An official website of the United States government
Multiplexed fluorescence detection has become increasingly important in the fields of biosensing and bioimaging. Although a variety of excitation/detection optical designs and fluorescence unmixing schemes have been proposed to allow for multiplexed imaging, rapid and reliable differentiation and quantification of multiple fluorescent species at each imaging pixel is still challenging. Here we present a pulsed interleaved excitation spectral fluorescence lifetime microscopic (PIE-sFLIM) system that can simultaneously image six fluorescent tags in live cells in a single hyperspectral snapshot. Using an alternating pulsed laser excitation scheme at two different wavelengths and a synchronized 16-channel time-resolved spectral detector, our PIE-sFLIM system can effectively excite multiple fluorophores and collect their emission over a broad spectrum for analysis. Combining our system with the advanced live-cell labeling techniques and the lifetime/spectral phasor analysis, our PIE-sFLIM approach can well unmix the fluorescence of six fluorophores acquired in a single measurement, thus improving the imaging speed in live-specimen investigation.
more »
« less
Toward implantable devices for angle-sensitive, lens-less, multifluorescent, single-photon lifetime imaging in the brain using Fabry–Perot and absorptive color filters
Abstract Implantable image sensors have the potential to revolutionize neuroscience. Due to their small form factor requirements; however, conventional filters and optics cannot be implemented. These limitations obstruct high-resolution imaging of large neural densities. Recent advances in angle-sensitive image sensors and single-photon avalanche diodes have provided a path toward ultrathin lens-less fluorescence imaging, enabling plenoptic sensing by extending sensing capabilities to include photon arrival time and incident angle, thereby providing the opportunity for separability of fluorescence point sources within the context of light-field microscopy (LFM). However, the addition of spectral sensitivity to angle-sensitive LFM reduces imager resolution because each wavelength requires a separate pixel subset. Here, we present a 1024-pixel, 50 µm thick implantable shank-based neural imager with color-filter-grating-based angle-sensitive pixels. This angular-spectral sensitive front end combines a metal–insulator–metal (MIM) Fabry–Perot color filter and diffractive optics to produce the measurement of orthogonal light-field information from two distinct colors within a single photodetector. The result is the ability to add independent color sensing to LFM while doubling the effective pixel density. The implantable imager combines angular-spectral and temporal information to demix and localize multispectral fluorescent targets. In this initial prototype, this is demonstrated with 45 μm diameter fluorescently labeled beads in scattering medium. Fluorescent lifetime imaging is exploited to further aid source separation, in addition to detecting pH through lifetime changes in fluorescent dyes. While these initial fluorescent targets are considerably brighter than fluorescently labeled neurons, further improvements will allow the application of these techniques to in-vivo multifluorescent structural and functional neural imaging.
more »
« less
- Award ID(s):
- 1706207
- PAR ID:
- 10341932
- Date Published:
- Journal Name:
- Light: Science & Applications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2047-7538
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Colorimetric sensors offer the prospect for on-demand sensing diagnostics in simple and low-cost form factors, enabling rapid spatiotemporal inspection by digital cameras or the naked eye. However, realizing strong dynamic color variations in response to small changes in sample properties has remained a considerable challenge, which is often pursued through the use of highly responsive materials under broadband illumination. In this work, we demonstrate a general colorimetric sensing technique that overcomes the performance limitations of existing chromatic and luminance-based sensing techniques. Our approach combines structural color optical filters as sensing elements alongside a multichromatic laser illuminant. We experimentally demonstrate our approach in the context of label-free biosensing and achieve ultrasensitive and perceptually enhanced chromatic color changes in response to refractive index changes and small molecule surface attachment. Using structurally enabled chromaticity variations, the human eye is able to resolve ∼0.1-nm spectral shifts with low-quality factor (e.g., Q ∼ 15) structural filters. This enables spatially resolved biosensing in large area (approximately centimeters squared) lithography-free sensing films with a naked eye limit of detection of ∼3 pg/mm 2 , lower than industry standard sensors based on surface plasmon resonance that require spectral or angular interrogation. This work highlights the key roles played by both the choice of illuminant and design of structural color filter, and it offers a promising pathway for colorimetric devices to meet the strong demand for high-performance, rapid, and portable (or point-of-care) diagnostic sensors in applications spanning from biomedicine to environmental/structural monitoring.more » « less
-
Schmidt, Dirk; Schreiber, Laura; Vernet, Elise (Ed.)Inner working angle is a key parameter for enabling scientific discovery in direct exoplanet imaging and characterization. Approaches to improving the inner working angle to reach the diffraction limit center on the sensing and control of wavefront errors, starlight suppression via coronagraphy, and differential techniques applied in post-processing. These approaches are ultimately limited by the shot noise of the residual starlight, placing a premium on the ability of the adaptive optics system to sense and control wavefront errors so that the coronagraph can effectively suppress starlight reaching the science focal plane. Photonic lanterns are attractive for use in the science focal plane because of their ability to spatially filter light using a finite basis of accepted modes and effectively couple the results to diffraction-limited spectrometers, providing a compact and cost-effective means to implement post-processing based on spectral diversity. We aim to characterize the ability of photonic lanterns to serve as focal-plane wavefront sensors, allowing the adaptive optics system to control aberrations affecting the science focal plane and reject additional stellar photon noise. By serving as focal-plane wavefront sensors, photonic lanterns can improve sensitivity to exoplanets through both direct and coronagraphic observations. We have studied the sensing capabilities of photonic lanterns in the linear and quadratic regimes with analytical and numerical treatments for different lantern geometries (including non-mode-selective, mode-selective, and hybrid geometries) as a function of port number. In this presentation we report on the sensitivity of such lanterns and comment on the relative suitability and sensitivity impacts of different lantern geometries for focal-plane wavefront sensing.more » « less
-
Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well-developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite the development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes, and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that are solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several S. pombe mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in S. cerevisiae, with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.more » « less
-
Förster resonance energy transfer (FRET) is a valuable tool for measuring molecular distances and the effects of biological processes such as cyclic nucleotide messenger signaling and protein localization. Most FRET techniques require two fluorescent proteins with overlapping excitation/emission spectral pairing to maximize detection sensitivity and FRET efficiency. FRET microscopy often utilizes differing peak intensities of the selected fluorophores measured through different optical filter sets to estimate the FRET index or efficiency. Microscopy platforms used to make these measurements include wide-field, laser scanning confocal, and fluorescence lifetime imaging. Each platform has associated advantages and disadvantages, such as speed, sensitivity, specificity, out-of-focus fluorescence, and Zresolution. In this study, we report comparisons among multiple microscopy and spectral filtering platforms such as standard 2-filter FRET, emission-scanning hyperspectral imaging, and excitation-scanning hyperspectral imaging. Samples of human embryonic kidney (HEK293) cells were grown on laminin-coated 28 mm round gridded glass coverslips (10816, Ibidi, Fitchburg, Wisconsin) and transfected with adenovirus encoding a cAMP-sensing FRET probe composed of a FRET donor (Turquoise) and acceptor (Venus). Additionally, 3 FRET “controls” with fixed linker lengths between Turquoise and Venus proteins were used for inter-platform validation. Grid locations were logged, recorded with light micrographs, and used to ensure that whole-cell FRET was compared on a cell-by-cell basis among the different microscopy platforms. FRET efficiencies were also calculated and compared for each method. Preliminary results indicate that hyperspectral methods increase the signal-to-noise ratio compared to a standard 2-filter approach.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1038/s41377-022-00708-9
