WO2005052559A1 - Procede d'identification de pigments d'une seule cellule par spectrophotometrie d'image confocale dans des communautes phototrophiques - Google Patents

Procede d'identification de pigments d'une seule cellule par spectrophotometrie d'image confocale dans des communautes phototrophiques Download PDF

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Publication number
WO2005052559A1
WO2005052559A1 PCT/ES2004/000527 ES2004000527W WO2005052559A1 WO 2005052559 A1 WO2005052559 A1 WO 2005052559A1 ES 2004000527 W ES2004000527 W ES 2004000527W WO 2005052559 A1 WO2005052559 A1 WO 2005052559A1
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Prior art keywords
laser
sample
fluorescence
pigments
emission
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PCT/ES2004/000527
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English (en)
Spanish (es)
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María Concepción HERNÁNDEZ MARINÉ
Mónica ROLDÁN MOLINA
Susana Castel Gil
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Universidad De Barcelona
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Publication of WO2005052559A1 publication Critical patent/WO2005052559A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/019—Biological contaminants; Fouling

Definitions

  • This invention relates to the field of environmental technologies, and particularly to the identification of living phototrophic organisms, that is, organisms that use light as an energy source.
  • Identification of a complex living community and discrimination between phylogenetic groups are particularly useful in the fields of aquaculture management and the sciences involved in the study of ecosystems, such as ecology, ecophysiology, oceanography, and limnology. Such identification and discrimination can be used, for example, to analyze incidence and to develop control strategies against problematic algal blooms that appear within aquatic and aerophytic ecosystems (organisms that live at the air interface and a nonliving structure such as a building or a wall) in natural and artificial conditions.
  • aquatic and aerophytic ecosystems organisms that live at the air interface and a nonliving structure such as a building or a wall
  • complex problem communities have been difficult to predict in the past, mainly due to the lack of appropriate methodologies and technologies. The nature of the samples and the need to analyze them without prior isolation from the organisms are other important obstacles to overcome.
  • algal pigments are often heterogeneously distributed within each cell, affecting the extent of absorption. Although pigments can be extracted, the extraction procedures carry the risks of the presence of artifacts in the preparation and removal of the original microenvironment from the chromophore.
  • Another known limitation of differentiating algae by absorbance / fluorescence measurements is related to the need for solid empirical approaches to statistically discriminate both the algal component of the composite signal for a given mass of water, and a single algal component by itself (cf. DF Millie et al., "Using absorbance and fluorescence spectra to discriminate microalgae", Eur. J. Phvcol. 2002, vol. 37, pp. 313-22).
  • Phototrophic organisms produce various types of photosynthetic pigments, each of which captures photons in a narrow range of the spectrum. A fraction of the energy absorbed by the pigments can be emitted immediately at a longer wavelength, a phenomenon known as fluorescence. The emitted fluorescence originates primarily from the photosystem II subantenna pigments (PSII) and is the result of the photosystem's inability to use all of the absorbed energy. Since photosynthesis and fluorescence are competitive processes, changes in photosynthetic activity are reflected in variations in fluorescence emission.
  • PSII photosystem II subantenna pigments
  • fluorescence is an indicator of photosynthetic processes in plants, algae and cyanobacteria, being a powerful analysis tool that allows the description of a complex community in terms of its physiological state, transfer energy, evolution cell and discrimination between phylogenetic groups.
  • Fluorescence microscopy techniques have allowed the description of the structure and organization of the samples in two dimensions (2D). These techniques have been used to study photosynthetic microorganisms and various systems have been described both at the microscopic level (cf. L. Ying et al., "Fluorescence emission and absorption spectra of single Anabaena sp. Strain PCC7120 cells", Photochemistrv and Photobiology 2002, vol. 76, pp. 310-3) as at the macroscopic level (cf. HK Lichtenthaler et al., "Detection of vegetation stress via a new high resolution fluorescence imaging system ", __ Plant Phvsiologv 1996. vol. 148, pp. 559-612).
  • CSLM confocal scanning laser microscope
  • Some CSLMs comprise a selective spectrophotometric prism for detecting emission fluorescence.
  • the combination of a CSLM and spectrophotometric detection is referred to herein as "confocal imaging spectrophotometry" (CIS).
  • CIS confocal imaging spectrophotometry
  • the inventors have surprisingly found a new method for identifying fluorescent signals in individual cells based on the combination of the power of the confocal scanning laser microscope (CSLM). with the capabilities of spectrophotometric methods.
  • the new method allows the unequivocal in vivo identification of the taxonomic group of an individual cell, based on its fluorescence signal, without manipulating the phototrophic communities.
  • the invention provides a non-invasive method for analyzing a sample using a CSLM coupled to a spectrophotometer detector, comprising the steps of: (i) selecting a particular area of the sample; (ii) obtaining spectral scanning images of the emission fluorescence from the selected sample area at selected system settings; and (iii) processing said spectral scanning images according to definable algorithms to obtain an emission fluorescence spectrum of said area; where the sample fluoresces under laser excitation, without any previous labeling (that is, without the addition of fluorescent chemicals).
  • Spectral scan images are the result of the spectral scan function ("lambdascan" function) of CIS devices.
  • the fluorescence signal is captured at a predefined interval for each section, moving along the spectrum.
  • Each individual measurement is based on the detection of real confocal images.
  • the method of the present invention uses the selected variables x-y- ⁇ , that is, the optical plane is recorded at different wavelengths.
  • the method uses the variables x-z- ⁇ , x-y- ⁇ -t (t stands for time), and x-y- ⁇ -z.
  • bandwidth as used herein means the range of transmitted frequencies of a given signal.
  • ⁇ steps is meant here the number of individual images detected in a specific range of wavelengths, from a single optical section. Images are recorded within a wavelength range, which is limited by their start and end points.
  • the "step size ⁇ " is the magnitude (expressed in nm) between the lower and upper limits of the wavelength range at which an image is recorded.
  • the appropriate software supplied with the specific CIS apparatus controls the detector during spectral scanning and aids in calculations, using definable algorithms, of the emission spectrum after scanning an image.
  • the emission fluorescence spectrum of the selected area is obtained by processing said spectral scanning images.
  • the selected area corresponds substantially to a single cell.
  • the selected area comprises at least one pixel of phototrophic organisms.
  • pixel based on the words "picture” and "element" represents the smallest, indivisible element of an image in a two-dimensional system. In this description, both the sample points of a specimen and the points of an image are qualified as pixels.
  • phototrophic organisms are selected from the group consisting of plants, algae, cyanobacteria, and mixtures thereof. Plants and algae can be macroscopic or microscopic.
  • the sample has a thickness equal to or less than the detection limit thickness of the used confocal scanning laser microscope.
  • the system settings for obtaining the emission fluorescence sample data from the selected area of the sample are set in order to minimize photobleaching.
  • Photobleaching is the loss of emission fluorescence intensity of the sample due to destruction of fluorescent substances by intense illumination.
  • the step size ⁇ is set between 5 and 40 nm, and the bandwidth is set from 360 to 800 nm.
  • performance and fit are held constant.
  • the performance value (“gain valué”) modifies the amplification of the detected signal and, consequently, the brightness and contrast of the image change.
  • the adjustment value (“offset valué”) defines a threshold value and, therefore, only those signals that are above the threshold value are detected and represented in the image.
  • the laser excitation wavelength is set to one or more of the following values: 351nm (UV Ar laser), 364nm (UV Ar laser), 458nm (Ar laser), 476nm (Ar laser) , 488 nm (Ar laser), 514 nm (Ar laser), 543 nm (HeNe laser) and 633 nm (HeNe laser).
  • the sample data is processed according to definable algorithms that provide two-dimensional plots of the mean fluorescence intensity versus the emission fluorescence wavelengths.
  • the method of the present invention provides the in vivo and three-dimensional localization of each community and the direct analysis of the fluorescent pigments of a single cell in situ. in coarse intact samples. The relationship between these two determinations allows the identification of the pigments, the subsequent identification of the groups and species present in the sample, and the knowledge of the physiological state of each particular organism.
  • the main improvements achieved with the new method are: (i) the analysis of single or multiple fluorescent pixels; (ii) 3D localization in vivo: (iii) direct in situ analysis of single cell fluorescent pigments in coarse samples without prior isolation; (iv) establishing the relationship between fluorescent properties and position within the specific microbial assembly; (v) the possible application of eight excitation wavelengths to obtain spectra of a single cell, providing high-resolution detection and detailed sample information; (vi) rapid access to statistical information on the number of cells and spectral properties of a community; (vii) discrimination of cells with particular fluorescent signals within the colony and correlation with individual cell states; and (viii) the free choice of the emission wavelength, which allows the discovery of new pigment signals.
  • FIG 1. shows the in vivo spectrophotometric analysis of the BF1 biofilm of the Roman catacomb of St. Callistus.
  • FIG. 1A shows the extended focus pseudocolor 3D projection in the xy planes and the orthogonal view in the z direction of the biofilm (49 optical sections).
  • FIGs. 1 BD. I know they represent the in vivo spectral profiles derived from ⁇ exc of 488, 514 and 543 nm, and the standard error (n 5 cells).
  • FIG 2. shows the spectrophotometric analysis of the BF2 biofilm in vivo of the Roman catacomb Domitilla.
  • FIG. 2A shows the extended focus 3D pseudocolor projection in the xy planes and the orthogonal view in the z direction of the biofilm (66 optical sections).
  • Analysis was performed with a CIS microscope, using either the 63x (NA 1.32, oil) or 100x (NA 1.4, oil) objectives (magnification range 1-4).
  • Spectral scanning was performed using the 351 and 364 nm lines of an Ar UV laser; lines 458, 476, 488, and 514 nm from an Ar laser; the 543 nm line from a green HeNe laser and the 633 nm line from a red HeNe laser.
  • the microscope uses spectrophotometric detection that allows the system to perform different scans from 360 to 800 nm of the spectrum using a motorized slit in front of the photomultiplier.
  • each image sequence (that is, the spectral scan or "lambda-scan" function of the system) was obtained by scanning the Same optical section xy using as step size 20nm for detection ( ⁇ coordinate of a data set xy- ⁇ ) to avoid photobleaching.
  • the emission detection was placed 4-9nm further from the excitation wavelength to avoid reflections from the laser beam.
  • the scans were performed using the beam filter, the substrate filter (for UV) or the triple dichroic filter (488/543/633). Data series x, y, ⁇ was acquired at the z position where fluorescence was highest. Background noise in areas without a sample was measured and then used to correct the primary spectra in the thin sections. The laser struck the sample perpendicularly and, to avoid interference with background radiation (light from the laboratory or light from excitation sources), the images were captured in the dark. Performance and contrast were the same for each field at each excitation wavelength and were unaltered throughout the scanning process.
  • the mean fluorescence intensity (MFI) of the xy- ⁇ data series was obtained using the software supplied in conjunction with the microscope.
  • the region of interest (RO ⁇ ) function of the software was used to determine the spectral signal of a selected area of the captured image.
  • An ROI can also be specified to determine the spectrum of each sample and the software displays the average intensity of all pixels within the ROI versus the wavelength.
  • Numerical data were processed with Microsoft Excel ® 97 or 2000. The mean and standard error were calculated for all regions or cells examined in each ⁇ exc . The maximums of the pigments corresponded to their dispersion interval in the different ⁇ exc .
  • the extracted pigments show variations in the fluorescence spectra when compared with the in vivo pigments, therefore a control with pure pigments was performed to compare them with the published studies.
  • Water-soluble pigments such as R-phycoerythrin (R-PE) from Porphyra te ⁇ era and C-phycocyanin (C-PE) from Spirulina sp. they were dissolved in filtered distilled water.
  • Biofilms are made up of populations or communities of microorganisms that adhere to environmental surfaces. These microorganisms are normally immersed in an extracellular polysaccharide that they synthesize themselves. Two aerophytic biofilms were selected in the CSLM observations to test the method with complex natural communities.
  • the biofilms which were described and identified, contained different phylogenetic groups (Cyanobacteria and Bacillariophyta).
  • the first biofilm (BF1) obtained from the catacomb of St. Callistus (Rome, Italy), was mainly made up of Scvtonema julianum and Leptolvngbva sp.
  • the second biofilm (BF2) obtained from the Domitilla catacomb (Rome, Italy), consisted of the Bacillariophyta Diadesmis gallica and an unidentified cyanobacterium from the Chroococcales group. Both biofilms were obtained from artificially lit surfaces. Fragments of biofilms were separated from their substrates (plaster, mortar or speleothems) or, rarely, were taken together with small pieces of their support. Biofilms were maintained in a 2mm layer of 10% agarose BG11 medium (1%, Merck), and processed for the first week. Biofilms and cultures were mounted in Nunc Lab-Tek TM glass bottom chambers. The samples were processed at room temperature in the dark.
  • the extended focus images (that is, the image is divided into three frames representing the maximum fluorescence intensity projection for the xy, xz and yz planes) of the two stratified biofilms showed a differential distribution of the microorganisms in depth in the biofilm (FIG. 1 A and 2A). Emission spectra for 488, 514 and 543 nm of ⁇ exc are shown for each biofilm (FIG. 1 BD and 2B-D).
  • FIG. 1A shows an extended focus pseudocolor 3D projection in the xy and orthogonal planes in the z direction of the biofilm (49 optical sections).
  • the image represents the maximum autofluorescence emitted in the 590-775 nm range when excited at 543 nm.
  • the volume under observation was 465.03 x 465.03 x 398.73 ⁇ m 3 .
  • Z-step 0.4 ⁇ m.
  • Magnification factor 1.
  • Thickness 19.54 ⁇ m.
  • MFI stands for Medium Fluorescence Intensity.
  • FIG. 2A shows optical sections at 66 xy of the laminated biofilm, which consists of two layers, the upper layer consisting of Diadesmis gallica colonies and the lower layer consisting of Chroococcales colonies.
  • the volume under observation was 75.82 x 75.82 x 98.51 ⁇ m 3 .
  • Z-step 0.1 ⁇ m.
  • Magnification factor 3.86.
  • Thickness 6.6 ⁇ m.
  • the image represents the maximum autofluorescence emitted in the 590-775 nm range when excited at 543 nm.
  • Diadesmis gallica (Bacillariophyta) was mainly concentrated at the top of the biofilm while the unidentified Chroococcal formed a discontinuous layer at the bottom (FIG. 2A).
  • D. gallica 3 ⁇ m in diameter, showed less fluorescence than the cyanobacterium.
  • Their ⁇ ma ⁇ , at 676.2 ⁇ 5 nm did not coincide with the ⁇ max of the other groups due to the presence of Chl c.
  • To avoid photobleaching, caused when these cells were consecutively excited with different ⁇ exc different optical fields were used to obtain the emission spectra in all the ⁇ exc .
  • the unidentified Chroococcal presented the same spectral shape as Leptolvngbva sp.
  • TABLE 2 shows the results of the fluorescence emission acquired from biofilms after excitation at four wavelengths ( ⁇ e ⁇ ) (351, 488, 514 and 543 nm). Each ⁇ max value was obtained from 5 cells. TABLE 2. Comparison of ⁇ ma ⁇ for each ⁇ _ * _ of the two aerophytic biofilms.
  • the cyanobacteria showed the highest MFI in the 640-740 nm range at any of the ⁇ exc , when compared with the Bacillariophyta (FIG. 1 BD and 2B-D). Cyanobacteria also had a high MFI at 577-580 nm ⁇ max , due to C-PE.
  • Each photosynthetic pigment present in species absorbs light of a certain wavelength, but in general, pigments pick up photons from a wide range of wavelengths when excited at wavelengths of 351-633 nm. Only at 364 nm (UV) and 458 nm (blue) ⁇ exc was a smaller emission response observed in all microorganisms. In the case of cyanobacteria, this indicates that the minimum efficiency of the photoreceptors is 430-460 nm.

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Abstract

L'invention concerne un procédé non effractif destiné à l'analyse d'un échantillon à émission fluorescente sous l'effet d'une excitation laser sans aucun marquage préalable, au moyen d'un microscope confocal à balayage laser couplé à un détecteur spectrophotomètre. Ce procédé permet de localiser in vivo et en trois dimensions chaque communauté et de procéder à l'analyse directe in situ des pigments fluorescents d'une cellule individuelle, dans des gros échantillons intacts. La relation entre ces deux déterminations permet d'identifier les pigments, d'identifier de manière postérieure les espèces et les groupes présents dans l'échantillon, et de connaître l'état physiologique de chaque organisme particulier. Ce procédé s'avère utile pour identifier des proliférations problématiques d'algues dans des écosystèmes aquatiques et aérophytiques.
PCT/ES2004/000527 2003-11-27 2004-11-26 Procede d'identification de pigments d'une seule cellule par spectrophotometrie d'image confocale dans des communautes phototrophiques WO2005052559A1 (fr)

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ES200302905A ES2237319B1 (es) 2003-11-27 2003-11-27 Metodo de identificacion de pigmentos de una sola celula mediante espectrofotometria de imagen confocal en comunidades fototroficas.
ESP200302905 2003-11-27

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102005058185A1 (de) * 2005-12-01 2007-06-14 Friedrich-Schiller-Universität Jena Verfahren und Anordnung zur Detektion von Fluoreszenz- oder Reflexionsspektren beliebig wählbarer Bereiche und Strukturen eines vom Fremdlicht überlagerten Objekts unter geringer Strahlenbelastung
DE102005058184A1 (de) * 2005-12-01 2007-06-14 Friedrich-Schiller-Universität Jena Verfahren und Anordnung zur Detektion von Fluoreszenz- bzw. Reflexionsspektren beliebig wählbarer Bereiche und Strukturen eines Objektes unter geringer Strahlenbelastung

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US5886784A (en) * 1993-09-08 1999-03-23 Leica Lasertechink Gmbh Device for the selection and detection of at least two spectral regions in a beam of light
JP2000097857A (ja) * 1998-09-21 2000-04-07 Olympus Optical Co Ltd 走査型サイトメータ
FR2817346A1 (fr) * 2000-11-29 2002-05-31 Edouard Nau Procede de detection et imagerie de polluants notamment en milieu liquide par fluorescence et/ou absorption induites par laser et dispositifs associes
US6510001B1 (en) * 1998-02-19 2003-01-21 Leica Microsystems Heidelberg Gmbh Optical arrangement with a spectrally selective element

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Publication number Priority date Publication date Assignee Title
WO1994016313A2 (fr) * 1993-01-18 1994-07-21 Evotec Biosystems Gmbh Procede et dispositif permettant d'evaluer l'aptitude a l'emploi de biopolymeres
US5886784A (en) * 1993-09-08 1999-03-23 Leica Lasertechink Gmbh Device for the selection and detection of at least two spectral regions in a beam of light
US6510001B1 (en) * 1998-02-19 2003-01-21 Leica Microsystems Heidelberg Gmbh Optical arrangement with a spectrally selective element
JP2000097857A (ja) * 1998-09-21 2000-04-07 Olympus Optical Co Ltd 走査型サイトメータ
FR2817346A1 (fr) * 2000-11-29 2002-05-31 Edouard Nau Procede de detection et imagerie de polluants notamment en milieu liquide par fluorescence et/ou absorption induites par laser et dispositifs associes

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DATABASE WPI Week 200033, Derwent World Patents Index; Class B04, AN 2000-379002 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102005058185A1 (de) * 2005-12-01 2007-06-14 Friedrich-Schiller-Universität Jena Verfahren und Anordnung zur Detektion von Fluoreszenz- oder Reflexionsspektren beliebig wählbarer Bereiche und Strukturen eines vom Fremdlicht überlagerten Objekts unter geringer Strahlenbelastung
DE102005058184A1 (de) * 2005-12-01 2007-06-14 Friedrich-Schiller-Universität Jena Verfahren und Anordnung zur Detektion von Fluoreszenz- bzw. Reflexionsspektren beliebig wählbarer Bereiche und Strukturen eines Objektes unter geringer Strahlenbelastung

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