A photo-convertible protein is found in several species of the coral genus Lobophyllia. Its green fluorescence is
converted to red by irradiation in the 340-400nm range. It also exhibits a wider range of photo-reactive properties,
including a reversible photo-bleaching when in a partially-converted state. We present data on its behaviour under
single and multiphoton irradiation.
Liver fibrosis has many causes, including hepatitis C, alcohol abuse, and non-alcoholic steatohepatitis. It is characterized by abnormal deposition of extracellular matrix proteins, mainly collagen. The deposition of these proteins results in impaired liver function caused by distortion of the hepatic architecture by fibrous scar tissue. The unique triple helix structure of collagen and high level of crystallinity make it very efficient for generating second harmonic signals. In this study we have set out to see if second harmonic imaging of collagen can be used as a non-biased quantitative tool for classification of fibrosis levels in liver biopsies and if it can detect early fibrosis formation not detected by current methods.
A variety of fluorescent and chromophoric proteins homologous to the green fluorescent protein (GFP) has been recently discovered and cloned from non-bioluminescent marine animals, such as corals, and now provide a multitude of colors for use in fluorescence imaging applications. Recently, a novel fluorescence imaging methodology has emerged that utilizes the unique photoactivatory property of several GFP-like proteins, which respond to irradiation by altering their optical properties, thereby providing a new spatio-temporal capability to the GFP-based imaging applications. During our studies of GFP-like proteins from the Great Barrier Reef corals, several novel photoactivatable (PA) GFP-like proteins have been discovered. These include fluorescence photo-amplifiers and reversible photoswitchers, similar to PA jelly-fish derived PA-GFP and Dronpa, that greatly increase their emissions following ultraviolet-A (UVA) irradiation; the red-to-green (R-to-G) converters, similar to DsRed, that rapidly change to green color following single- or 2-photon irradiation; the green-to-red (G-to-R) converters, that acquire bright red fluorescence following UV-violet irradiation, similar to Kaede-like proteins; and the kindling GFP-like proteins, that are non fluorescent, but rapidly acquire bright fluorescence after green light irradiation. We report on the various optical characteristics of these coral PA proteins that may be used to expand the scope of the available fluorescence bio-imaging technologies.
We report the two photon luminescence (TPL) and second harmonic generation (SHG) characteristics of Zinc Oxide (ZnO) in ceramic, thick film and nano-rod. All samples were prepared from commercially available analytical pure ZnO powder. Sintering, physical vapour deposition (PVD), and hydrothermal methods were used in preparing the three types of samples respectively. The comparison among the three showed, while a degree of similarities between the ceramic and the nano-rod, a significant difference in the film. Possible reasons for the wavelength downshift in the film sample are discussed. Images acquired by TPL and SHG microscopy are presented, both ceramic and film samples show granular structure and a reverse bright-dark contract was observed from TPF to SHG image between the grain region and the granular boundaries.
Reef-building corals are dependent on dinoflagellate algal symbionts (zooxanthellae). Within the range of habitats of any one coral species there can be huge variations in light intensities, so there is a risk of photoinhibition from excess light. In extremes of light and heat, senescent algae are expelled en masse, a phenomenon known as coral bleaching. In freshly isolated tissue the chlorophyll fluorescence has a lifetime of ~1.1ns. 6 hours and 15 hours after isolation the zooxanthellae looked visually healthy, but the lifetimes had increased to 2ns after 6 hours and 2.2ns after 15 hours. Zooxanthellae which were visibly damaged or necrotic had a mean lifetime of 3ns. Lifetime of chlorophyll fluorescence is thus a sensitive indicator, revealing effects in cell metabolism before any structural changes are evident. The occurrence of FRET between fluorescent proteins in corals has already been reported and time-resolved spectra have shown the effect on fluorescent lifetime, but without any spatial resolution. Lifetime confocal microscopy offers lower time resolution but excellent spatial resolution. Lifetimes of the isolated A. millepora pigments amilFP490, amilFP504 and amilFP593 (names indicate emission peaks) were 2.8ns, 2.9ns and 2.9ns respectively. In the coral sample, imaging the entire emission spectrum from 420nm, the mean lifetime was reduced to 1.5ns, implying that FRET was occurring. Looking just at the fluorescence from FRET donors the lifetime was even shorter, at 1.3ns, supporting this interpretation.
The application of second-harmonic generation (SHG) microscopy to plant materials has been neglected hitherto even though it would seem to have promise for identification and characterization of biologically and commercially important plant polysaccharides. We find that imaging of cellulose requires rather high laser powers, which are above optimal values for live cell imaging. Starch, however, is easily imaged by the technique at laser fluences compatible with extended cell viability. This also has useful applications in imaging plant-derived starchy food products. Lignin in plant cell walls shows a strong three-photon excited fluorescence, which may be enhanced by resonance effects.
Second harmonic generation (SHG) results from molecules being polarized by an external electric field, often provided by an intense laser beam. The polarizability depends on firstly the intrinsic structural properties of the substance and hence the second order nonlinear susceptibility, and secondly the intensity and polarization direction of the incident light. The polarization characteristics of the beam are therefore of interest. In this paper, we discuss some considerations in SHG microscopy when the incoming beam is circularly polarized, and present some supporting results as well as a question for future work.
The application of second harmonic generation (SHG) microscopy to plant materials has been neglected hitherto even though it would seem to have promise for identification and characterisation of biologically and commercially important plant polysaccharides. We have found that imaging of cellulose requires rather high laser powers which are above optimal values for live cell imaging. Starch, however is easily imaged by the technique at laser fluences compatible with extended cell viability. This also has useful applications in imaging plant-derived starchy food products. Lignin in plant cell walls shows a strong 3-photon excited fluorescence which may be enhanced by resonance effects.
The polarization dependence of the second harmonic emission of purified in-vitro reconstituted fibrils of collagen has been examined. The results confirmed the quasi-hexagonal crystalline structure within the fibrils. Interesting different polarization behaviours were seen between collagen types I and II, which can be utilized as an experimental technique for differentiation.
In recent years, a variety of Green Fluorescent Protein (GFP)-like pigments have been discovered from corals and other marine organisms. They are widely used to expand the range of available GFP-type proteins in imaging applications, such as in vivo markers for gene expression and protein localization studies, FRET-based (Förster resonance energy transfer) multicolor imaging and biosensors. They have known diverse optical and biochemical properties but their in vivo spectral properties and biological function in marine organisms is only beginning to be understood. We have investigated their spectral diversity, optical properties and cellular microstructure in corals of the Great Barrier Reef with the aim of elucidating their photo-biological function/s as well as to identify novel proteins suitable for GFP-based technologies. We found numerous spectral variants, with emissions covering almost the full range of the visible spectrum. Many of these GFP-like proteins, especially in corals from the more extreme habitats, such as sun-exposed shallows or in deep water, showed a range of light-related spectral characteristics: high photostability, spectral tuning for energy transfer and dynamic photo-induced transformation properties. Intra-cellularly they were organized into spectral donor-acceptor pairs or even arrays, tuned for FRET. Coral color proteins thus offer an exciting potential to expand the use of the available GFPs in bio-imaging applications and as a basis for improved protein engineering.
Tissues of many marine invertebrates of class Anthozoa contain intensely fluorescent or brightly colored pigments. These pigments belong to a family of photoactive proteins closely related to Green Fluorescent Protein (GFP), and their emissions range from blue to red wavelengths. The great diversity of these pigments has only recently been realized. To investigate the role of these proteins in corals, we have performed an in vivo fluorescent pigment (FP) spectral and cellular distribution analyses in live coral cells using single and multi-photon laser scanning imaging and microspectroscopy. These analyses revealed that even single color corals contain spectroscopically heterogeneous pigment mixtures, with 2-5 major color types in the same area of tissue. They were typically arranged in step-wise light emission energy gradients (e.g. blue, green, yellow, red). The successive overlapping emission-excitation spectral profiles of differently colored FPs suggested that they were suited for sequential energy coupling. Traces of red FPs (emission = 570-660 nm) were present, even in non-red corals. We confirmed that radiative energy transfer could occur between separate granules of blue and green FPs and that energy transfer was inversely proportional to the square of the distance between them. Multi-photon micro-spectrofluorometric analysis gave significantly improved spectral resolution by restricting FP excitation to a single point in the focal plane of the sample. Pigment heterogeneity at small scales within granules suggested that fluorescence resonance energy transfer (FRET) might be occurring, and we confirmed that this was the case. Thus, energy transfer can take place both radiatively and by FRET, probably functioning in photoprotection by dissipation of excessive solar radiation.
Collagen is known to be a very effective generator of the second harmonic of incident light from 700 to 1100nm, and second harmonic generation (SHG) microscopy is coming into use as a tool for studying the distribution of collagen in tissue. It also shows promise as a technique for characterizing collagen - both in distinguishing different collagen types and their packing and in identifying degradation of collagen in pathologic conditions. However many aspects of image formation in SHG microscopy of collagen remain imperfectly understood, and we have commenced a rigorous study of these factors. The present paper presents the first results from this program.
It has recently been demonstrated that collagen is a very effective upconverter of light by second harmonic generation (SHG) but hitherto the potential this offers for biomedical imaging has not been realized. We show that bright SHG images van be obtained over a wide excitation range at illumination levels comparable to or lower than those required for two-photon excitation of fluorescent labels, with no damage to the collagen structure. Both paraffin and cryostat sections have been used, and medically significant results have been obtained in several fields. We show that the signal is easily distinguished from single and two-photon excited fluorescence by its forward propagation and narrow spectral width; in principle it could also be distinguished by lifetime. Key microscope requisites are: immersion objectives and condensers, high-efficiency PMT detectors for transmitted light, suitable filters, and effective blocking of stray light, especially from the mercury lamp.
Using confocal and multiphoton microscopy we have mapped the three-dimensional arrangements of chromosomes, microtubules and gamma tubulin during cell division in plants. We have also for the first time imaged diivision in living, intact plant tissue. These results are preliminary, but exciting and we anticipate considerable further progress will be possible with advances in hardware which are now becoming available.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.