Fluorescence imaging is complicated by the spectral proximity of fluorophores 5 and the use of many fluorophores in a single scene 6,7. Hyperspectral fluorescence microscopy based on compressed sensing. Hyperspectral imaging, like other spectral imaging, collects and processes information from across the electromagnetic spectrum. The goal of hyperspectral imaging is to obtain the spectrum for each pixel in the image of a scene, with the purpose of finding objects, identifying materials, or detecting processes. Description nist has developed a laboratory dedicated to measuring the optical properties of materials through the use of commercial and custom hyperspectral imagers. Sonification of hyperspectral fluorescence microscopy datasets summary. Elemental analysis integrated raman and hyperspectral. Based on the fluorescence emission spectra, the representative hyperspectral fluorescence images of the cherry tomatoes with defects are illustrated in fig. Pdf hyperspectral fluorescence microfluidic hfm microscopy. Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescentlylabeled cells in highly autofluorescent tissue. Department of biomedical engineering, washington university in st. Thinfilm tunable filters for hyperspectral fluorescence. Realtime hyperspectral fluorescence imaging of pancreatic bcell. Compressive fluorescence microscopy for biological and hyperspectral imaging vincent studer y, jerome bobinz, makhlad chahid,s.
Compressive fluorescence microscopy for biological and hyperspectral imaging article pdf available in proceedings of the national academy of sciences 10926. Sandia national laboratories will demonstrate a new hyperspectral confocal fluorescence microscope friday, aug. Journal of cell science realtime hyperspectral fluorescence imaging of pancreatic bcell dynamics with the image mapping spectrometer amicia d. Objects flowing in a microfluidic device are excited by a 50 mw diodepumped solid state laser emitting at. The user can interrogate these data for pure component. Multimodal hyperspectral optical microscopy sciencedirect. Hyperspectral microscopy utilises 18 spectral channels. Hyperspectral fluorescence microscopy hfm acquires many spectral bands of a scene to create hyperspectral datacubes x,y. The hyperspectral fluorescence imaging experiments were implemented on a nikon. Detection of cuticle defects on cherry tomatoes using. Osa lenslet array tunable snapshot imaging spectrometer. Understanding the principles underlying fluorescence microscopy is useful when. Na, which determines the spatial resolution of a fhmi system.
Biophysical comparison of four silver nanoparticles coatings. The analytical tools that could identify and quantitatively map metabolites in unprocessed human tissues with submicrometer resolution are highly desired. Elemental analysis integrated raman and hyperspectral microscopy. Confocal fluorescence imaging of biological systems is an important method by which researchers can investigate molecular processes occurring in live cells.
Principles of hyperspectral microscope imaging techniques and. Read hyperspectral fluorescence microscopy based on compressed sensing, proceedings of spie on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. The completely integrated system rapidly maps diffuse reflectance, transmittance, photoluminescence, electroluminescence and fluorescence in the visnirswir spectral range. The fluorescence signal is descanned onto a single line with a second scan mirror. Fluorescence microscopy an overview sciencedirect topics. Hyperspectral microscopy for luminescence and raman imaging. While confocal microscopy is moving to spectral and fastscanning architectures in terms of. In fluorescence microscopy, the spectra for the differently colored fluorescent chemical compounds in the specimen fluorophores of interest are the endmembers. We investigate the linear mixing model as a special case and define a linear unmixing performance bound by using the cramerrao inequality. Abstract hyperspectral imaging hsi techniques play an important role in the. Here, hyperspectral confocal fluorescence microscopy was employed to noninvasively probe the locale of tolyporphins within live cells under. While hyperspectral imaging techniques have been demonstrated in widefield fluorescence microscopy,911 confocal microscopy,1215 and in in vivo fluorescence imaging,1621 a thorough and quantitative comparison between singleband and hyperspectral fluorescence imaging has not been performed. Jun 26, 2012 in this paper, we describe compressive fluorescence microscopy cfm, a unique modality for fluorescence biological and hyperspectral imaging based on the concepts of cs theory.
Thinfilm tunable filters for hyperspectral fluorescence microscopy. Fluorescence microscopy is a technique most commonly applied to biological samples, many of which. For instance, the spatial resolution of a hyperspectral confocal fluorescence microscopy used by graus, neumann, and timlin was 0. Deep learning enhanced hyperspectral fluorescence lifetime. A hyperspectral fluorescence imaging system for biological. We present a general stochastic model for hyperspectral imaging data and derive analytical expressions for the fisher information matrix for the underlying spectral unmixing problem. Hyperspectral microscopy and corresponding spectral analysis are powerful methods for detecting weak fluorescent signals within a high autofluorescence background. Hyperspectral fluorescence and reflectance imaging instrument a single system contains spatialscanning, illumination, and spectralimaging subsystems. Because of its high spectral resolution less than lonm, hfm has found many applications in spectral imaging of living cells 69. Deep learning enhanced hyperspectral fluorescence lifetime imaging marien ochoa 1, alena rudkouskaya 2, ruoyang yao 1, pingkun yan 1, margarida barroso 2, xavier intes 1 acquiring dense highdimensional optical data in biological applications remains a challenge due to the very low levels of light typically encountered. Such a set of images carries information pro pixel close to those collected by drs method in scanning mode, for instance, dimensional maps of hemoglobin oxygen saturation so 2 or total hemoglobin concentration. Cytoviva hyperspectral imaging can be acquired in either the visible nearinfrared vnir 400 nm 1,000 nm wavelength ranges as well as in the short wave infrared swir 900 nm 1,700 nm wavelength ranges. The microscope features a fully confocal design to ensure high spatial resolution and high quality optical.
In vivo 3d molecular imaging of mice and multifluorophore imaging of 2d tissue samples. Osa information theoretic analysis of hyperspectral. We have developed a new, high performance, hyperspectral microscope for biological and other applications. Hyperspectral fluorescence imaging is gaining popularity for it enables multiplexing of spatiotemporal dynamics across scales for molecules, cells and tissues with multiple fluorescent labels. The hfm microscope has been tested on a mixture of fluorescent beads and cells that have been labeled with one and two different fluorophores. Numerical aperture na is an important parameter for microscope objective, and the minimal resolvable distance is 0. Abstract we are developing a hyperspectral imaging system aimed at imaging fluorescent molecules in two different contexts. Hyperspectral fluorescence microfluidic hfm microscopy giuseppe di caprio,1, diane schaak, 1 and ethan schonbrun 1rowland institute at harvard, harvard university, 100 e. Snapshot imagemapping spectrometer for hyperspectral fluorescence microscopy article pdf available in optics and photonics news 2112. Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum.
Hyperspectral confocal fluorescence microscopy, when combined with multivariate curve resolution mcr, provides a powerful new tool for improved quantitative imaging of. Hyperspectral fluorescence and reflectance imaging instrument. As an application, we consider fluorescence imaging and show how. Fluorescence microscopy is widely used in biology and medicine, as well as in other fields. Additionally, we provide a framework for prototyping and testing new sonification methods and a mathematical model.
Hyperspectral fluorescence and reflectance imaging. Pdf compressive fluorescence microscopy for biological. Optimizing imaging parameters for the separation of multiple labels in a fluorescence image. Multicolor fluorescent imaging by spaceconstrained. In both imaging modalities the acquisition is independent of the information. Hyperspectral imagery hsi microscopy allows for vegetation and geologic material to be examined at the subcentimeter spatial scale. Ima is an ultrafast and allinone customizable hyperspectral microscopy platform of high spatial and spectral resolution. The resulting hyperspectral fluorescence image contains both spatial and spectral information about the sample. Hyperspectral fluorescence imaging for molecular biology applications a major qualifying project submitted to the faculty of the worcester polytechnic institute. Hyperspectral light sheet microscopy nature communications. Bordeaux, interdisciplinary institute for neuroscience, umr 5297, f33000, bordeaux, france.
Excitationscanning hyperspectral imaging microscopy to. Realtime hyperspectral fluorescence imaging of pancreatic. A diffractive unit separates the spectral components of the incoming light spatially. As an application, we consider fluorescence imaging and show how the.
Our results indicate that hyperspectral microscopy has both increased sensitivity and specificity, compared to singleband microscopy methods. Fluorescence microscopy is a powerful and widely used technique in cellular and molecular biology research. Spring national institutes of health, bethesda, maryland, u. Figure 2 the spectral and structural properties of fluorophores. One of the major drawbacks with the current technology is the inability of the uv filter cubes in microscopes to separate and discriminate between fluorophores. Edgepreserving nonnegative deconvolution of hyperspectral. Compressive fluorescence microscopy for biological and hyperspectral imaging. We have developed a new 3d hyperspectral confocal fluorescence microscope that can further enhance the usefulness of fluorescence microscopy in studying biological systems. Biophysical comparison of four silver nanoparticles. Realtime hyperspectral fluorescence imaging of pancreatic b. In both imaging modalities the acquisition is independent of the information content of the image. With the spectral information, hyperspectral imaging approaches are capable of unambiguously identifying fluorophores with overlapping spectra and permitting.
Hyperspectral imaging fluorescence excitation scanning spectral characteristics of remodeled mouse arteries. Hyperspectral fluorescence imaging for molecular biology. Hyperspectral imaging, fluorescent biosensors, image mapping spectrometer, ims. Snapshot hyperspectral imaging augments pixel dwell time and acquisition speeds over existing scanning systems, making it a powerful tool for fluorescence microscopy. In cfm, the sample is excited with a patterned illumination and its fluorescence is collected on a point detector. Additionally, we provide a framework for prototyping and testing new sonification methods and a mathematical model to point out scenarios where.
As a compliment to highresolution hyperspectral sensing technology, we have been actively investigating the use of companion fluorescence probes and taggants to provide a versatile and expanded resolution of information with regards to such targets. Download fulltext pdf download fulltext pdf download fulltext pdf hyperspectral light sheet microscopy article pdf available in nature communications 6. Hyperspectral fluorescence microfluidic hfm microscopy. One of the major drawbacks with the current technology is the inability of the uv filter cubes in microscopes to separate and discriminate between fluorophores that emit fluorescence within the same color channel. Although elegant demonstrations of hyperspectral fluorescence,,, and dark field,, microscopy have been previously reported, hyperspectral optical absorption microscopy experiments are scarce, particularly in the realm of biological imaging. In fluorescence microscopy, one can distinguish two kinds of imaging approaches, wide field and raster scan microscopy, differing by their excitation and detection scheme. While hyperspectral imaging techniques have been demonstrated in widefield fluorescence microscopy,911 confocal microscopy,1215 and in in vivo fluorescence imaging,1621 a thorough and quantitative comparison between singleband and hyperspectral fluorescence imaging has. Fluorescence microscopy bio 5068 molecular cell biology. As an alternative, hyperspectral approaches have been developed in confocal microscopy, where a diffraction grating or prism is used to acquire the full spectrum at each point of the image 2. Hyperspectral imaging an overview sciencedirect topics.
Information theoretic analysis of hyperspectral imaging. Hamed shams mousavi, emmanuel candesxand maxime dahan univ. In this paper, we have demonstrated a hyperspectral microscope, called the hfm microscope, for analyzing fluorescence in cells travelling through microfluidic channels. However, the need to acquire images with everincreasing size and spatial resolution and spectral information puts considerable constraints on the acquisition process, particularly on. Sandia to demonstrate hyperspectral confocal fluorescence. Introduction when organic or inorganic specimens absorb and subsequently reradiate light, the process is typically a result of fluorescence or phosphorescence. Fluorescence microscopy is a crucial tool for fundamental and applied biomedical research.
Hyperspectral confocal fluorescence microscopy, when combined with multivariate curve resolution mcr, provides a powerful new tool for improved quantitative imaging of multifluorophore samples. Hyperspectral imaging was originally developed for remote sensing applications. Hyperspectral imaging hsi is a spectral imaging acquisition where each pixel of the image was employed to acquire a set of images within certain spectral bands. To learn more about enhanced darkfield hyperspectral microscopy and how it can improve the development of apis and nano to micronscale delivery vectors or provide an effective quality control tool for existing formulations, you.
Biophysical parameters of cellular uptake and effects included flow cytometry side scatter ssc intensity, nuclear light scatter, cell cycle distributions, surface plasmonic resonance spr, fluorescence microscopy of mitochondrial gross structure, and darkfield hyperspectral imaging. Vincent studera,b, jerome bobinc, makhlad chahida,b. Hyperspectral confocal fluorescence imaging of cells. For each voxel within a threedimensional specimen, the microscope simultaneously records the emission spectrum from 500 nm to 800 nm, with better than 3 nm spectral resolution. Stennis space center, mississippi the system is a single hyperspectral imaging instrument that has the unique capability to acquire both fluorescence and reflectance highspatialresolution. The system employs a unique hyperspectral fluorescence camera to acquire images. However, in the case of hyperspectral fluorescence microscope images acquired with ccdtype technologies, cosmic spikes and the presence of detector artifacts in the spectral data can make the. Compressive fluorescence microscopy for biological and. Preprocessing visualization of hyperspectral fluorescent. We concede the limitations of the approach described in this work. Elliott2, liang gao1, alessandro ustione2, noah bedard1, robert kester1, david w.
Larger wavelength bands were selected for the hyperspectral images to cover the full emission band of each type of singlewalled carbon nanotube swcnt. Here, we implemented analytical hyperspectral stimulated raman scattering microscopy to map the lipid metabolites in situ in normal and cancerous liver tissues from 24 patients. Fluorescence emission is nearly simultaneous with the absorption of the. See below a system configuration for hyperspectral imaging of macroscale samples that do not require microscopy level imaging. Hyperspectral imaging microscopy for identification and. Sonification of hyperspectral fluorescence microscopy. The main concept is to add high resolution spectral information to the intensity data to improve imaging capabilities. In fluorescence microscopy, differently coloured fluorophores are used to selectively label structures. Pdf hyperspectral reflectance and fluorescence imaging system. Lenslet array tunable snapshot imaging spectrometer latis. Pdf hyperspectral fluorescence microscopy based on.
Pdf this article presents a laboratorybased hyperspectral imaging system designed and developed by the instrumentation and sensing laboratory, u. Hyperspectral fluorescence microscopy hfm is an emerging field based on hyperspectral or multispectral imaging concepts, which often borrow from remote sensing techniques 4, 5. Hyperspectral fluorescence microscopy based on compressed. The new microscope can increase the information content. For example, a hyperspectral image can contain a variety of dyed or stained tissues or fluorescent cell components. One of the major drawbacks with the current technology is the inability. Fhmi, fluorescence hyperspectral microscope imaging. The development of multicolored fluorescent proteins, nanocrystals and organic fluorophores, along with the resulting engineered biosensors, has revolutionized the study of protein localization and dynamics in living cells.
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