Intravital microscopy using multiphoton processes is the standard tool for deep tissue imaging inside of biological specimens. Usually, near-infrared and infrared light is used to excite the sample, which enables imaging several mean free path inside a scattering tissues. Using longer wavelengths, however, increases the width of the effective multiphoton Point Spread Function (PSF). Many features inside of cells and tissues are smaller than the diffraction limit, and therefore not possible to distinguish using a large PSF. Microscopy using high refractive index microspheres has shown promise to increase the numerical aperture of an imaging system and enhance the resolution. It has been shown that microspheres can image features ~λ/7 using single photon process fluorescence. In this work, we investigate resolution enhancement for Second Harmonic Generation (SHG) and 2-photon fluorescence microscopy. We used Barium Titanate glass microspheres with diameters ∼20–30 μm and refractive index ∼1.9–2.1. We show microsphere-assisted SHG imaging in bone collagen fibers. Since bone is a very dense tissue constructed of bundles of collagen fibers, it is nontrivial to image individual fibers. We placed microspheres on a dense area of the mouse cranial bone, and achieved imaging of individual fibers. We found that microsphere assisted SHG imaging resolves features of the bone fibers that are not readily visible in conventional SHG imaging. We extended this work to 2-photon microscopy of mitochondria in mouse soleus muscle, and with the help of microsphere resolving power, we were able to trace individual mitochondrion from their ensemble.
more »
« less
Refractive index dispersion measurement in the short-wave infrared range using synthetic phase microscopy
Refractive index is an optical property explored in the light scattering measurement of micro- and nano-particles as well as in label-free imaging of cells and tissues. Because the refractive index value is a major input to the characterization and quantification of the analyzed specimens, various methods have been developed targeting at different sample types. In this paper, we demonstrate a technique for the refractive index measurement of homogeneous microspheres and liquids in the short-wave infrared (SWIR) range. We use synthetic phase microscopy (SPM), which records a scattering-corrected projection of the 3D refractive index distribution, in combination with a least-squares fitting to a theoretical model of a sphere. Using the method, we determine the refractive index dispersion of two polymer microspheres (polymethyl methacrylate and polystyrene), two glass microspheres (silica and soda lime), and three microscopy mounting media (glycerol, FluorSave, and Eukitt) in the SWIR range of 1100–1650 nm.
more »
« less
- Award ID(s):
- 1808331
- PAR ID:
- 10453018
- Date Published:
- Journal Name:
- Physical Chemistry Chemical Physics
- Volume:
- 25
- Issue:
- 34
- ISSN:
- 1463-9076
- Page Range / eLocation ID:
- 23141 to 23149
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Spectroscopic microtomography provides the ability to perform 4D (3D structural and 1D chemical) imaging of a thick microscopic specimen. Here, we demonstrate spectroscopic microtomography in the short-wave infrared (SWIR) wavelength using digital holographic tomography, which captures both the absorption coefficient and refractive index. A broadband laser in tandem with a tunable optical filter allows us to scan the wavelength from 1100 to 1650 nm. Using the developed system, we measure human hair and sea urchin embryo samples. The resolution estimated with gold nanoparticles is 1.51 μm (transverse) and 1.57 μm (axial) for the field of view of 307 × 246 μm2. The developed technique will enable accurate and efficient analyses of microscopic specimens that have a distinctive absorption or refractive index contrast in the SWIR range.more » « less
-
null (Ed.)Microsphere Photolithography (MPL) is a nanopatterning technique that utilizes a self-assembled monolayer of microspheres as an optical element to focus incident radiation inside a layer of photoresist. The microspheres produces a sub-diffraction limited photonic-jet on the opposite side of each microsphere from the illumination. When combined with pattern transfer techniques such as etching/lift-off, MPL provides a versatile, low-cost fabrication method for producing hexagonal close-packed metasurfaces. This article investigates the MPL process for creating refractive index (RI) sensors on the cleaved tips of optical fiber. The resonant wavelength of metal elements on the surface is dependent on the local dielectric environment and allows the refractive index of an analyte to be resolved spectrally. A numerical study of hole arrays defined in metal films shows that the waveguide mode provides good sensitivity to the analyte refractive index. This can be readily tuned by adjusting the MPL exposure and the simulation results guide the fabrication of a defect tolerant refractive index sensor on the tip of a fiber tip with a sensitivity of 613 nm/RIU. The conformal nature of the microsphere monolayer simplifies the fabrication process and provides a viable alternative to direct-write techniques such as Focused Ion Beam (FIB) millingmore » « less
-
Holographic particle characterization uses in-line holographic microscopy and the Lorenz-Mie theory of light scattering to measure the diameter and the refractive index of individual colloidal particles in their native dispersions. This wealth of information has proved invaluable in fields as diverse as soft-matter physics, biopharmaceuticals, wastewater management, and food science but so far has been available only for dispersions in transparent media. Here, we demonstrate that holographic characterization can yield precise and accurate results even when the particles of interest are dispersed in turbid media. By elucidating how multiple light scattering contributes to image formation in holographic microscopy, we establish the range conditions under which holographic characterization can reliably probe turbid samples. We validate the technique with measurements on model colloidal spheres dispersed in commercial nanoparticle slurries.more » « less
-
Abstract A sensing platform is presented that uses dielectric Huygens source metasurfaces to measure refractive index changes in a microfluidic channel with experimentally measured sensitivity of 323 nm RIU−1, a figure of merit (FOM) of 5.4, and a response of 8.2 (820%) change in transmittance per refractive index unit (T/RIU). Changes in the refractive index of liquids flown through the channel are measured by single‐wavelength transmittance measurement, requiring only a simple light source and photodetector, significantly reducing device expense in comparison to state‐of‐the‐art refractive index sensing technologies. A technoeconomic analysis predicts a device costing ≈$2400 that is capable of detecting refractive index changes of the order of 2*10−8. The metasurfaces utilized are low profile, scalable, and use materials and fabrication processes compatible with CMOS and other technologies making them suitable for device integration. The Huygens metasurface system, characterized by spectrally overlapping electric and magnetic dipole modes, offers a high degree of customizability. Interplay between the two resonances may be controlled via metasurface geometry, leading to tunability of device sensitivity and measurement range. Ultrahigh sensitivity of 350 nm RIU−1with FOM of 219, corresponding to single‐wavelength sensitivity of 360 RIU−1, is demonstrated computationally through use of antisymmetric resonances of a Huygens metasurface illuminated at small incidence angles.more » « less
An official website of the United States government

