Fluorescence Spectral Imaging Research Articles

Multimodal near infrared spectral imaging as an exploratory tool for dysplastic esophageal lesion identification

We explore nine different combinations of fluorescence, light scattering, and polarization spectral imaging approaches in the near-infrare spectral region toward the diagnosis of pathologic and normal esophageal lesions. The combinations of all the imaging techniques were evaluated for maximal sensitivity and specificity. The results suggest that this multimodal approach is capable of highly accurate detection of the presence of pathologic tissue.

Combined AFM and confocal fluorescence microscope for applications in bio‐nanotechnology

We present a custom-designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near-infrared spectral region. Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high-resolution topographical imaging of AFM with the reliable (bio)-chemical identification capability of optical methods. The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects. The modular design allows easy switching between different modes of operation such as tip-scanning, sample-scanning or mechanical manipulation, all of which are combined with synchronous optical detection. We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution. Examples of several modes of operation of the AFFM are shown using two-dimensional crystals and membranes containing light-harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides.

Nondestructive Fingerprinting of Ivories Using Fourier Transform Imaging Fluorescence and Raman Spectral Imaging

Fingerprinting of ivory products is of considerable importance for both commercial and forensic reasons. An attempt at achieving nondestructive fingerprinting using a combination of optical methods is presented. It is based on Fourier transform imaging fluorescence (FTIF) and on micro‐Raman imaging. Although, fluorescence and Raman spectra of ivories have been studied, this is the first application of the imaging versions of these techniques. It is shown that the combination of the proposed spectral imaging methods may provide unique fingerprinting of the studied samples. The large range of the Raman spectra and the unique method that provides full fluorescence spectrum at each pixel, ensures proper fingerprinting of a variety of ivory samples.

Spectral Fluorescence Imaging for Quantitative Diagnosis and Mapping of Dental Fluorosis Affected Tooth Surfaces

A new analytical method for quantitative diagnosis of dental fluorosis is suggested. The method is based on the spectral modifications in the fluorescence light emitted from affected enamel surfaces, relative to normal dental enamel. Imaging of the tooth surface is obtained using an imaging Fourier transform fluorescence spectrophotometer. This technique provides simultaneous fluorescence spectra at all pixels of the examined area. Images and the corresponding spectra were acquired at various tooth regions, and it was shown that normal, white opaque, brown discolored, and pitted tooth surfaces have different distinct spectral features which characterize the different degrees of the observed pathology of dental fluorosis. Criteria for quantitative assessment of the dental fluorosis stages were suggested. These were based on the measured spectral emission ratio at two wavelengths and comparison with the spectrum of normal enamel. The suggested parameters were validated against clinical observation of numerous samples affected by dental fluorosis at various pathological stages. Besides the introduction of the quantitative assessment, the method is suggested for detection of early changes in the characteristics of the tooth surface in dental fluorosis. The associated mapping capability allows for morphological characterization, which provides additional important clinical information.

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.

Distribution of metal-free sulfonated phthalocyanine in subcutaneously transplanted murine tumors

Metal-free sulfonated phthalocyanine with the average number of sulfonate groups per molecule 2.4 (H 2PcS 2.4) was recently proved to be an efficient photosensitizer for the photodynamic therapy. Fluorescence spectral imaging microscopy was applied here to study localization and relative concentration of H 2PcS 2.4 with micron-scale resolution in subcutaneously transplanted murine tumors: Ehrlich mammary gland carcinoma (EC), Lewis lung carcinoma (LLC), P388 lymphoid leukemia (P388) and B16 melanoma (B16). The study of cryogenic tissue sections prepared 24 h after H 2PcS 2.4 intravenous injection revealed that H 2PcS 2.4 was present in all tissue structures in the monomeric photoactive state. The preferential accumulation of H 2PcS 2.4 was documented in tumor cells and adjacent non-tumor tissues (skin structures, fatty tissue, connective tissue enriched in fibrous component and infiltrated with fibroblasts and macrophages) for all the studied tumor models. P388 and B16 were stained with H 2PcS 2.4 less than adjacent skin structures, whereas EC and LLC accumulated H 2PcS 2.4 alike or higher than particular skin structures. Staining of EC and LLC was similar and ca. 1.4 and 2 times higher than that of B16 and P388, respectively, thus revealing the differences in ability of particular tumor strains to H 2PcS 2.4 accumulation. The H 2PcS 2.4 concentration in remote healthy tissues (skin, muscles and connective tissue) was 2–3 times lower as compared with the analogous tissue structures from the tumor area, whereas subcutaneous fatty tissue staining did not depend on the tissue-to-tumor distance. The tissue distribution of H 2PcS 2.4 predefines the combined action of two photodynamic damage mechanisms: eradication of tumor due to the direct tumor cell destruction and suppression of tumor growth due to the injury of growth supporting system.

Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy.

Summary Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region of a small maize GTPase (ROP7) and yellow fluorescent protein (YFP) was fused to the N-myristoylation motif of the calcium-dependent protein kinase 1 (LeCPK1) of tomato. Upon co-expressing in cowpea protoplasts a perfect co-localization at the plasma membrane of the constructs was observed. Acceptor-photobleaching FRET microscopy indicated a FRET efficiency of 58% in protoplasts co-expressing CFP-Zm7hvr and myrLeCPK1-YFP, whereas no FRET was apparent in protoplasts co-expressing CFP-Zm7hvr and YFP. Fluorescence spectral imaging microscopy (FSPIM) revealed, upon excitation at 435 nm, strong YFP emission in the fluorescence spectra of the protoplasts expressing CFP-Zm7hvr and myrLeCPK1-YFP. Also, fluorescence lifetime imaging microscopy (FLIM) analysis indicated FRET because the CFP fluorescence lifetime of CFP-Zm7hvr was reduced in the presence of myrLeCPK1-YFP. A FRET fluorescence recovery after photobleaching (FRAP) analysis on a partially acceptor-bleached protoplast co-expressing CFP-Zm7hvr and myrLeCPK1-YFP revealed slow requenching of the CFP fluorescence in the acceptor-bleached area upon diffusion of unbleached acceptors into this area. The slow exchange of myrLeCPK1-YFP in the complex with CFP-Zm7hvr reflects a relatively high stability of the complex. Together, the FRET data suggest the existence of plasma membrane lipid microdomains in cowpea protoplasts.

Facile and Stable Dispersion of Carbon Nanotubes into a Hydrogel Composed of a Low Molecular-weight Gelator Bearing a Tautomeric Dye Group

Abstract It was found that single-walled carbon nanotubes are stably dispersed into a low molecular-weight hydrogel (β-d-glucopyranoside–azonaphthol conjugate). From absorption and fluorescence spectral observations and microscopic images, it was shown that this gelator tends to orientate on the surface of SWNTs and specifically emits fluorescence light.

Thermal Imaging for the Study of Plant Water Relations

The use of infrared thermography for the study of plant water relations is reviewed. Increases in leaf temperature detected by thermography largely reflect stomatal closure as a measure of “stress.” Increases in leaf temperature can therefore be used as an indicator for irrigation scheduling. Most other uses of thermography in plant and crop science, for example as a diagnostic tool for a number of plant diseases and environmental pollutants, also depend on the indirect effects of these stressors on stomatal aperture and transpiration rate. Advances in digital image analysis are providing new ways automating image collection and analysis of specific areas within images and for large numbers of images. In spite of the power of infrared thermography, its value for diagnosis and quantification of plant stress will be greatly aided by its use in combination with other sensing techniques such as spectral reflectance or fluorescence imaging.

Fluorescence spectral imaging of dihydroxyacetone on skin in vivo.

Dihydroxyacetone (DHA) has been proposed as a potential alternative to dansyl chloride for use as a fluorescence marker on skin to assess stratum corneum turnover time in vivo. However, the fluorescence from DHA on skin has not been adequately studied. To address this void, a noninvasive, noncontact spectral imaging system is used to characterize the fluorescence spectrum of DHA on skin in vivo and to determine the optimal wavelengths over which to collect the DHA signal that minimizes the contributions from skin autofluorescence. The DHA-skin fluorescence signal dominates the 580-680 nm region of the visible spectrum when excited with ultraviolet radiation in the 320-400 nm wavelength region (UVA). An explanation of the time-dependent spectral features is proposed in terms of DHA polymerization and binding to skin.

Fluorescence spectral imaging for characterization of tissue based on multivariate statistical analysis.

A novel spectral imaging method for the classification of light-induced autofluorescence spectra based on principal component analysis (PCA), a multivariate statistical analysis technique commonly used for studying the statistical characteristics of spectral data, is proposed and investigated. A set of optical spectral filters related to the diagnostically relevant principal components is proposed to process autofluorescence signals optically and generate principal component score images of the examined tissue simultaneously. A diagnostic image is then formed on the basis of an algorithm that relates the principal component scores to tissue pathology. With autofluorescence spectral data collected from nasopharyngeal tissue in vivo, a set of principal component filters was designed to process the autofluorescence signal, and the PCA-based diagnostic algorithms were developed to classify the spectral signal. Simulation results demonstrate that the proposed spectral imaging system can differentiate carcinoma lesions from normal tissue with a sensitivity of 95% and specificity of 93%. The optimal design of principal filters and the optimal selection of PCA-based algorithms were investigated to improve the diagnostic accuracy. The robustness of the spectral imaging method against noise in the autofluorescence signal was studied as well.

Spectral imaging fluorescence microscopy.

The spectral resolution of fluorescence microscope images in living cells is achieved by using a confocal laser scanning microscope equipped with grating optics. This capability of temporal and spectral resolution is especially useful for detecting spectral changes of a fluorescent dye; for example, those associated with fluorescence resonance energy transfer (FRET). Using the spectral imaging fluorescence microscope system, it is also possible to resolve emitted signals from fluorescent dyes that have spectra largely overlapping with each other, such as fluorescein isothiocyanate (FITC) and green fluorescent protein (GFP).

The Arabidopsis kinase-associated protein phosphatase controls internalization of the somatic embryogenesis receptor kinase 1.

The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does not interact with a catalytically inactive kinase mutant. Using mutant AtSERK1 proteins in which Thr 462, Thr 463, and Thr 468 in the A-loop of the AtSERK1 kinase domain were replaced by alanines, we show that phosphorylation status of the receptor is involved in interaction with KAPP. KAPP and AtSERK1 cDNAs were fused to two different variants of green fluorescent protein (GFP), the yellow fluorescent protein (YFP) or the cyan fluorescent protein (CFP). Both KAPP and AtSERK1 proteins are found at the plasma membrane. Our results show that AtSERK1-CFP becomes sequestered into intracellular vesicles when transiently coexpressed with KAPP-YFP proteins. AtSERK1(T463A)-CFP and AtSERK1(3T-->A)-CFP proteins were partially sequestered intracellularly in the absence of KAPP-YFP protein, suggesting an active role for KAPP dephosphorylation of threonine residues in the AtSERK1 A-loop in receptor internalization. The interaction between the KAPP-CFP/YFP and AtSERK1-CFP/YFP fusion proteins was investigated with fluorescence spectral imaging microscopy (FSPIM). Our results show that AtSERK1-CFP and KAPP-YFP proteins are colocalized at the plasma membrane but only show fluorescence energy transfer (FRET) indicative of physical interaction in intracellular vesicles. These results suggest that KAPP is an integral part of the AtSERK1 endocytosis mechanism.

Subcellular Localization and Oligomerization of the Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase 1 Protein

The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 ( AtSERK1) gene is expressed in developing ovules and early embryos. AtSERK1 is also transiently expressed during somatic embryogenesis. The predicted AtSERK1 protein contains an extracellular domain with a leucine zipper motif followed by five leucine-rich repeats, a proline-rich region, a single transmembrane region and an intracellular kinase domain. The AtSERK1 cDNA was fused to two different variants of green fluorescent protein (GFP), a yellow-emitting GFP (YFP) and a cyan-emitting GFP (CFP), and transiently expressed in both plant protoplasts and insect cells. Using confocal laser scanning microscopy it was determined that the AtSERK1-YFP fusion protein is targeted to plasma membranes in both plant and animal cells. The extracellular leucine-rich repeats, and in particular the N-linked oligosaccharides that are present on them appear to be essential for correct localization of the AtSERK1-YFP protein. The potential for dimerization of the AtSERK1 protein was investigated by measuring the YFP/CFP fluorescence emission ratio using fluorescence spectral imaging microscopy. This ratio will increase due to fluorescence resonance energy transfer if the AtSERK1-CFP and AtSERK1-YFP fusion proteins interact. In 15 % of the cells the YFP/CFP emission ratio for plasma membrane localized AtSERK1 proteins was enhanced. Yeast-protein interaction experiments confirmed the possibility for AtSERK1 homodimerization. Elimination of the extracellular leucine zipper domain reduced the YFP/CFP emission ratio to control levels indicating that without the leucine zipper domain AtSERK1 is monomeric.

Photoangioplasty with local motexafin lutetium delivery reduces macrophages in a rabbit post-balloon injury model.

Motexafin lutetium (Lu-Tex, Antrin Injection) is a photosensitizer that selectively accumulates in atheromatous plaque where it can be activated by far-red light. The localization and retention of intra-arterially administered Lu-Tex and its efficacy following activation by endovascularly delivered light (photoangioplasty) was evaluated. Bilateral iliac artery lesions were induced in 17 rabbits by balloon denudation, followed by a high cholesterol diet. Lu-Tex distribution within the atheroma was examined (n=8) following local injection. Fluorescence spectral imaging and chemical extraction techniques were used to measure Lu-Tex levels within the atheroma and adjacent normal tissue. Photoactivation was performed 15 min following Lu-Tex administration (180 J/cm fiber at 200 mW/cm fiber). Two weeks post photoangioplasty, vessels were harvested and hematoxylin and eosin (H&E) and RAM11 (macrophages) staining was performed. Local delivery of Lu-Tex achieved immediate high concentrations within plaque (mean 40x control iliac atheroma). Mean percent plaque area in the treated segments was significantly lower than in the non-treated contralateral lesions (73 vs. 82%, P<0.01). No medial damage was observed. Quantitative analysis using RAM11 positive cells revealed significant reduction of macrophages in treated lesions in both the intima (5 vs. 22%, P<0.01) and in media (8 vs. 23%, P<0.01) compared to untreated contralateral segments. Local delivery provides high levels of Lu-Tex selectively within atheroma. Photoactivation results in a significant decrease in macrophage and a small decrease in atheroma burden without damage to the normal vessel wall.

Optical Absorption and Fluorescence Spectral Imaging Using Fiber Bundle Image Compression

Optical absorption and fluorescence spectral imaging is performed by using the recently developed fiber-bundle image compression (FIC) technique in which an entire spectral image is collected in a single scan of a charged-coupled device (CCD) detector. Absorption imaging is demonstrated by mapping the optical absorbance of a stained microscopic lily plant stem section. Fluorescence imaging is demonstrated by mapping shifts in the ruby R1 fluorescence line to determine the pressure distribution in a microcrystalline ruby powder squeezed between two diamond surfaces. The advantages and limitations of the FIC method, relative to tunable filter and line imaging techniques, are discussed.

High-speed spectral imager for imaging transient fluorescence phenomena

We describe fluorescence spectral imaging results with the microscope computed-tomography imaging spectrometer (muCTIS). This imaging spectrometer is capable of recording spatial and spectral data simultaneously. Consequently, muCTIS can be used to image dynamic phenomena. The results presented consist of proof-of-concept imaging results with static targets composed of 6-mum fluorescing microspheres. Image data were collected with integration times of 16 ms, comparable with video-frame-rate integration times. Conversion of raw data acquired by the muCTIS to spatial and spectral data requires postprocessing. The emission spectra were sampled at 10-nm intervals between 420 and 710 nm. The smallest spatial sampling interval presented is 1.7 mum.

Spatial Distribution of Phenolic Materials in Durum Wheat Grain as Probed by Confocal Fluorescence Spectral Imaging

Microspectrofluorometry has been employed to study the spatial distribution of phenolic material in cereal grain. Transverse sections of the grain were used for the spectral characterisation of different molecular species present inTriticum durumgrains. Auto-fluorescence emission spectra were recorded on micro regions of each section. The analysis of the whole set of spectra permitted the characterisation of three principal spectral features; indicators of phenolics in specific regions of wheat sections. The comparison with model reference spectra showed that spectral components could be attributed to ferulic and p-coumaric acids. Using these spectral components, spectral fluorescence imaging was performed allowing the relative fluorescence intensity of each phenolic feature to be mapped. Images generated were used for the generation of the 3D organisation of auto-fluorescent phenolic materials within the grain. This new and rapid method was further used for the spectral characterisation of the various aleurone cell walls with high sensitivity. Analysis of the data showed that outer and inner aleurone cell walls exhibited similar fluorescence profiles but with significantly different intensities.

The kinetics of protoporphyrin fluorescence during ALA-PDT in human malignant skin tumors

Fluorescence monitoring during photodynamic therapy (PDT) with the use of topical 5-aminolevulinic acid (ALA) was carried out in patients bearing superficial and nodular basal cell carcinomas (BCC), squamous cell carcinomas (SCC) and Kaposi's sarcomas. A new diagnostic-therapeutic system based on an incoherent CW light source was used for fluorescence spectral measurements and imaging. The results showed that photoirradiation reduced ALA-induced protoporphyrin IX (PP) fluorescence in all tumors. The rate of PP photobleaching in superficial BCC and SCC tumors was significantly higher than in large nodular BCC tumors. The results showed that the differences in kinetics of fluorescence reduction could be attributed to the tumor thickness. One hour after photoirradiation with a light dose of 170 J/cm 2 a phenomenon of re-appearance and recovery of PP fluorescence was observed in the large deeply penetrating BCC tumors and Kaposi's sarcoma lesions. In such cases an additional light treatment was performed. The results of the study demonstrated that fluorescence monitoring is very appropriate for the definition of an optimal ALA-PDT clinical protocol.

Microspectroscopic imaging of nodulation factor-binding sites on living Vicia sativa roots using a novel bioactive fluorescent nodulation factor

A novel bioactive fluorescent nodulation (Nod) factor, NodRlv-IV(BODIPY FL-C16), has been synthesized by attaching a BODIPY FL-C16 acyl chain to the primary amino group of chitotetraose deacetylated at the nonreducing terminus by recombinant NodB. The binding of the fluorescent Nod factor to root systems of Vicia sativa was investigated with fluorescence spectral imaging microscopy (FSPIM) and fluorescence ratio imaging microscopy (FRIM). Spatially resolved fluorescence spectra of living and labeled Vicia sativa root systems were measured by FSPIM. Strong autofluorescence, inherent to many plant systems when excited at 488 nm, was corrected for by utilizing the difference in fluorescence emission spectra of the autofluorescence and NodRlv-IV(BODIPY FL-C16). A methodology is presented to break down the in situ fluorescence emission spectra into spatially resolved autofluorescence and BODIPY FL fluorescence spectra. Furthermore, an FRIM method was developed for correcting autofluorescence in fluorescence micrographs for this system. After autofluorescence correction it was shown that NodRlv-IV(BODIPY FL-C16) was concentrated in the root hairs, but was also bound to other parts of the root surface.