess primary particle sizes at the nanometric scale in particle mixtures, however, synchrotron-based X-ray fluorescence investigations have been performed at both the micro (μ-XRF) and nano (ν-XRF) range. Furthermore, we assessed biomolecular changes in the respective tissues at the micrometric scale and in the proximity of tattoo particles using synchrotron-based Fourier transform infrared (μ-FTIR) spectroscopy.

Results

Organic pigments translocate from skin to lymph nodes

Providing analytical evidence of tattoo particles being distributed inside the human body was a key objective of this investigation. To this end, tissue samples of four individuals tattooed with orange, red, green or black and two non-tattooed control donors were analyzed for the presence of organic pigments. Detection of the same pigment species in both skin and regional lymph nodes revealed the drainage of tattoo particles in two out of four tattooed donors (Fig. 2).
Figure 2
figure2
Organic pigments translocate from skin to lymph nodes. Organic pigments in lysed skin and lymph nodes were identified by means of LDI-ToF-MS. Adjacent skin and lymph tissue specimens (about 5–10 mm) are displayed in cryo-matrix after preparing thin sections for μ-FTIR and μ-XRF analyses. Skin specimens are oriented with its surface on the right side. Identified organic pigments are indicated below each sample. Chemical structures of the organic pigments identified in the samples are displayed on the right.
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Identification of organic pigments using LDI-ToF-MS has mostly been described using inks21,22,23. This technique is mainly based on isotope distributions and the molecular mass (see Supplementary Fig. S1). In the lysed tissues presented here, color-giving pigments were found to be copper phthalocyanines with either hydrogen, chlorine or bromine residues in three out of four skin samples. Reddish parts of the tattoos contained the azo group-containing pigments red 170 and orange 13 (Fig. 2).
For donors 1 and 2, the absence of organic pigments in the lymph nodes suggests either concentrations below the limit of detection (approx. 0.1–1% w/w pigment per extract), possible metabolic decomposition or drainage to alternative lymph nodes. The general ability for azo pigment translocation to lymph nodes was proven in additional skin and lymph node samples of donor 2 (Supplementary Table 1). On the other hand, carbon black particles possibly responsible for the black color in skin and lymph nodes (Fig. 2) were not accessible with the analytical methods used in this investigation. No xenobiotic pigment particles were detected in either skin or lymph tissue of the control samples.

Tattoos contribute to the elemental load of lymph nodes

A central aim of this study was to assess to what extent tattooing increases the proportion of toxic elements in the body. We found Al, Cr, Fe, Ni and Cu quantitatively elevated in skin and lymph node specimens using ICP-MS analysis (Table 1 and Supplementary Table S2). For donor 4, Cd and Hg concentrations were found increased only in the lymph nodes, but not in the analyzed skin sections. These elements probably result from other tattoos that were not part of this study or other routes of exposure drained through the same lymphatic tissue. Non-quantitative evaluation of the survey scans revealed the presence of Ti, presumably derived from TiO2, in all tattooed skin samples but not in controls.
Table 1 Element concentrations per tissue weight (ppm) in human skin and lymph node samples analyzed by ICP-MS.
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The microwave digestion used in this investigation is not suitable to fully dissolve Fe and Ti, although no residual particles were visible. Therefore Fe concentrations might not represent the total amount in the samples, but they enable the distinction between physiological concentrations in controls and samples containing extrinsic Fe. The elevated levels of Fe found in the skin and lymph nodes of donor 4 imply an additional use of iron-based pigments. In donors 1, 2 and 3, Fe concentrations were only increased in adjacent lymph nodes and not in the corresponding skin samples (Table 1). Fe concentrations can also be affected by residual blood within the tissue samples.
In donor 4, the use of pigment copper phthalocyanine green 36, as identified with LDI-ToF-MS, is reflected by high amounts of Cu in skin and lymph nodes as well as the non-quantitative detection of Br (Table 1). By contrast, although pigment copper phthalocyanine green 7 was well detectable with our LDI-ToF-MS approach in the skin of donor 2, it was not in the corresponding regional lymph node. Increased Cu levels measured by ICP-MS in this adjacent sample, however, suggest the presence of this copper phthalocyanine pigment. In light of the other two copper phthalocyanines applied in donor 2 (green 7) and 3 (blue 15) elevated Cu levels in skin came without surprise (Table 1). In donor 2, Cu levels in lymph nodes are strongly increased despite the fact that green 7 could not be detected with LDI-ToF-MS. However, adjacent samples of tissue were used for each analysis. Given the nature of the samples, pigment deposition within skin and lymph nodes is not homogeneous and therefore explaining the different findings. Interestingly, the non-tattooed control donor 1 also had slightly elevated levels of about 13 ppm Cu in the lymph nodes which is still in the range of the average 5.89 ± 8.03 ppm of Cu detectable in lymph nodes of female cadavers (Table 1)24.
Additionally, Ni and Cr were found in the human specimens. Since Ni levels were increased in the skin and lymph nodes of donor 2 and 3, the likely source is the tattoo. In different studies, both elements were linked to adverse reactions occurring in tattooed patients25,26,27,28. Ni and Cr are known to be allergenic as well as carcinogenic. Ni concentrations of 0.28–10.05 ppm total tissue weight found here are within the range of 0.8–3.7 ppm dry weight Ni in hilar lymph nodes in previous studies29. Cd was drastically elevated only in the lymph node of donor 4. For all other samples, Cd tissue burdens lie within normal values24.
Finally, Al was also present in skin and lymph node tissues of the three tattooed donors 2, 3 and 4 (Table 1). Since auxiliary lymph nodes have been investigated in the case of donor 2 and 3, co-exposure from antiperspirants containing various aluminum salts cannot be excluded, neither in tattooed nor control samples. However, Al concentrations in the controls were lower. The light metal Al has recently attracted attention because of its accumulation in breast cancer tissue30. While its role in the emerging of neoplasia is currently highly disputed, its contribution to the occurrence of hypersensitivity granulomas associated with tattoos has been proven since decades31.

μ-XRF mapping links metallic elements to tattoo particles

In order to link elements found with ICP-MS in tattoo pigment particles and to locate them inside the tissues, μ-XRF imaging was carried out with sub-micrometric probes over skin and lymph node sections (Fig. 3a–d). The location of particles can be altered by sample preparation. Since transversal sections were made by moving the knife parallel to the skin surface, the depth profile of the pigments should remain unaffected. Thin sections of skin and lymph nodes from donors 1, 3 and 4 were analyzed at the ESRF beamline ID21, with an exciting energy of 5.05 keV (Fig. 3a–d and Supplementary Fig. S2). Since the thin sections were deposited on BaF2 windows for further μ-FTIR analyses, the energy was chosen to avoid excitation of Ba L-lines (<5 .24="" 4="" a="" are="" data-track-action="figure anchor" data-track-label="link" data-track="click" displayed="" donor="" fig.="" href="https://www.nature.com/articles/s41598-017-11721-z#Fig3" in="" kev="" nbsp="" of="" results="">3
as an example.
Figure 3
figure3
μ-XRF mapping identifies and locates tattoo particle elements in skin and lymph node tissue sections. Sections of skin and lymph node tissue from donor 4 were analyzed by means of synchrotron μ-XRF. (a) Visible light microscopy (VLM) images of the area mapped by μ-XRF. Tattoo pigments are indicated by a red arrow. (b) DAPI staining of adjacent sections showing the cell nuclei. (c) μ-XRF maps of P, Ti, Cl and/or Br. For the lymph node, areas of similar size are marked in (a) and (b). (d) Average μ-XRF spectra over the full area displayed in (c) *diffraction peak from sample support; **scatter peak of the incoming beam. (e) Ti K-edge μ-XANES spectra of skin and lymph node compared to transmission XANES spectra of reference material of rutile, anatase and an 80/20 rutile/anatase mixture calculation.
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The majority of particles in the skin tissue were surrounded by phosphor-rich nuclei visualized by DAPI staining in fluorescence light microscopy (Fig. 3b) and integration of the element P in μ-XRF analysis (Fig. 3c). It was previously shown that tattoo particles can primarily be found around vessels10 which might account for the high cell density in the dermis co-localized with the pigments.
Intensities of Ti K-lines and Br L-lines were extracted to map the distribution of TiO2 and the highly brominated pigment copper phthalocyanine green 36 (Fig. 3c). Since the Br L-lines completely overlap with the Al K-lines, both may contribute to the intensity of the peak. However, LDI-ToF-MS analysis revealed the presence of pigment green 36 (Fig. 2) and the following ν-XRF results from ID16B acquired at 17.5 keV, i.e. above Br K-edge (13.47 keV) undermined the primarily Br-related contribution (see Supplementary Fig. S3).
Tattoo particles containing Ti and Br are adjacent to each other with only a slight overlap in skin and seem to be more evenly co-localized in lymph tissue (Fig. 3c). Both elements were found in the dermis of donor 4 directly beneath the cell nuclei-rich epidermis and up to a few hundred micrometers deep in the skin. In the lymph nodes, some particles were deposited in the stroma directly beneath the capsule. The bulk of Ti and Br containing particles, however, became visible as pigment agglomerates at a distance of about 250 µm to the lymph node capsule. Conversely, Cl concentrations are highest in the lymph node capsule and lower concentrations can be found in the particle region as part of the pigment phthalocyanine green 36.
All analyzed samples from the tattooed donors contained Ti. It is unlikely that other sources, e.g. sun screens, would explain the high amounts found in this investigation. Elevated amounts of Ti are only expected in lung and hilar lymph nodes from respiratory exposure32. Other highly abundant elements are K and Ca as they are physiologically present in cells (Fig. 3d).
We also investigated if the Ti present is the expected white pigment TiO2 and whether the stable rutile and/or the more photoreactive anatase crystal phases were used in tattoo inks. Micro X-ray absorption near edge structure (μ-XANES) spectra at the Ti K-edge were collected for the skin and lymph nodes of donors 1, 3 and 4. The spectra of donor 4 showed more qualitative correlation with the reference spectrum of rutile than with that of anatase (Fig. 3e). A clear switch of peak maxima between 4.99–5 keV occurs as a difference of both types of crystal structures. A calculated spectrum of 20% anatase and 80% rutile mixture is not clearly distinguishable from pure rutile, but shows a pre-edge at around 4.97 keV, similar to the μ-XANES spectra of the tattooed samples. Therefore, mostly rutile TiO2 is present in all tattooed donors, with minor amounts of anatase (Fig. 3e and Supplementary Fig. S2).

Particle size varies between pigment species

The obtained μ-XRF maps of skin and lymph node sections show large tattoo particle agglomerates up to several micrometers (Fig. 3c). However, it is known that small-sized particles are preferentially transported to lymph nodes. The 0.3 × 0.7 µm² beam size for μ-XRF mapping at ID21 was therefore a limiting factor for the determination of particle sizes. To assess the primary particle sizes, we additionally performed ν-XRF investigations by applying a beam of 50 × 50 nm² at 17.5 keV in order to excite the Br K-lines. Experiments were carried out in adjacent sections of skin and lymph node from donor 4, prepared on ultralene foil (Fig. 4). We detected three different pigment particles, each showing a different elemental composition and distribution within the same area (Fig. 4b,e). The average particle size of TiO2 in both skin and lymph nodes was 180 nm with a standard deviation of 23 nm and a standard error of 7 nm. Therefore this rather large particle size does not prevent distribution via the lymph fluid.
Figure 4
figure4
Particle mapping and size distribution of different tattoo pigment elements. Skin and lymph node of donor 4 were analyzed by means of synchrotron ν-XRF. (a,d) Ti and the Br containing pigment phthalocyanine green 36 are located next to each other. Average XRF spectra over the full area displayed in the regions of interest reveal the presence of Br, Si, S, Cl, Ca, Ti, Cr, Fe, Ni, Cu, and Zn. (b,e) Log scale mappings of Ti, Br and Fe in the same areas as displayed in (a) and (d) reveal primary particle sizes of different pigment species. (c,f) Magnifications of the indicated areas in (b) and (e), respectively.
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In contrast, the pigment phthalocyanine green 36 analyzed by ν-XRF mapping of Br was much more polydisperse, with particles presumably smaller than the resolution of 50 nm and up to the µm range in skin. In lymph node tissue, particles containing Br were smaller, with fewer particles of a larger size (Fig. 4c,f). Hence it can be assumed that the transport of smaller particles is preferential.
With the chosen energy, Br can be unequivocally identified from its K-lines emission. The skin and lymph node of donor 4 also contained Cu, related to the identified copper phthalocyanine pigments, and its maps show perfect co-localization with Br (see Supplementary Fig. S3). Additionally, Fe particles were present in the lymph node but not skin tissue and therefore possibly originate from another tattoo or route of exposure (Fig. 4c,f and Supplementary Fig. S3).

Tattoo particles induce biomolecular changes

The synchrotron-based μ-FTIR end-station at ID21 was used to monitor changes in protein conformation as well as in the overall protein and lipid contents in the proximity of tattoo particles. Synchrotron μ-FTIR analyses allow the assumption that tattoo pigments became located in a lipid-rich β-sheet protein environment.
The very same sections investigated by means of μ-XRF at ID21 were analyzed by means of μ-FTIR, prior to X-ray analyses, to facilitate exact site matching (cf. Figures 3 and 5). Thus, μ-FTIR results were not altered by μ-XRF radiation of the tissue sections. The high synchrotron photon flux allowed for high spatial resolution. Accordingly, the beam and pixel sizes were reduced to 10 × 10 µm² and 8 × 8 µm², respectively. This resolution is sufficient to distinguish regular dermis from pigment containing areas in the dermis, but remains insufficient to unambiguously separate the stratum corneum from the epidermis, which were analyzed here as a single domain (see below). Specific spectral changes related to the modification of biomolecule composition and conformation are displayed using donor 4 as an example, on the basis of two μ-FTIR maps obtained in a single section at two different locations, for the skin and regional lymph node (Fig. 5). The absorption band which peaks at 2920 cm−1 corresponds to the –CH2 stretching mode, which is much more intense in lipids than in proteins. It can be used to qualitatively map the distribution of lipids over thin sections (Fig. 5a). It shows a higher intensity in the stratum corneum, as expected33. These maps also qualitatively show a higher intensity in the areas of dermis containing tattoo pigments compared to pigment-free control regions. Based on the microscopic images and the μ-XRF maps described earlier, three regions were selected on each map. For the skin section, we divided the obtained map into stratum corneum and epidermis (SC), dermis without pigment (D) and dermis around pigment particles (DP) (Fig. 5a). Spectra in the second derivative of these areas were statistically analyzed by means of Principal Component Analysis (PCA). Distribution of points along the PC-1 axis confirms that D and DP have fewer lipid-related long alkyl chains (–CH2 stretching mode, asym. at 2920 cm−1 and –CH2 sym. at 2854 cm−1) and ester (–C = O stretching mode, peak at 1745 cm−1) vibrations than SC, and that DP regions contain higher levels of lipids than D (Fig. 5b–d). PC-2 separates DP from D and SC since the latter two have higher protein concentrations.
Figure 5
figure5
Changes of the biological composition and structure in the cellular proximity of tattoo pigment particles. Section of donor 4 analyzed by means of synchrotron μ-FTIR at ID21, ESRF. (a,e) Maps in second derivative obtained at 2920 cm−1 (–CH2 asymmetric vibration) of two different areas in either the skin or lymph node of donor 4 in overlay with a visible light microscopy image. Single points for PCA analysis in (c) and (g) were picked from the indicated areas. (b,f) Mean spectra from each region marked in (a,e) in second derivative. (c,g) PCA score plot of PC-1 vs. PC-2. (d,h) Loading plots of PC-1 and PC-2. Abbreviations: SC = stratum corneum and epidermis; D = dermis; DP = dermis with particles; P1, P2 = particle-containing regions; C1, C2 = control regions without particles.
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In addition to determining the component distribution, μ-FTIR can be used to identify and map the protein secondary structures across skin sections33. In the epidermis, keratinocytes differentiate to finally form the dead, protein- and lipid-rich stratum corneum. In the designated SC area of our investigation—comprising also the epidermal layer—the amide I peak maximum at ~1655 cm−1 (Fig. 5b) derives from α-helices present in keratin33. In the dermis, the peak maximum located at 1660 cm−1 corresponds to triple helices present in collagen, while the β-sheet shoulder at 1635 cm−1 can be assigned to crosslinked collagen fibers33. In the proximity of the pigment particles (DP), the protein content is lower compared to other parts of the collagen-rich dermis. However, the β-sheet shoulder at 1635 cm−1 becomes more pronounced close to the particles (Fig. 5b). The –CH2 and –C = O vibrations related to lipids are also higher in the proximity of particles compared to other parts of the dermis. Both findings suggest the presence of denatured β-sheet-rich protein and lipid membranes surrounding the pigment particles. Other investigations have shown that when in contact with foreign surfaces, protein structures can be altered towards the formation of β-sheets34. In the skin of donor 2, a similarly enhanced lipid content and the presence of β-sheet structures in the dermis around particles were also noticed (see Supplementary Fig. S4). A statistical comparison of particle-containing and particle-free areas in the lymph node tissue of donor 4 showed a similar increase of lipid contents in the former (Fig. 5e–h). However, no consistent differences in the kind of protein folding could be observed in lymph nodes.

Discussion

In this investigation, we found a broad range of tattoo pigment particles with up to several micrometers in size in human skin but only smaller (nano)particles transported to the lymph nodes. The exact size limit preventing this translocation is unknown yet. The deposit of particles leads to chronic enlargement of the respective lymph node and lifelong exposure. With the detection of the same organic pigments and inorganic TiO2 in skin and lymph nodes, we provide strong analytical evidence for the migration of pigments from the skin towards regional lymph nodes in humans. So far, this only has been assumed to occur based on limited data from mice and visual observations in humans13, 35. We also were able to prove the presence of several toxic elements, such as Cr and Ni, derived from tattooing. However, elemental deposits in lymph nodes which were not found in the corresponding skin revealed that tattooing might not have been the only route of exposure in these particular individuals whose tissues were removed after their demise.
It is known that pigments reside in lysosomes or stay attached to membranes of dermal fibroblasts8, 36, an observation that supports our μ-FTIR findings of concentrated lipid levels in the proximity of pigment particles. Long alkyl chains and ester groups which we assigned to lipids may also derive from components of tattoo inks, e.g. thickening polymers, surfactants and pigment coatings. However, the frequently used polyethylene glycol37 and polyvinyl pyrrolidone polymers below 20 kDa38 are known to be metabolized and secreted. In addition, the strong lysosomal and reactive oxygen species-driven reaction of macrophages against foreign material was shown to alter even the highly stable polyurethane39. We therefore assume these additives being biodegradable in vivo and thus not anymore present in healed tattooed skin. Since the initially used coatings and surfactants of the particles were unknown, interferences in μ-FTIR cannot be fully excluded though.
In cases where foreign hydrophobic material is introduced into the body, fibrinogen and other proteins are likely to become adsorbed and denatured at its surface, thus leading to the generation and presentation of pro-inflammatory matter and subsequent recruitment of immune cells as the initial step in the triggered foreign body reaction40, 41. This assumption becomes supported by our μ-FTIR data on β-sheet associated conformational changes of proteins in the proximity of hydrophobic, insoluble tattoo pigments. Foreign body reactions are known from subcutaneous injections of TiO2 42. Despite the hydrophobic nature of pigment surfaces and the here confirmed β-sheet protein conformation in the proximity of tattoo pigments in skin, most tattooed individuals including the donors analyzed here do not suffer from chronic inflammation though. Yet, granulomatous foreign material reactions are among the main non-infectious side effects occurring upon tattooing43. Factors preventing the progression towards adverse foreign body reactions in most tattooed individuals despite a β-sheet conformation need to be further investigated.
In future experiments we will also look into the pigment and heavy metal burden of other, more distant internal organs and tissues in order to track any possible biodistribution of tattoo ink ingredients throughout the body. The outcome of these investigations not only will be helpful in the assessment of the health risks associated with tattooing but also in the judgment of other exposures such as, e.g., the entrance of TiO2 nanoparticles present in cosmetics at the site of damaged skin.

Methods

Human sample preparation

Samples of tattooed skin and regional lymph nodes as well as skin and lymph node samples of two additional donors without any tattoos were taken postmortem at the Institute of Forensic Medicine at the Ludwig-Maximilians University of Munich (court-ordered autopsies without any additional cosmetic impairment to the skin). The experiments were performed according to the Helsinki Declaration of 1975. All samples were obtained anonymously without information on the patients disease status, causes of death or demographies. Ethical approval of human biopsy samples was granted by the Ethics committee of the Medical Faculty of the Ludwig-Maximilians University of Munich. We selected specimens with tattoos other than black and which are more likely to contain TiO2 and organic pigments. The sample size was limited by the availability of specimens and the beamtime at ESRF. Tissue samples were stored in plastic bags at −20 °C directly after excision and further processed for analysis within a year. Subsamples were cut using a standard scalpel and frozen in TissueTek O.C.T. matrix (Sakura Finetek, Staufen, Germany) for cryo-microtome sectioning. Sections of 5 or 6 µm were obtained and mounted on BaF2 substrates (Crystal GmbH, Berlin, Germany) for μ-FTIR and μ-XRF measurements at ID21. Sections for fluorescence light microscopy had a thickness of 6–10 µm and were deposited on standard glass slides, while ν-XRF analyses at ID16B were performed on 12–14 µm sections on 4 µm Ultralene window films (Spex Sample Prep, Metuchen, NJ, USA) mounted on Si3N4 windows. Sections were inactivated using 4% formaldehyde buffer for 10 min and subsequently washed with deionized water (2 times, 2–5 min). For μ-FTIR and μ-XRF analyses, samples were freeze-dried and stored in a dehydrated environment. Sections on microscopic glass slides were mounted in DAPI-Fluoromount G (Southern Biotech, Birmingham, AL, USA) for cell nucleus staining.

ICP-MS analysis

Elemental compositions of in total 20 skin and 25 lymph node samples of tattooed donors as well as 2 skin and 2 lymph node samples of non-tattooed donors were analyzed using a nitric acid microwave digestion (Ultraclave, MLS, Leutkirch, Germany). Samples were directly adjacent to those used in other parts in this investigation. Five milliliter of 69% nitric acid was added to 50–200 mg tissue samples in Teflon vessels and heated in the microwave with the following steps: 20–80 °C (3.5 min, 100 bar, 700 W); 80–130 °C (10 min, 120 bar, 1000 W); 130–200 °C (6.5 min, 150 bar, 1000 W), 200 °C (30 min, 150 bar, 1000 W). Elemental concentrations given in ppm are calculated in relation to the weight of digested tissue. Nitric acid was purified using a duoPUR quartz sub-boiling distillation system (MLS, Leutkirch, Germany). Ultrapure water was obtained using a Milli-Q Advantage A10 water purification system equipped with a Millipore Q-POD Element Unit (both from Merck, Darmstadt, Germany). Standards for ICP were purchased either from Sigma Aldrich (Munich, Germany; i.e. Sc, Al, Cu, Ni, Hg) or Merck (Darmstadt, Germany) in the case of In. For Cr, Fe and Cd 1000 mg/l standard solutions in diluted nitric acid were obtained from VWR (Darmstadt, Germany).
A 20-fold dilution of each sample was prepared including 10 ppb of the elements In and Sc as internal standards. XSeries II ICP-MS (Thermo Fischer Scientific, Bremen, Germany) together with an ESI SC2 autosampler (Elemental Service & Instruments, Mainz, Germany) were used for sample analysis. Sample analysis was carried out in triplicate with 100 sweeps each. Resolution was set to 0.02 amu and the dwell time for all elements was 10 ms. Measurements were carried out with collision cell in either −3.0 V mode (In, Sc, Cr, Fe, Ni, Cu, Cd) or 0.0 V mode (Sc, Al). H2/He (7% v/v) was used as the collision gas with 5 ml/min flow rate. Data were processed with PlasmaLab 2.5.11.321 (Thermo Scientific, Bremen, Germany).

LDI-ToF-MS identification of organic pigments

In total 8 skin and 8 lymph node samples of tattooed donors as well as 2 skin and 2 lymph node samples of non-tattooed donors were analyzed. Samples between 50–200 µg were lysed using 1 mg/ml collagenase from Clostridium histolyticum Type IA (Sigma Aldrich, Munich, Germany) with an incubation time of at least 24 hours at 37 °C. Lysates were heat-inactivated at 90–95 °C for at least 12 hours. Precipitated pigment particles were washed twice with PBS. Centrifugation was carried out with 500× g for 10 min. Precipitates were applied as thin films to a ground steel target plate with a plastic pipette tip and measured using an UltrafleXtreme MALDI-ToF/ToF (Bruker Daltonik, Bremen, Germany). Spectra were obtained by averaging 3000 individual spectra, with a laser rate of 1000 Hz in positive reflector mode. The instrument was calibrated prior to each measurement with an external ProteoMass™ MALDI Calibration Kit (Sigma Aldrich, Munich, Germany). Data were processed using the flexControl 3.4 and flexAnalysis 3.4 software (Bruker Daltonik, Bremen, Germany).

Synchrotron FTIR microscopy

FTIR microscopy analyses were performed at beamline ID21 at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France44. The beamline is equipped with a Thermo Nicolet Continuum (Thermo Scientific, Madison, WT, USA) microscope coupled to a Thermo Nicolet Nexus FTIR spectrometer (Thermo Scientific, Madison, WT, USA) with a 32x objective, a motorized sample stage, and a liquid nitrogen-cooled 50 µm HgCdTe detector. Maps were acquired in transmission mode using a 10 × 10 µm² beam, step size of 8 µm. Spectra were recorded as an average of 64 scans per spectrum, over a range of 4000 to 850 cm−1 and with a spectral resolution of 4 cm−1.
The OMNIC software was used to transform spectra from maps of skin and lymph node samples to second derivatives using Savitsky-Golay of second polynomial order with 21 smoothing points45, 46. Unscrambler X software (Version 10.3, CAMO Software, Oslo, Norway) was used for further statistical analysis. Principal component analysis (PCA) was performed on the mean-centered data using the spectral regions from: 1800 to 1350 cm−1 (related to proteins) and 3200 to 2800 cm−1 (related to lipids)47, 48. PCA was performed separately for skin and lymph node samples. Score plots and loading plots obtained by PCA analysis as well as mean values from the regions of interest were used for data interpretation.

Synchrotron μ-XRF and μ-XANES

μ-XRF and μ-XANES analyses were carried out at the beamline ID2149. Here, X-rays were generated by an U42 undulator operated in “gap-tracking” mode, i.e. the gap value was optimized for each energy. A fixed exit double-crystal Si(111) Kohzu-monochromator was used in combination with a Ni-coated flat double-mirror rejecting high-energy harmonics and allowed for energy selection with about 0.4 eV resolution of the primary radiation at Ti K-edge (5.1 keV). Downstream of the monochromator, the beam was focused down to 0.4 × 0.8 µm2 (vertical × horizontal) using a fixed-curvature Kirkpatrick-Baez (KB) mirror system. The flux was 1.6 × 1010 photons/s (~180 mA SR current in multi-bunch mode). A 30 µm Al attenuator was used to reduce the photon flux by one order of magnitude to keep the XRF detector dead time within its linear range. A photodiode collecting the XRF from a thin Si3N4 membrane inserted in the beam path was used to continuously monitor the incoming beam intensity. XRF and scattered radiation were collected with a dispersive energy silicon drift detector with an active area of 80 mm² (Bruker Daltonik, Bremen, Germany). Acquisition time per point was 100 ms. The pixel size for collecting the XRF maps was adjusted to the regions of interest and varied from 0.5 µm to 5 µm. Scans were performed in continuous (zap) mode and an energy of 5.05 keV was selected for μ-XRF mapping. For collecting Ti XANES spectra, the energy of the incoming beam was scanned from 4.95 to 5.1 keV in increments of 0.5 eV, with acquisition times of 100 ms per energy. Depending on the concentration of the probed region, between 1 and 10 μ-XANES spectra were collected per point and subsequently averaged. Full-field XANES maps were also collected to total the XANES spectra over multiple pixels.

Synchrotron ν-XRF

The analysis on an adjacent section of skin and lymph node tissue from donor 4 was performed by means of ν-XRF at ID16B at the ESRF. The experimental set-up is described elsewhere50. A pink beam with an energy of 17.5 keV with ΔE/E = 1% was focused down to 50 × 50 nm² using KB mirrors. The flux of >1 × 1011 photons/s was subsequently reduced using gold and silicon attenuators to keep the dead time on the XRF detectors within the linear range. Two three-element silicon drift detector arrays (SGX Sensortech, Buckinghamshire, UK) were used. The two ν-XRF maps were recorded with a step size of 50 × 50 nm² and 100 ms dwell time. In contrast to the set-up installed at ID21, ID16B operates in air. For estimating the particle size of TiO2, analysis was performed on 10 particles by computing the full width at half maximum of line profiles through the particles.

Availability of materials and data

XRF and FTIR data sets can be provided by the authors upon individual request.

Ethical approval of human biopsy samples

Samples of tattooed skin and regional lymph nodes were taken postmortem and anonymously at the Institute of Forensic Medicine at the Ludwig-Maximilians University of Munich in the frame of court-ordered autopsies without information on the patients disease status, causes of death or demographies. Experiments were performed according to the Helsinki Declaration of 1975 (see: http://www.wma.net/en/30publications/10policies/b3/17c.pdf). Ethical approval of human biopsy retrieval was granted by the Ethics committee of the Medical Faculty of the Ludwig-Maximilians University of Munich, Germany (confirmation by R.P., member of the ethics committee).

References

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Acknowledgements

The authors thank the ESRF for allocated beamtimes on ID21 and ID16B. N. Dommershausen is acknowlegded for technical help with LDI-ToF-MS analyses. We are grateful to Prof. Dr. Bäumler for valuable discussions. This work was supported by the intramural research project (SFP #1322–604) at the German Federal Institute for Risk Assessment (BfR).

Author information

Author notes

  1. Ines Schreiver and Bernhard Hesse contributed equally to this work.

Affiliations

  1. German Federal Institute for Risk Assessment (BfR), Department of Chemical and Product Safety, Max-Dohrn-Strasse 8-10, 10589, Berlin, Germany

    • Ines Schreiver
    • , Peter Laux
    • , Nadine Dreiack
    •  & Andreas Luch

  2. European Synchrotron Radiation Facility (ESRF), 38043, Grenoble, Cedex 9, France

    • Bernhard Hesse
    • , Hiram Castillo-Michel
    • , Julie Villanova
    • , Remi Tucoulou
    •  & Marine Cotte

  3. Physikalisch-Technische Bundesanstalt, Department of X-ray Spectrometry, Abbestrasse 2-12, 10587, Berlin, Germany

    • Christian Seim

  4. Technische Universität Berlin, Institute for Optics and Atomic Physics, Hardenbergstrasse 36, 10623, Berlin, Germany

    • Christian Seim

  5. Institute of Forensic Medicine, Ludwig-Maximilians University, Munich, Germany

    • Randolf Penning

Contributions

A.L., M.C., R.T. and P.L. designed and supervised the study, including the interpretation of analytical data. I.S., B.H., H.C.-M. and C.S. planned the experiments. B.H., H.C.-M., I.S. and C.S. performed the experiments at ID21. J.V. performed the experiments at ID16B. I.S. analyzed the samples by means of LDI-ToF-MS. I.S. and N.D. carried out ICP-MS analysis. I.S., B.H., H.C.-M. and C.S. critically reviewed the data and drafted the manuscript. C.S. graphically designed the figures. R.P. selected and provided human specimens suitable for these experiments. A.L. and M.C. analyzed the overall results and finalized the manuscript.

Corresponding author

Correspondence to Andreas Luch.

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Competing Interests

The authors declare that they have no competing interests.

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