Hyperspectral fluorescence microscopy pdf

Detection of cuticle defects on cherry tomatoes using. 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. Description nist has developed a laboratory dedicated to measuring the optical properties of materials through the use of commercial and custom hyperspectral imagers. Compressive fluorescence microscopy for biological and hyperspectral imaging. Compressive fluorescence microscopy for biological and. Fluorescence microscopy is a technique most commonly applied to biological samples, many of which. Stennis space center, mississippi the system is a single hyperspectral imaging instrument that has the unique capability to acquire both fluorescence and reflectance highspatialresolution. In both imaging modalities the acquisition is independent of the information. See below a system configuration for hyperspectral imaging of macroscale samples that do not require microscopy level imaging. Elliott2, liang gao1, alessandro ustione2, noah bedard1, robert kester1, david w. Hyperspectral imaging, like other spectral imaging, collects and processes information from across the electromagnetic spectrum. Excitationscanning hyperspectral imaging microscopy to. Hyperspectral imaging fluorescence excitation scanning spectral characteristics of remodeled mouse arteries.

Abstract hyperspectral imaging hsi techniques play an important role in the. Hyperspectral confocal fluorescence imaging of cells. Fluorescence microscopy an overview sciencedirect topics. Hyperspectral microscopy for luminescence and raman imaging. Realtime hyperspectral fluorescence imaging of pancreatic bcell.

For instance, the spatial resolution of a hyperspectral confocal fluorescence microscopy used by graus, neumann, and timlin was 0. 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. In cfm, the sample is excited with a patterned illumination and its fluorescence is collected on a point detector. In fluorescence microscopy, differently coloured fluorophores are used to selectively label structures. 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. Osa information theoretic analysis of hyperspectral. As an application, we consider fluorescence imaging and show how. 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. 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.

We have developed a new, high performance, hyperspectral microscope for biological and other applications. Understanding the principles underlying fluorescence microscopy is useful when. A hyperspectral fluorescence imaging system for biological. The microscope features a fully confocal design to ensure high spatial resolution and high quality optical. We concede the limitations of the approach described in this work. Hyperspectral fluorescence and reflectance imaging instrument. The hyperspectral fluorescence imaging experiments were implemented on a nikon. 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. However, the need to acquire images with everincreasing size and spatial resolution and spectral information puts considerable constraints on the acquisition process, particularly on. Hyperspectral imagery hsi microscopy allows for vegetation and geologic material to be examined at the subcentimeter spatial scale. Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescentlylabeled cells in highly autofluorescent tissue.

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. Abstract we are developing a hyperspectral imaging system aimed at imaging fluorescent molecules in two different contexts. The hfm microscope has been tested on a mixture of fluorescent beads and cells that have been labeled with one and two different fluorophores. Hyperspectral fluorescence microscopy based on compressed sensing. Na, which determines the spatial resolution of a fhmi system. The main concept is to add high resolution spectral information to the intensity data to improve imaging capabilities. Optimizing imaging parameters for the separation of multiple labels in a fluorescence image. The user can interrogate these data for pure component. For example, a hyperspectral image can contain a variety of dyed or stained tissues or fluorescent cell components.

Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum. Ima is an ultrafast and allinone customizable hyperspectral microscopy platform of high spatial and spectral resolution. Sandia to demonstrate hyperspectral confocal fluorescence. Numerical aperture na is an important parameter for microscope objective, and the minimal resolvable distance is 0. Multimodal hyperspectral optical microscopy sciencedirect. Fluorescence microscopy is a powerful and widely used technique in cellular and molecular biology research. We investigate the linear mixing model as a special case and define a linear unmixing performance bound by using the cramerrao inequality. Hyperspectral imaging microscopy for identification and. 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.

Compressive fluorescence microscopy for biological and hyperspectral imaging vincent studer y, jerome bobinz, makhlad chahid,s. In both imaging modalities the acquisition is independent of the information content of the image. 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. Lenslet array tunable snapshot imaging spectrometer latis. Fluorescence emission is nearly simultaneous with the absorption of the. Introduction when organic or inorganic specimens absorb and subsequently reradiate light, the process is typically a result of fluorescence or phosphorescence. Department of biomedical engineering, washington university in st. Additionally, we provide a framework for prototyping and testing new sonification methods and a mathematical model. Snapshot hyperspectral imaging augments pixel dwell time and acquisition speeds over existing scanning systems, making it a powerful tool for fluorescence microscopy.

Pdf hyperspectral fluorescence microfluidic hfm microscopy. Thinfilm tunable filters for hyperspectral fluorescence. Larger wavelength bands were selected for the hyperspectral images to cover the full emission band of each type of singlewalled carbon nanotube swcnt. Fluorescence microscopy is a crucial tool for fundamental and applied biomedical research. 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. Hyperspectral fluorescence imaging for molecular biology. Because of its high spectral resolution less than lonm, hfm has found many applications in spectral imaging of living cells 69. Hyperspectral fluorescence and reflectance imaging instrument a single system contains spatialscanning, illumination, and spectralimaging subsystems. 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.

The resulting hyperspectral fluorescence image contains both spatial and spectral information about the sample. Sonification of hyperspectral fluorescence microscopy datasets summary. 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. The analytical tools that could identify and quantitatively map metabolites in unprocessed human tissues with submicrometer resolution are highly desired. 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. Pdf hyperspectral fluorescence microscopy based on. Osa lenslet array tunable snapshot imaging spectrometer.

Confocal fluorescence imaging of biological systems is an important method by which researchers can investigate molecular processes occurring in live cells. One of the major drawbacks with the current technology is the inability. Multicolor fluorescent imaging by spaceconstrained. Hyperspectral fluorescence microscopy hfm acquires many spectral bands of a scene to create hyperspectral datacubes x,y. Spring national institutes of health, bethesda, maryland, u. The system employs a unique hyperspectral fluorescence camera to acquire images. Our results indicate that hyperspectral microscopy has both increased sensitivity and specificity, compared to singleband microscopy methods. Information theoretic analysis of hyperspectral imaging. Biophysical comparison of four silver nanoparticles coatings. Pdf hyperspectral fluorescence microscopy based on compressed sensing maxime dahan academia.

Objects flowing in a microfluidic device are excited by a 50 mw diodepumped solid state laser emitting at. 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 microscopy is widely used in biology and medicine, as well as in other fields. In fluorescence microscopy, the spectra for the differently colored fluorescent chemical compounds in the specimen fluorophores of interest are the endmembers. Hyperspectral imaging, fluorescent biosensors, image mapping spectrometer, ims. Pdf compressive fluorescence microscopy for biological. 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. Hyperspectral fluorescence microscopy based on compressed. Fluorescence imaging is complicated by the spectral proximity of fluorophores 5 and the use of many fluorophores in a single scene 6,7. Download fulltext pdf download fulltext pdf download fulltext pdf hyperspectral light sheet microscopy article pdf available in nature communications 6. Bordeaux, interdisciplinary institute for neuroscience, umr 5297, f33000, bordeaux, france.

The macrophor hyperspectral fluorescence imaging system is optimized for samples that are too large for standard microscope analysis. Sonification of hyperspectral fluorescence microscopy. As an application, we consider fluorescence imaging and show how the. Journal of cell science realtime hyperspectral fluorescence imaging of pancreatic bcell dynamics with the image mapping spectrometer amicia d. 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. Additionally, we provide a framework for prototyping and testing new sonification methods and a mathematical model to point out scenarios where. Hyperspectral fluorescence and reflectance imaging.

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. Hyperspectral imaging was originally developed for remote sensing 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. Elemental analysis integrated raman and hyperspectral microscopy. Realtime hyperspectral fluorescence imaging of pancreatic b. Pdf hyperspectral reflectance and fluorescence imaging system. Hyperspectral confocal fluorescence microscopy, when combined with multivariate curve resolution mcr, provides a powerful new tool for improved quantitative imaging of multifluorophore samples. Hyperspectral fluorescence microfluidic hfm microscopy giuseppe di caprio,1, diane schaak, 1 and ethan schonbrun 1rowland institute at harvard, harvard university, 100 e. Compressive fluorescence microscopy for biological and hyperspectral imaging article pdf available in proceedings of the national academy of sciences 10926. Hyperspectral stimulated raman scattering microscopy unravels. Hyperspectral microscopy utilises 18 spectral channels. Preprocessing visualization of hyperspectral fluorescent. In vivo 3d molecular imaging of mice and multifluorophore imaging of 2d tissue samples. Here, hyperspectral confocal fluorescence microscopy was employed to noninvasively probe the locale of tolyporphins within live cells under.

Here, we present a hyperspectral light sheet microscope that acquires spectral information at. Vincent studera,b, jerome bobinc, makhlad chahida,b. Figure 2 the spectral and structural properties of fluorophores. We have developed a new 3d hyperspectral confocal fluorescence microscope that can further enhance the usefulness of fluorescence microscopy in studying biological systems.

Pdf this article presents a laboratorybased hyperspectral imaging system designed and developed by the instrumentation and sensing laboratory, u. Realtime hyperspectral fluorescence imaging of pancreatic. Deep learning enhanced hyperspectral fluorescence lifetime. Hyperspectral light sheet microscopy nature communications. Hyperspectral fluorescence microfluidic hfm microscopy. 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. Based on the fluorescence emission spectra, the representative hyperspectral fluorescence images of the cherry tomatoes with defects are illustrated in fig.

The new microscope can increase the information content. 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. Hyperspectral fluorescence microscopy hfm is an emerging field based on hyperspectral or multispectral imaging concepts, which often borrow from remote sensing techniques 4, 5. A diffractive unit separates the spectral components of the incoming light spatially. Hyperspectral fluorescence imaging for molecular biology applications a major qualifying project submitted to the faculty of the worcester polytechnic institute. With the spectral information, hyperspectral imaging approaches are capable of unambiguously identifying fluorophores with overlapping spectra and permitting. Hyperspectral confocal fluorescence microscopy, when combined with multivariate curve resolution mcr, provides a powerful new tool for improved quantitative imaging of. The completely integrated system rapidly maps diffuse reflectance, transmittance, photoluminescence, electroluminescence and fluorescence in the visnirswir spectral range.

In fluorescence microscopy, one can distinguish two kinds of imaging approaches, wide field and raster scan microscopy, differing by their excitation and detection scheme. Edgepreserving nonnegative deconvolution of hyperspectral. The fluorescence signal is descanned onto a single line with a second scan mirror. Snapshot imagemapping spectrometer for hyperspectral fluorescence microscopy article pdf available in optics and photonics news 2112. Elemental analysis integrated raman and hyperspectral. Hyperspectral microscopy and corresponding spectral analysis are powerful methods for detecting weak fluorescent signals within a high autofluorescence background. Thinfilm tunable filters for hyperspectral fluorescence microscopy. Fluorescence microscopy bio 5068 molecular cell biology. Principles of hyperspectral microscope imaging techniques and. 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. In this paper, we have demonstrated a hyperspectral microscope, called the hfm microscope, for analyzing fluorescence in cells travelling through microfluidic channels.