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On the contrary, fluorophores are vulnerable to photobleaching at relatively low fluence To demonstrate this, we performed repeated serial imaging of reflectance and fluorescence on a fluorescent microsphere. By contrast, fluorescence decreased exponentially with a fluence half-life of 3.

We further questioned whether reflectophores are practically applicable in scattering biological media. We compared reflectance with fluorescence in a scattering polymer matrix encapsulating fluorescent reflectophores Fig. The SNR was obtained from the average intensity divided by the standard deviation of background noise. In agreement with our previous characterization Fig.

Interferometer(Principle And Working)

Notably, reflectophores showed higher SNRs, conceivably because of the concentrated signal at the centroid Fig. To further evaluate the applicability in biological tissues, we mounted reflectophores beneath a cortical brain slice of a Thy1-YFP mouse and performed a SpeRe measurement Fig. The results collectively indicated that reflectophores can be adapted in turbid biological media. Reflectophore in turbid media. Dashed lines indicate the best-fit to the exponential decay R 2 : 0. The fibrous structures in the reflectance image are myelinated axons because of their unique multilayered cytoarchitecture.

To compensate the depth-dependent signal attenuation, intensity was normalized for each slice.

Interferometry

Due to their micron-scale size, conventional intracellular delivery methods, such as passive diffusion or electrophoresis, cannot be used with reflectophores. Alternatively, it has been reported that both phagocytic and non-phagocytic cells can actively uptake microparticles via endocytosis 33 , 34 , We validated that serial phase-contrast images provide a robust quantification of intracellular uptake by showing that the cell-impermeant fluorescent streptavidin does not stain intracellular biotinylated reflectophores Supplementary Fig.

To our surprise, we observed that the surface coating significantly affected uptake efficiency: amine-functionalized reflectophores showed an over two-fold higher uptake compared to bare and biotinylated reflectophores Fig. Intracellular reflectophores. Arrowheads indicate the reflectophore under endocytosis.

The error bar represents standard error of the mean. Hela cells loaded with reflectophores were subsequently cultured over several days to form a tumor spheroid. The solid sinusoidal curves are the best-fit simulation spectra R 2 : 0. The dotted lines indicate the means. Grayscale image indicates MitoTracker fluorescence and pseudo-colored traces represent time-series tracking of individual reflectophores over time.

As expected, cell division introduced the segregation of intracellular reflectophores into two daughter cells. Although rare, we also observed that reflectophores escaped the cell i. We did not observe any signs of cellular damage such as membrane permeabilization, blebbing, or apoptosis by intracellular reflectophore in three cell lines Supplementary Fig. Collectively, these results illustrate that reflectophores can be rationally designed to be internalized into cells, and intracellular reflectophores do not significantly interfere with cellular physiology.

Picometre level displacement interferometry

Next, we questioned whether differently sized reflectophores could serve as multiplexed labeling for tracing individual cells, as also proposed in intracellular microlasers To address this, we prepared multicellular spheroids using reflectophore-loaded cells Fig. Last, we tested the potential of optical barcoding for tracking migrating cells Fig.

We observed no significant change in migration activity by intracellular reflectophores Supplementary Fig. We next tracked individual migrating cells in the process of forming capillary-like structures with reflectophore barcoding. We reasoned that the nanoscale precision realized may enable the sensing of macromolecules adhered to the reflectophores via specific biological interactions.

Molecular adsorption forms an additional dielectric thin-film layer with a thickness corresponding to the size of the macromolecule, which can be detected by SpeRe Fig. In our numerical simulation, we estimated that the binding of streptavidin leads to a spectral redshift i. We used fluorescent streptavidin to independently confirm its binding to the reflectophore Fig. After binding, we observed a robust redshift of 0.


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Microenvironmental sensing. Note that fluorescence was localized specifically at the surface of the reflectophore. The arrows indicate the spectral peaks. Data are represented as mean open circle and the standard deviation shaded region was acquired from 30 repeated SpeRe measurements. The information encoded by reflectophores is the optical distance, thus, the change in refractive index, as well as size, can be detected at high precision. To demonstrate this alternative sensing principle, we prepared a liquid-crystal reflectophore via self-assembly of a nematic liquid crystal into a droplet in a polymer matrix Fig.

The sample was then sandwiched between conductive indium-tin-oxide glasses and connected to a function generator to control the external electric field during optical sensing. When there was no external electric field, liquid-crystal reflectophores exhibited radially ordered structures, which was apparent in bright-field and polarization microscopy Fig. In SpeRe measurements, we observed that the measured spectra shifted toward longer wavelengths at higher electric fields corresponding to the higher refractive index, consistent with the theoretical prediction Fig.

We reported on the reflectophore, a novel interferometric optical probe that encodes information by optical distance i. Furthermore, the variable refractive index of a liquid-crystal reflectophore enabled the sensing of local electric fields. Our theoretical and experimental studies demonstrated that reflectophores have several compelling advantages. First, reflectophores have orders-of-magnitude higher brightness compared to typical fluorophores because of the high reflective cross-sections of their spherical geometry. The bright signal localized at the centroid provides high SNR for reliable spectral detection even in scattering media Fig.

The spherical shape also ensures orientation-independent readouts for applications in three-dimensional tissues, which is in stark contrast to previously reported barcodes requiring a certain readout angle Second, reflectophores exhibit negligible photobleaching. This enables a practically unlimited number of readouts as well as long-term information storage.

Changes in medium composition, which are often unavoidable in applications in living systems, only affect the magnitude of reflectivity but not its spectral shape, thereby providing robust readouts Supplementary Fig. Last, as shown in our studies using a fluorescent microsphere, reflectophores can be synergistically integrated with existing fluorophores to take advantage of both modalities.

The signals are easily separable in the spectral domain because of the Stokes shift in fluorophores and also in the spatial domain because of the center localization in reflectophores. This feature provides an additional degree-of-freedom in design, for example, to increase the multiplexing capacity for barcoding or to incorporate multiple sensors.

Reflectophores do, however, have shortcomings. Thus, reflectophores are not suitable for labeling individual proteins or subcellular organelles. In addition, the large size may interfere with cellular physiology when it is introduced into cells. Although we observed that intracellular reflectophores do not significantly interfere with viability, migration, and proliferation in multiple cell lines, interference to the biological process of interest should be verified for each experimental context.

Applications in extracellular space in tissues or cell-free systems are relatively free from this issue. Next, for high-degree multiplexing, fabrication of various microspheres at nanoscale precision are required and is technically challenging. Ranges of nanotechnologies are available but may have low throughput and are costly 46 , As a potential solution, bulk polydisperse microspheres may be sorted based on our SpeRe-based readout, like the widely adapted fluorescent-assisted cell sorter.

Last, our current readout system is rather slow as it involves volumetric scanning for locating the geometric center of a reflectophore. Since off-centered acquisitions are ignored, an advanced scanning algorithm to efficiently find the centers will dramatically accelerate acquisition speed. Alternatively, reflectophores could be physically aligned through optical or magnetic tweezers. Although our studies only exemplified reflectophores made with certain materials e.

Recently, a plethora of hydrogels equipped with various functionalities were developed for bio-integration Notably, stimuli-responsive hydrogels can change their physical size in response to specific chemical or physical milieu such as pH, temperature, and electric field.

Introduction

Reflectophores made with these stimuli-responsive materials could function as bio-integrable sensors. It may also be possible to fabricate multilayered reflectophores to incorporate multi-functional sensing capabilities. Moreover, chemical engineering on the surface offers an additional route for functionalization such as affinity-based molecular sensing, improved biocompatibility, and targeting of a specific cell type We envision that the adoption of advanced knowledge in material science and chemistry will vastly expand the utilities of reflectophores.

The refractive index spectra for water and silica was provided by the toolbox and that of polystyrene was adopted from a previous study SpeRe measurements were performed on a customized galvanometer-based laser scanning microscope H, Cambridge Technology; Supplementary Fig. For the input source, a supercontinuum white light laser EXB-6, NKT photonics was attenuated using a neutral density filter 3.


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  7. The reflected light was spatially filtered by a confocal pinhole 0. Prairie software Bruker was used for controlling the scanner and triggering the spectrometer. The spectrum corresponding to the geometric center of the microsphere was acquired at the pixel with the maximum intensity Supplementary Fig. If the geometric center was not within the scanning volume, the dataset was excluded from the analysis.

    The spectrum was subsequently normalized by the reference spectrum obtained with a protected silver mirror PF05—P01, Thorlabs at the sample stage. Fluorescent microspheres , Thermo Fisher Scientific immersed in 0. To gauge fluorescence levels, we compared the fluorescence obtained with the microsphere with that of a known dye, fluorescein isothiocyanate-dextran , Sigma , at the same optical gain and excitation intensity.

    A standard calibration kit —01C, Ted Pella was used for scale calibration. To compare the intensity and SNR attenuation of SpeRe and fluorescent, a depth-dependent image was acquired. The SNR was obtained by the mean intensity divided by the standard deviation of the background signal. All animal experiments were performed in compliance with institutional guidelines and approved by the subcommittee for research animal care at Sungkyunkwan University. For the biotin-coated microspheres, we afterwards confirmed their intracellular localization by taking fluorescence images after adding 0.

    For the migration assay, 3. Negative staining of fluorescent streptavidin 0. The microspheres were placed within the wells of the micromesh. We typically chose the microspheres adhered near the edge of the micromesh because of their positional stability even during solvent exchange.

    After the baseline spectral acquisition at a spectral resolution of 0. We used GraphPad Prism for statistical analyses. Unpaired t -tests two-sided were used for group comparisons by assuming normality, based on previous literature. Neither randomization nor blinding was applied. For regression analysis, we present the correlation coefficient R 2 along with the sample size. All relevant data are available from the corresponding author upon request.

    A reporting summary for this Article is available as a Supplementary Information file. The source data underlying Figs. Chalfie, M. Green fluorescent protein as a marker for gene expression. Science , — Specht, E. A critical and comparative review of fluorescent tools for live-cell imaging. Tsien, R. The green fluorescent protein. Lichtman, J. Fluorescence microscopy. Methods 2 , — Garland, M.

    A bright future for precision medicine: advances in fluorescent chemical probe design and their clinical application. Cell Chem. Germond, A. Design and development of genetically encoded fluorescent sensors to monitor intracellular chemical and physical parameters. Vendrell, M. Combinatorial strategies in fluorescent probe development. Resch-Genger, U. Quantum dots versus organic dyes as fluorescent labels. Methods 5 , — Jaiswal, J. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Zimmermann, T.

    Spectral imaging and linear unmixing in light microscopy. Lu, Y. Tunable lifetime multiplexing using luminescent nanocrystals. Photonics 8 , 32—36 Submicrometer metallic barcodes. Lee, H. Colour-barcoded magnetic microparticles for multiplexed bioassays. Lee, J. Universal process-inert encoding architecture for polymer microparticles. Braeckmans, K. Encoding microcarriers by spatial selective photobleaching. Pregibon, D.

    Multifunctional encoded particles for high-throughput biomolecule analysis. Han, M. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Lin, C. Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA. McGloin, D. Biophotonics: cellular lasers. Photonics 9 , — Humar, M. Intracellular microlasers. Heylman, K. Optical microresonators for sensing and transduction: a materials perspective.

    Schubert, M. Lasing within live cells containing intracellular optical microresonators for barcode-type cell tagging and tracking.


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    6. Nano Lett. Lasing in live mitotic and non- phagocytic cells by efficient delivery of microresonators. Silicon chips detect intracellular pressure changes in living cells. Shambat, G. There the two beams overlap and constructive and destructive interference occurs depending on the relative phase shift between the two plane waves.

      Figure 1: Schematic of the Michelson interferometer. Each beam experiences a phase shift of when they are reflected at the mirror surfaces M1 and M2 and an additional phase shift due to the propagation through the glass of the beam splitter and the partial reflection at the semi-transparent surface. The total phase shift between the two beams therefore is given by:. Here is the wave number and the wave length of the light.

      You will observe constructive interference when the with. The number of changes is given by:.

      Fabry and Perot's interferometer | Opinion | Chemistry World

      Therefore this instruments allows you to observe distance changes of the order of the wavelength of light. If light travels in a medium its speed is smaller than in vacuum. The ratio between the speed of light in vacuum and the speed of light in a medium is give by the index of refraction.

      As a consequence if light travels a distance in a medium it experiences a phase shift where and , the wavelength of light in a medium is shorter that in vacuum. This property is later used to determine the index of refraction of air. The main parts are shown in Fig. If you have to tighten set screws use only two fingers and do not apply a lot of force or you might crack a lens or a mirror. The various optical parts are labeled. If you are unsure ask your instructor or learning assistant for help. Figure 3: Setup of the lens and mirror supports. The index of refraction of air is very close to one.

      It is a good approximation to assume that the deviation from one is proportional to the density of air:. Assuming that the temperature is constant and using and one obtains:.