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Orientador: Carlos Lenz Cesar Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin Resumo: A comunidade científica concorda que há grandes chances que a próxima revolução tecnológica virá do controle dos processos biológicos. Grandes mudanças são esperadas, desde como produzimos alimentos até como combatemos as doenças. O controle dos processos biológicos nos permitirá produzir carne sintética para alimentação, produzir biocombustíveis retirando CO2 da atmosfera, produzir órgãos inteiros para transplante e combater de forma eficiente doenças como câncer, por exemplo. Está claro para o nosso grupo que para se obter esses resultados é necessário entender a biologia na sua unidade mais básica: a célula. A partir do entendimento e domínio das reações químicas que acontecem dentro da célula, e mais especificamente do controle do DNA, é que vamos conseguir atingir essas previsões e revolucionar a maneira como vivemos hoje. Com esse pensamento em mente, o objetivo dessa tese foi desenvolver uma plataforma fotônica integrada para estudos de processos celulares. Nós acreditamos que as ferramentas fotônicas são as ferramentas que preenchem todos os requisitos para os estudos de processos celulares, pois possibilitam o acompanhamento dos processos em tempo real sem causar dano as células. As técnicas presentes são: fluorescência excitada por 1 ou 2 fotons, geração de segundo ou terceiro harmônico, pinças ópticas, imagem por tempo de vida da fluorescência e "fluorescence correlation spectroscopy" (FCS). Nesta tese demonstramos como montar essa plataforma integrada e mostramos sua versatilidade com resultados em várias áreas da biologia e também para o estudo de quantum dots. Abstract: The scientific community believes there is a great chance that the next technological revolution is coming from the control of biological processes. Great changes are expected, from the way we produce food up to the way we fight diseases. The control of biological processes will allow us to produce synthetic meat as food, to produce biofuels extracting CO2 directly from the atmosphere, to produce whole synthetic organs for transplant and to fight diseases, like cancer, in more efficient ways. It is clear to our group that in order to obtain these results it is necessary to understand biology from its most basic unity: the cell. Only from understanding and controlling chemical reactions inside a cell, and more specifically from the DNA controlling, it will be possible to achieve these predictions and cause a revolution in the way we live nowadays. Bearing these thoughts in mind, the objective of this thesis was to develop an integrated photonic platform for study of cellular processes. We believe that photonic tools are the only tools that fulfill all the requeriments for studies of cellular processes because they are capable to follow processes in real time without any damage to the cells. The techniques integrated are: 1 or 2 photon excited fluorescence, second or third harmonic generation, optical tweezers, fluorescence lifetime imaging and fluorescence correlation spectroscopy. In this thesis we demonstraded how to assemble this integrated plataform and we showed its versatility with results from different areas of biology and quantum dots. Doutorado Física Doutor em Ciências

Single-wall carbon nanotubes (SWCNTs) are used in diverse applications that require chemical tailoring of the SWCNT surface, including optical sensing, imaging, targeted drug delivery and single-photon generation. SWCNTs have been noncovalently modified with (bio)polymers to preserve their intrinsic near-infrared fluorescence. However, demanding applications (e.g., requiring stability in biological fluids) would benefit from a stable covalent linkage between the SWCNT and the functional unit (e.g., antibody, fluorophore, drug). Here we present how to use diazonium salt chemistry to introduce sp3 quantum defects in the SWCNT carbon lattice to serve as handles for conjugation while preserving near-infrared fluorescence. In this protocol, we describe the straightforward, stable (covalent), highly versatile and scalable functionalization of SWCNTs with biomolecules such as peptides and proteins to yield near-infrared fluorescent SWCNT bioconjugates. We provide a step-by-step procedure covering SWCNT dispersion, quantum defect incorporation, bioconjugation, in situ peptide synthesis on SWCNTs, and characterization, which can be completed in 5-7 d.

Subject of Research. The paper presents qualitative analysis of terahertz time-domain spectroscopy application for cancer diagnosis by measurement of the optical properties and spectral characteristics of cancer cells. For this purpose, the cultivation of two cancer cells, U-251 (glioblastoma brain) and A549 (lung adenocarcinoma), were carried out, then their refractive index, absorption coefficient and dielectric constant were measured, and the optical properties of tumor cells were compared with the optical properties of healthy cells (fibroblasts). Tumor cells contain more OH-components in comparison with healthy cells. Since terahertz radiation is heavily absorbed by water, there are differences in the spectra of healthy and oncological cells. Because of the demand for rapid and effective diagnostics of oncology (including intraoperative), the obtained results show that terahertz time-domain spectroscopy can be used for this purpose in the present time.Method. To obtain the optical properties and spectral characteristics of the researched objects, a terahertz time-domain spectroscopy method in the transmission mode was used. The researched cell lines were cultured in vitro. Optical properties and spectral characteristics of the samples were calculated by the thin film method and Fourier transform.Main Results. The results show the differences of refractive index, absorption coefficient and dielectric permittivity between the oncological cell lines U-251, A549 and the healthy cells in the frequency range 0.2-1 THz. It was found that cancer cells have higher values of refractive indices and absorption coefficients than those of healthy cells. Brain glioblastoma (U-251) has a transmission peak at the frequency of 0.24 THz.Practical Relevance. The results obtained in this work can form the basis for the intraoperative diagnosis of brain and lung cancer with the use of terahertz time-domain spectroscopy, and it is also useful in the other studies, for example, the development of biotissue phantoms in the THz frequency range

The photochemical core of every photosynthetic apparatus is the reaction center, a transmembrane enzyme that converts photons into charge-separated states across the biological membrane with an almost unitary quantum yield. We present a light-driven organic transistor architecture, which converts light into electrical current by exploiting the efficiency of this biological machinery. Proper surface tailoring enables the integration of the bacterial reaction center as photoactive element in organic transistors, allowing the transduction of its photogenerated voltage into photomodulation of the output current up to two orders of magnitude. The device architecture, termed Light-driven Electrolyte-Gated Organic Transistor (LEGOT), is the prototype of a new generation of low-power hybrid bio-optoelectronic organic devices.

Key Clinical Message Treating solar lentigines using picosecond‐switched lasers that selectively remove the excess pigment was combined with Kleresca® biophotonic treatment. This therapy uses fluorescent light energy to stimulate healing by increasing collagen production and reducing inflammation. Combining these therapies successfully removed solar lentigines and achieved normalized and rejuvenated treated skin.

Unfertilised eggs (oocytes) release chemical biomarkers into the medium surrounding them. This provides an opportunity to monitor cell health and development during assisted reproductive processes if detected in a non-invasive manner. Here we report the measurement of pH using an optical fibre probe, OFP1, in 5 μL drops of culture medium containing single mouse cumulus oocyte complexes (COCs). This allowed for the detection of statistically significant differences in pH between COCs in culture medium with no additives and those incubated with either a chemical (cobalt chloride) or hormonal treatment (follicle stimulating hormone); both of which serve to induce the release of lactic acid into the medium immediately surrounding the COC. Importantly, OFP1 was shown to be cell-safe with no inherent cell toxicity or light-induced phototoxicity indicated by negative DNA damage staining. Pre-measurement photobleaching of the probe reduced fluorescence signal variability, providing improved measurement precision (0.01-0.05 pH units) compared to previous studies. This optical technology presents a promising platform for the measurement of pH and the detection of other extracellular biomarkers to assess cell health during assisted reproduction. Refereed/Peer-reviewed

This thesis reports on studies of plasmonic nanoparticles and particularly gold nanostars as signal enhancers and contrast agents for biophotonic applications including visualisation, treatment of living cells and chemical sensing. In this thesis, the optical properties of nanoparticles of different size and morphology and their silica composites were compared. Because they are the most suitable plasmonic nanostructures, gold nanostars were utilised for optical imaging modalities such as confocal microscopy and Doppler optical coherence tomography. The ability of gold nanoparticles to enhance the signal in surface-enhanced vibrational spectroscopy, including Raman and Fourier transform infrared spectroscopy was additionally studied. Finally, various gold nanoparticles were applied for cell optoporation to increase the penetration ability of exogeneous substances. In summary, significant advantages of nanostars such as their low-toxicity, high scattering and contrast abilities, in addition to a broad, tunable, plasmon resonance wavelength range, as well as the capability to enhance the signal of analyte molecules in vibrational spectroscopy were demonstrated in this thesis. The results of this study on the effectiveness of nanostars have a broad scope of utility and open a wide perspective for their utilisation in nanobiophotonics and biomedicine. Tiivistelmä Tämä opinnäytetyö kertoo tutkimuksista, joissa plasmoninanopartikkeleita ja erityisesti kultananotähtiä on käytetty signaalinvahvistimina biofotoniikan sovelluksissa, kuten visualisointi, elävien solujen käsittely ja kemiallinen tunnistus. Tässä työssä verrattiin eri kokoisten ja muotoisten nanopartikkeleiden ja niiden piioksidikomposiittien optisia ominaisuuksia. Sopivimpina plasmoninanorakenteina kultananotähtiä käytettiin optisiin kuvantamismenetelmiin, kuten konfokaalimikroskopiaan ja Doppler-optiseen koherenssitomografiaan. Lisäksi kuvattiin myös kultananopartikkelien kykyä parantaa pinta-aktivoidun värähtelevän spektroskopian signaalia, mukaan lukien Raman- ja Fourier-muunnos-infrapuna-spektroskopia. Lopuksi, eri kultananopartikkeleita käytettiin soluoptoporaatioon eksogeenisten aineiden läpäisevyyden lisäämiseksi. Yhteenvetona, työssä osoitettiin nanotähtien merkittävät edut, kuten matala-myrkyllisyys, suuret sironta- ja kontrastiominaisuudet, laaja plasmoniresonanssin aallonpituusalue ja sen viritettävyys, sekä kyky parantaa analyyttimolekyylien signaalia värähtelyspektroskopiassa. Niinpä tutkimustulokset nanotähtien tehokkuudesta ovat laajasti käyttökelpoisia ja ne avaavat laajan näkökulman niiden hyödyntämiseen nanobiofotoniikassa ja biolääketieteessä.

International audience; In biology, molecules and macromolecules such as sugars, proteins, DNA, RNA, etc., are of utmost importance. Detecting their presence as well as getting information on their actual structure is still a challenge in many cases. The vibrational states of such molecules correspond to a spectral range extending from infrared to terahertz. Spectroscopy is used for the detection and the identification of such compounds and their structure. Terahertz spectroscopy of a biosample is challenging for two main reasons: the high terahertz absorption by water molecules in the sample; and the small size of the sample - its volume is usually smaller than the cube of the terahertz wavelength, thus the light-matter interaction is extremely reduced. In this paper, we present the design, fabrication, characterization, and first typical use of a biophotonic device that aims to increase the light?matter interaction to enable terahertz spectroscopy of very small samples over a broad band (0.2-2 THz). Finally, we demonstrate the validity of our approach by time-domain spectroscopy of samples of a few µL.

In the last decade silk fibroin (SF), the protein extracted from the silk fibers, emerged as an attractive material for biophotonic applications due to its biocompatibility combined with unique mechanical and optical properties. A strategy to enhance the optical properties of silk, and to simultaneously introduce specific functionalities, is to combine silk with some specific inorganic, organic or biological compounds. The work here presented reports the results achievable by combining silk with two different inorganic nanoparticles for the fabrication of optical nanostructured devices. The first strategy investigated was to combine silk with titanate nanosheets (TNSs), a 2D precursor of TiO2, in order to significantly increase the refractive index of silk while preserving all its specific properties. The structural and functional characterizations of the SF-TNSs composites were performed to correlate the material structure with its properties. In particular, the ion exchange process was investigated as a strategy to easy functionalize post-process the material. A second strategy that was investigated exploits the combination of gold nanoparticles within the silk matrix in order to introduce plasmonic functionalities in the material. Specifically, in this first phase, the inclusion of gold nanoparticles was exploited to induce localized heating thanks to the excitation of the nanoparticles plasmon resonance, an effect that can be potentially used in biomedical applications in the treatment of bacterial infections. Finally, the fabrication of simple optical and photonic devices with the nanocomposites, such as multilayer Bragg reflectors and inverse opals, was demonstrated. In particular, a bioinspired multilayer optical structured was fabricated with the SF-TNSs material, showing a stimuli-responsive behavior, which results in a reversible change of structural coloration in response to humidity.

Lycaenid butterflies from the genera Callophrys, Cyanophrys and Thecla have evolved remarkable biophotonic gyroid nanostructures within their wing scales that have only recently been replicated by nanoscale additive manufacturing. These nanostructures selectively reflect parts of the visible spectrum to give their characteristic non-iridescent, matte-green appearance, despite a distinct blue–green–yellow iridescence predicted for individual crystals from theory. It has been hypothesized that the organism must achieve its uniform appearance by growing crystals with some restrictions on the possible distribution of orientations, yet preferential orientation observed in Callophrys rubi confirms that this distribution need not be uniform. By analysing scanning electron microscope and optical images of 912 crystals in three wing scales, we find no preference for their rotational alignment in the plane of the scales. However, crystal orientation normal to the scale was highly correlated to their colour at low (conical) angles of view and illumination. This correlation enabled the use of optical images, each containing up to 104–105 crystals, for concluding the preferential alignment seen along the at the level of single scales, appears ubiquitous. By contrast, orientations were found to occur at no greater rate than that expected by chance. Above a critical cone angle, all crystals reflected bright green light indicating the dominant light scattering is due to the predicted band gap along the direction, independent of the domain orientation. Together with the natural variation in scale and wing shapes, we can readily understand the detailed mechanism of uniform colour production and iridescence suppression in these butterflies. It appears that the combination of preferential alignment normal to the wing scale, and uniform distribution within the plane is a near optimal solution for homogenizing the angular distribution of the band gap relative to the wings. Finally, the distributions of orientations, shapes, sizes and degree of order of crystals within single scales provide useful insights for understanding the mechanisms at play in the formation of these biophotonic nanostructures.

Orientador: Carlos Lenz César Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin Resumo: Técnicas de microscopias ópticas são as principais ferramentas capazes de observar células e tecidos biológicos em tempo real e com mínimo dano. Essa área foi revolucionada recentemente através das microscopias confocais de varredura a laser e as microscopias de óptica não linear, naturalmente confocais. Entre os processos não lineares temos, a fluorescência excitada por dois ou mais fótons, geração de segundo harmônico [Second Harmonic Generation - SHG] e terceiro harmônico [Third Harmonic Generation - THG]. SHG e THG são técnicas de óptica não linear coerentes, não necessitam de marcadores exógenos e permitem reconstrução de imagens em três dimensões com resolução espacial subcelular. As técnicas de fluorescência permitem visualizar estruturas específicas no espaço, mas não permitem discriminar as substâncias químicas nas estruturas celulares, e as técnicas de SHG e THG não possuem especificidade química. Espectroscopia Raman possui especificidade química através das propriedades vibracionais das moléculas e pode ser usada como mecanismo de contraste na aquisição de imagens. Comparada com a espectroscopia/microscopia infravermelho, a microscopia Raman traz a informação das vibrações moleculares do infravermelho para o visível, eliminando os problemas da baixa resolução espacial e opacidade das amostras. Entretanto a baixa sensibilidade dessa técnica implica em tempos de aquisição de imagens muito longos, da ordem de horas, inviabilizando acompanhar a dinâmica de processos celulares em tempo real. Como solução para essa baixa sensibilidade do espalhamento Raman espontâneo, surgiu a microscopia por espalhamento Raman Coerente anti-Stokes [Coherent Anti-Stokes Raman Scattering - CARS]. Comparado com Raman espontâneo, a microscopia CARS representa aumento de 4 a 5 ordens de grandeza na sensitividade da técnica, diminuindo os tempos de aquisição ao ponto de viabilizar a aquisição em taxas de vídeos (mais rápido do que 30 quadros por segundo) e estudos em tempo real. Essa tese é dedicada ao estudo experimental e teórico, assim como de algumas aplicações, das técnicas de óptica não linear, com destaque para processos de óptica não linear coerentes. Apresentamos de forma detalhada três sistemas experimentais para a aquisição de imagens de Raman coerente e um sistema integrado com várias técnicas de óptica não linear. Mostramos as primeiras imagens de CARS realizadas no Brasil. Além do CARS convencional, trabalhamos com outra técnica de CARS de ordem mais alta, o CARS cascata [cascade CARS - CCARS], e, no melhor do nosso conhecimento, apresentamos as primeiras imagens internacionais obtidas com essa metodologia. CCARS aumenta o contraste da técnica CARS, diminuindo o fundo não ressonante, um problema que aflige a comunidade científica dedicada ao uso dessa técnica. Além da diminuição do fundo não ressonante, a emissão do CCARS acontece em um comprimento de onda diferente de qualquer outro efeito não linear coerente, significando um acréscimo de complexidade mínimo para sua detecção quando comparado com o CARS. Por último mostramos algumas aplicações realizadas com o sistema experimental desenvolvido para integrar diversas modalidades ópticas em paralelo, especialmente da geração de harmônicos com a fluorescência excitada por dois fótons e suas variantes, como microscopia de tempo de vida de fluorescência (Fluorescence Lifetime Imaging ¿ FLIM) Abstract: Optical microscopies techniques are the main tools capable of observing cell and biological tissues in real time and with minimum damage. This area have recently been revolutionized by confocal laser scanning microscopies and non-linear microscopies, naturally confocal. Among the non-linear process we have, the two or more photons excited fluorescence, second harmonic generation [SHG] and third harmonic generation [THG]. SHG and THG are coherent nonlinear techniques, they do not require exogenous markers and allow three dimension imaging reconstruction with subcellular resolution. The fluorescence techniques allow visualizing specific structures in space, but do not allow discriminating the chemical substances in cellular structures, SHG and THG techniques do not have chemical specificity. Raman spectroscopy has chemical specificity through the vibrational properties of the molecules and can be used as a contrast mechanism for imaging acquisition. Compared to infrared spectroscopy/microscopy, Raman microscopy brings information about molecular vibration from infrared to visible, eliminating the low resolution and sample opacity problems. However, this technique low sensibility implies in very long imaging acquisition times, order of hours, making it not viable for following cellular process dynamics in real time. As an answer for the spontaneous Raman scattering low sensibility, the coherent anti-Stokes Raman scattering [CARS] emerged. Compared to spontaneous Raman, CARS microscopy presents an increase of 4 to 5 orders of magnitude in the sensitivity of the technique, lowering the acquisition times to the point of making video acquisition (faster than 30 frames per second) and real time studies possible. This thesis is dedicated to the experimental and theoretical study, as well as some applications, of the non-linear techniques, with emphasis on coherent non-linear optical processes. We present in detailed form three experimental systems for the acquisition of coherent Raman images, and a system with the integration of various non-linear techniques. We show the first CARS images acquired in Brazil. In addition to conventional CARS, we worked with other higher order CARS technique, the cascade CARS [CCARS], and, in the best of our knowledge, we present the first international image acquired with this methodology. CCARS increases the contrast from CARS technique, decreasing the non-resonant background, a problem that afflicts the scientific community dedicated to the use of this technique. Besides the decrease of the non-resonant background, the CCARS emission occurs in a different wavelength from any other non-linear coherent effect, meaning a minimum complexity increase for its detection when compared with CARS. Finally we show some applications performed with the experimental system developed to integrate several optical modalities in parallel, especially the generation of harmonics with two photons excitation fluorescence and their variants such as Fluorescence Lifetime Imaging [FLIM] Doutorado Física Doutor em Ciências CAPES 830406/2010

We report the generation of subdiffractive laser light focal spots in far-field regime, by means of the so-called Optical Eigenmodes (OEi) technique applied to the focusing properties of a single valve of Arachnoidiscus genus diatom. Diatoms are ubiquitous microalgae whose external walls are able to confine light in several hot-spots. We demonstrated that the presence of the valve brings a significant contribution to the OEi-induced light squeezing, improving its performance.

It is known that a properly arranged distribution of nanoholes on a metallic slab is able to produce, in far field conditions, light confinement at sub-diffraction and even sub-wavelength scale. The same effect can also be implemented by the use of Optical Eigenmode (OEi) technique. In this case, a spatial light modulator (SLM) encodes phase and amplitudes of N probe beams whose interference is able to lead to sub-wavelength confinement of light focused by an objective. The OEi technique has been already used in a wide range of applications, such as photoporation, confocal imaging, and coherent control of plasmonic nanoantennas. Here, we describe the application of OEi technique to a single valve of a marine diatom. Diatoms are ubiquitous monocellular algae provided with an external cell wall, the frustule, made of hydrated porous silica which play an active role in efficient light collection and confinement for photosynthesis. Every frustule is made of two valves interconnected by a lateral girdle band. We show that, applying OEi illumination to a single diatom valve, we can achieve unprecedented sub-diffractive focusing for the transmitted light.

Orientador: Cristiano de Mello Gallep Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Tecnologia Resumo: A emissão fotônica ultrafraca proveniente de plântulas de trigo foi mensurada em várias séries de testes sob condições constantes, no interior de câmara escura. Grãos brasileiros também foram transportados e testados na Alemanha, em séries simultâneas realizadas paralelamente a séries conduzidas tanto no Brasil quanto na Alemanha. As plântulas em ambos os países apresentaram ritmos semicircadianos, coincidentes e em sincronia com os ritmos da variação da aceleração gravimétrica local, produzidos pelos movimentos lunissolares. São apresentadas análises matemáticas a respeito destas sincronias e coincidências. Em mais de 80% do período de testes considerados o coeficiente local de correlação de Pearson entre os dois parâmetros revelou relação significante (P>0,7). Com o uso da transformada rápida de Fourier foram evidenciadas as similaridades entre as componentes temporais de oscilação do parâmetro fotônico e as encontradas no parâmetro gravimétrico. As fases da lua e a época do ano possivelmente produzem influências relevantes no desenvolvimento das plântulas. Os resultados encontrados sustentam a hipótese de que os processos envolvidos com a emissão fotônica em plântulas de trigo sejam regulados pelos ritmos de variação gravimétrica, produzidos pelos movimentos lunissolares. A técnica de detecção de emissão fotônica espontânea se mostrou uma ferramenta inovadora e útil para estudos biofísicos e cronobiológicos Abstract: The ultra-weak photon emission from wheat seedling was measured in several series of tests under constant conditions inside a dark chamber. Brazilian seeds were also transported and tested in Germany, with simultaneous series conducted both in Brazil and in Germany. The seedlings showed, in both countries, semi-circadian rhythms coincident and in synchrony with the rhythms of local gravimetric variations of acceleration produced by lunisolar movements. Mathematical analysis of these synchronies and coincidences are presented. The local Pearson correlation coefficient between the two parameters showed a significant relationship (P> 0.7) in more than 80% of the considered testing period. The rhythmic similarities between the biophotonic oscillation and the gravimetric parameter were done by fast Fourier transform. The phases of the moon and the time of year possibly produce relevant influences on development of seedlings. The results support the hypothesis that processes involved with biophoton emission in wheat seedlings may be regulated by variations on the gravimetric rhythms in accordance to lunisolar movements. Further studies are desirable in order to look for the physiological origin of this phenomenon. The spontaneous ultra-weak photon emission detection technique proved to be a new and useful tool for chrono-biological and biophysical studies Mestrado Tecnologia e Inovação Mestre em Tecnologia

Valves of Coscinodiscus wailesii diatoms, monocellular micro-algae characterized by a diameter between 100 and 200 μm, show regular pores patterns which confine light in a spot of few μm2. This effect can be ascribed to the superposition of diffracted wave fronts coming from the pores on the valve surface. We studied the transmission of partially coherent light, at different wavelengths, through single valves of Coscinodiscus wailesii diatoms. The spatial distribution of transmitted light strongly depends on the wavelength of the incident radiation. Numerical simulations help to demonstrate how this effect is not present in the ultraviolet region of the light spectrum, showing one of the possible evolutionary advantages represented by the regular pores patterns of the valves. Valves of Coscinodiscus wailesii diatoms, monocellular micro-algae characterized by a diameter between 100 and 200 mu m, show regular pores patterns which confine light in a spot of few mu m(2). This effect can be ascribed to the superposition of diffracted wave fronts coming from the pores on the valve surface. We studied the transmission of partially coherent light, at different wavelengths, through single valves of Coscinodiscus wailesii diatoms. The spatial distribution of transmitted light strongly depends on the wavelength of the incident radiation. Numerical simulations help to demonstrate how this effect is not present in the ultraviolet region of the light spectrum, showing one of the possible evolutionary advantages represented by the regular pores patterns of the valves.

A new approach to environmental monitoring is presented in this work, related to the use of a biophotonic instrument to support evaluation of lichens stress status. Lichens are widely used as bioindicators, that is organisms able to indicate the presence and amount of atmospheric pollutants. The proposed approach requires the measure of lichens fluorescence response by means of a self-developed biophotonic instrument, in order to obtain an objective evaluation of lichens stress status. The lichens rise fluorescence response was measured, following the OJIP procedure, a standardized laboratory method for plants health analysis. The experimental validation of this approach was done in two steps. Firstly, lichens of "Physcia Adscendens" species were collected on poplar trees, in tree areas: the first and the second areas near an industrial zone, direction of the prevailing winds, the third at a distance of about ten kilometers. Then the changes in fluorescence activity were evaluated, placing near an industrial area several samples of "Flavoparmelia Caperata (L.) Hale" lichens, collected in a non-polluted area. A fluorescence response modification was observed, demonstrating that changes in atmospheric pollution could be monitored by means of differential measurements on suitable samples. To validate the obtained results, the pollution degree was estimated using also a widely accepted method, which is the evaluation of lichen biodiversity. Lichen vegetation of the poplar trees was surveyed using a sampling grid of ten units. The Lichen Biodiversity Index (LBI) was calculated as the sum of the frequencies of all species within the grid. As a result of the comparison between fluorescence response shape and LBI evaluation, a real correlation was found, demonstrating the proposed approach effectiveness.

Nowadays biological sensing is representing a very active area of research due to the many possible applications in environmental control, food testing, pharmaceutical screening, and genetic analysis. A particular need exists for miniature biosensors for the detection of proteins, DNA, toxins and, more generally, infectious agents. Several optical techniques have proven to be quite effective, and biophotonics is a continuously growing discipline. Here we provide a quick overview of the recent progresses in the development of optical biosensors based on resonant cavities constituted by dielectric microspheres, where light propagation occurs through whispering-gallery modes (WGMs). The effect of any perturbation to the optical resonance structure of a microsphere is such that a very high sensitivity can be achieved.