Multicolor fluorescent imaging by space-constrained computational spectral imaging
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Spectral imaging is a powerful technique used to simultaneously study multiple fluorophore labels with overlapping emissions. Here, we present a computational spectral imaging method, which uses sample spatial fluorescence information as a reconstruction constraint. Our method addresses both the under-sampling issue of compressive spectral imaging and the low throughput issue of scanning spectral imaging. With simulated and experimental data, we have demonstrated the reconstruction precision of our method in two and three-color imaging. We have experimentally validated this method for differentiating cellular structures labeled with two red-colored fluorescent proteins, tdTomato and mCherry, which have highly overlapping emission spectra. Our method has the advantage of totally free wavelength choice and can also be combined with conventional filter-based sequential multi-color imaging to further improve multiplexing capability.Keywords:
Fluorescence-lifetime imaging microscopy
One important challenge for in-vivo imaging fluorescence in cancer research and related pharmaceutical studies is to discriminate the exogenous fluorescence signal of the specific tagged agents from the natural fluorescence. For mice, natural fluorescence is composed of endogenous fluorescence from organs like the skin, the bladder, etc. and from ingested food. The discrimination between the two kinds of fluorescence makes easy monitoring the targeted tissues. Generally, the amplitude of the fluorescence signal depends on the location and on the amount of injected fluorophore, which is limited in in-vivo experiments. This paper exposes some results of natural fluorescence analysis from in-vivo mice experiments using a time domain small animal fluorescence imaging system: eXplore OptixTM. Fluorescence signals are expressed by a Time Point Spread Function (TPSF) at each scan point. The study uses measures of similarity applied purposely to the TPSF to evaluate the discrepancy and/or the homogeneity of scanned regions of a mouse. These measures allow a classification scheme to be performed on the TPSF's based on their temporal shapes. The work ends by showing how the exogenous fluorescence can be distinguished from natural fluorescence by using the TPSF temporal shape.
Fluorescence-lifetime imaging microscopy
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We describe fluorescence spectral-imaging results with the computed-tomography imaging spectrometer (CTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, the CTIS can be used to image dynamic phenomena involving multiple, spectrally overlapping fluorescence probes. This system is also optimal for simultaneously monitoring changes in spectral characteristics of multiple probes from different locations within the same sample. This advantage will provide additional information about the physiological changes in function form populations of cells which respond in a heterogeneous manner. The results presented in this paper consist of proof-of-concept imaging results from the CTIS in combination with two different systems of fore- optics. In the first configuration, raw image data were collected using the CTIS coupled to an inverted fluorescence microscope. The second configuration combined the CTIS with a confocal microscope equipped with a fiber-optic imaging bundle, previously for in vivo imaging. Image data were collected at frame rates of 15 frame per second and emission spectra were sample at 10-nm intervals with a minimum of 29 spectral bands. The smallest spatial sampling interval presented in this paper is 0.7 micrometers .
Fluorescence-lifetime imaging microscopy
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Frame rate
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Ion concentrations in biological cells are widely studied with fluorescent probes. The probes have a high selectivity for specific ions and exhibit marked changes in their photophysical properties upon binding ions. The fluorescence decay behavior of the probes in the presence of ions can now be used as a contrast mechanism for imaging purposes. This technique can be further exploited for the quantitative determination of ion concentrations within living cells. Here we describe the fluorescence lifetime properties of the free calcium probe CalciumGreen and the pH probe carboxy SNAFL-1. The potential of fluorescence lifetime imaging is illustrated by the imaging of Ca2+ concentrations and pH in single cells. In the case of the emission ratio probe c.SNAFL-1, it was possible to determine the pH in the same cell using both the ratio and the fluorescence lifetime method. It turns out that no cumbersome in vivo calibration procedure is required when c.SNAFL-1 is used for quantitative fluorescence lifetime imaging of pH in single cells.
Fluorescence-lifetime imaging microscopy
Live cell imaging
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Multiplexed fluorescence detection has become an indispensable tool in modern biosensing and imaging. Although a variety of excitation/detection optics designs and unmixing schemes have been proposed to achieve multiplexed detection, successful differentiation and quantification of multiple fluorophores at each imaging pixel is still challenging. Recently, fluorescence lifetime imaging microscopy (FLIM) in combination with the phasor plot analysis has shown many advantages over other multiplexed detection methods. Being an intrinsic property of a fluorescent molecule, fluorescence lifetime measured by FLIM is not biased by excitation power or probe abundance and can reveal information on the probe's microenvironment (ions, pH, oxygen content, electrical signals, index of refractions, etc.). In addition, FLIM is one of the most robust ways of quantifying FRET for studying protein-protein interactions. Moreover, by combining lifetime information with a spectral reading at each pixel, spectral FLIM adds two more dimensions to the spatiotemporal information collected by a conventional confocal setup, resulting in a 6-dimenional (x, y, z, λ, τ, t) dataset. The major challenges to the spectral FLIM method lie in the data acquisition speed and the complexity in post processing and analysis. Here, we present a new time-resolved spectral detector with parallel 16-channel digital frequency domain FLIM (FastFLIM) readouts, for fast spectral FLIM data acquisition. The 16-channel FastFLIM can produce unbiased spectral FLIM data for phasor analysis that unambiguously discriminate and quantitate fluorescent species with unique spectral and lifetime features at each image pixel. Our spectral FastFLIM method offers new opportunities for monitoring multiple dynamic signaling events in live specimens, providing insights into complex biological systems.
Fluorescence-lifetime imaging microscopy
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Autofluorescence
Fluorescence-lifetime imaging microscopy
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Fluorescence is a very promising radioactive-free technique for functional imaging in small animals and, in the future, in humans. However, most commercial near-infrared dyes display poor optical properties, such as low fluorescence quantum yields and short fluorescence lifetimes. In this paper, we explore whether the encapsulation of infrared cyanine dyes within the core of lipid nanoparticles (LNPs) could improve their optical properties. Lipophilic dialkylcarbocyanines DiD and DiR are loaded very efficiently in 30-35-nm-diam lipid droplets stabilized in water by surfactants. No significant fluorescence autoquenching is observed up to 53 dyes per particle. Encapsulated in LNP, which are stable for more than one year at room temperature in HBS buffer (HEPES 0.02 M, EDTA 0.01 M, pH 5.5), DiD and DiR display far improved fluorescence quantum yields (respectively, 0.38 and 0.25) and longer fluorescence lifetimes (respectively, 1.8 and 1.1 ns) in comparison to their hydrophilic counterparts Cy5 (=0.28, =1.0 ns) and Cy7 (=0.13, =0.57 ns). Moreover, dye-loaded LNPs are able to accumulate passively in various subcutaneous tumors in mice, thanks to the enhanced permeability and retention effect. These new fluorescent nanoparticles therefore appear as very promising labels for in vivo fluorescence imaging.
Cyanine
Nile red
Fluorescence-lifetime imaging microscopy
Indocyanine Green
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Spectrally resolved fluorescence lifetime imaging microscopy (λFLIM) has powerful potential for biochemical and medical imaging applications.However, long acquisition times, low spectral resolution and complexity of λFLIM often narrow its use to specialized laboratories.Therefore, we demonstrate here a simple spectral FLIM based on a solidstate detector array providing in-pixel histrogramming and delivering faster acquisition, larger dynamic range, and higher spectral elements than stateof-the-art λFLIM.We successfully apply this novel microscopy system to biochemical and medical imaging demonstrating that solid-state detectors are a key strategic technology to enable complex assays in biomedical laboratories and the clinic.
Fluorescence-lifetime imaging microscopy
Spectral resolution
Imaging science
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We have developed a multimodal optical system for simultaneous optical coherence tomography (OCT) and fluorescence lifetime imaging microscopy (FLIM), and demonstrate its capability for high-speed co-registered micro-anatomical and biochemical tissue imaging.
Fluorescence-lifetime imaging microscopy
Diffuse optical imaging
Biological Imaging
Optical Tomography
Biological tissue
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We have developed a multimodal optical system for simultaneous optical coherence tomography (OCT) and fluorescence lifetime imaging microscopy (FLIM) imaging, and demonstrate its capability for high-speed co-registered micro-anatomical and biochemical tissue imaging.
Fluorescence-lifetime imaging microscopy
Diffuse optical imaging
Biological Imaging
Optical Tomography
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Modality (human–computer interaction)
Fluorescence-lifetime imaging microscopy
Molecular Imaging
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