ES2237319A1 - Method of identifying pigments from a single cell using confocal image spectrophotometry in phototrophic communities - Google Patents

Abstract

The invention relates to a non-invasive method of analysing a sample emitting low-excitation laser fluorescence without any prior labelling, using a confocal laser scanning microscope which is connected to a spectrophotometer detector. The inventive method can be used for in-vivo three-dimensional position finding in relation to each community and for direct in-situ analysis of the fluorescent pigments of an individual cell in intact bulk samples. The relationship between said two determinations enables the pigments to be identified, the groups and species present in the sample to be subsequently identified and the physiological state of each individual organism to be ascertained. The invention can be used to identify problematic proliferations of algae in aquatic and aerophytic ecosystems.

Description

Single pigment identification method cell using confocal imaging spectrophotometry in communities Phototrophic

This invention relates to the field of environmental technologies, and particularly with the identification of phototrophic living organisms, that is, organisms that use light as a source of energy.

Prior art

The identification of a complex living community and discrimination between phylogenetic groups are particularly useful in the fields of aquaculture and science management involved in the study of ecosystems, such as ecology, ecophysiology, oceanography and limnology. Such identification and discrimination can be used, for example, to analyze the incidence and to develop control strategies against problematic algal blooms that appear within the aquatic and aerophytic ecosystems (organisms that live in the air interface and a non-living structure such as a building or a wall) in natural and artificial conditions. Unfortunately, in the past it has been difficult to predict complex problem communities, mainly due to the absence of appropriate methodologies and technologies. The nature of the samples and the need to analyze them without prior isolation of the organisms are other important obstacles to overcome. Current efforts typically depend on microscopic evaluation, but this has limitations such as being laborious, generating variable data, requiring an intrusive sampling program with limited coverage areas, and lacking near real-time coverage. As an alternative to microscopic evaluation, analyzes based on measures of cellular absorbance and / or fluorescence have been proposed (cf. JJ Cullen et al ., "Optical detection and assessment of algal blooms", Limnol. Oceanogr . 1997, vol. 42, pp. 1223-39), but these analyzes are not without limitations. A limitation of discriminating algae by absorbance / fluorescence measures is the need that the spectra corresponding to the phylogenetic groups or species involved must be sufficiently different. In addition, algal pigments are often distributed heterogeneously within each cell, affecting the extent of absorption. Although the pigments can be extracted, the extraction procedures carry the risks of the presence of artifacts in the preparation and removal of the original chromophore microenvironment. Another known limitation of differentiating algae by absorbance / fluorescence measures 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 itself (cf. DF Millie et al ., "Using absorbance and fluorescent spectra to discriminate microalgae", Eur. J. Phycol . 2002, vol. 37, pp. 313-22). There have been attempts to improve the use of cellular absorbance / fluorescence techniques, but the improved techniques still cannot analyze three-dimensional (3D) samples, a fact that prevents discriminating morphologies, measuring emission spectra in a single pixel area and analyze 3D samples without altering them.

Phototrophic organisms produce several types of photosynthetic pigments, where each one captures photons in a narrow range of the spectrum. A fraction of the energy absorbed by pigments can be emitted immediately in a longer wavelength, phenomenon known as fluorescence. The  emitted fluorescence originates mainly in pigments photosystem II (PSII) subantena and is the result of inability of the photosystem to use all absorbed energy. Since photosynthesis and fluorescence are processes competitive, changes in photosynthetic activity are reflected in variations of fluorescence emission. So, the fluorescence It is an indicator of photosynthetic processes in plants, algae and cyanobacteria, being a powerful analysis tool which allows the description of a complex community so refers to its physiological state, the transfer energy, the cellular evolution and discrimination between groups phylogenetic

Fluorescence microscopy techniques have allowed the description of the structure and organization of samples in two dimensions (2D). These techniques have been used to study photosynthetic microorganisms and several 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", Photochemistry and Photobioloqy 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", J. Plant Physiology 1996, vol. 148, pp. 559-612). But all these methodologies are not useful enough when the samples are made up of complex communities, such as microbial mats or biofilms. In these cases, it would be desirable to make a first survey of the 3D microstructure and a subsequent in vivo location of the organisms of interest within the intact sample.

Thus, it is highly desirable to have a new method for rapid identification and discrimination of complex communities, which overcomes some of the limitations of the methods known in the art. In particular, it would be useful for this new method to be able to elucidate the 3D location of each community in vivo , and analyze the fluorescent pigments of a single cell in situ , within thick intact samples.

Explanation of the invention.

To date, the confocal laser microscope of scanning ("confocal scanning laser microscope", CSLM) has been Mainly used to obtain 3D images of samples with minimal preparation or alteration, given the capacity of the CSLM to use multiple excitations and detect wavelengths at Different depths of focus. The CSLM allows you to point to specific elements within the sample, such as molecules, structures (eg surfaces, matrices) and properties (eg phase cell division). Some CSLMs comprise a prism selective spectrophotometric for fluorescence detection of emission, like the one described and commercialized by Leica Microsystems (cf. US 5,886,784). The combination of a CSLM and detection spectrophotometric is referred to here as "spectrophotometry of confocal image "(" confocal imaging spectrophotometry ", CIS). Overcoming common CSLMs, the CIS device has the advantage additional to determine the optimal detection and to separate the emission spectra of fluorochromes to avoid overlap (i.e. signal channel overlap). The present invention provides a new application of the apparatus CIS.

The inventors have surprisingly found a new method to identify fluorescent signals in individual cells based on the combination of the power of the confocal laser scanning microscope (CSLM) with the capabilities of the 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.

Thus, the invention provides a method not invasive for the analysis of a sample using a CSLM coupled to a spectrophotometer detector comprising the steps from: (i) select a particular area of the sample; (ii) get spectral scan images of emission fluorescence from the selected sample area at settings of the selected system; and (iii) process said scan images spectral according to definable algorithms to obtain a spectrum of emission fluorescence of said area; where the sample emits fluorescence under laser excitation, without any previous marking (that is, without the addition of fluorescent chemical compounds). The spectral scan images are the result of the function of spectral scanning ("lambdascan" function) of CIS devices, as commercialized by Leica Microsystems. With the function of spectral scanning, the fluorescence signal is captured in a predefined interval for each section, moving along the spectrum. Each individual measurement is based on image detection real confocals In a particular embodiment, the method of present invention uses the selected variables x-y- \ lambda, that is, the plane Optical is recorded at different wavelengths. In others particular realizations, the method uses the variables x-z- \ lambda, x-y- \ lambda-t (t means time) and x-y- \ lambda-z. He term "bandwidth" as used here, means the range of transmitted frequencies of a given signal. By "steps \ lambda" means the number of images here individual detected in a specific range of lengths of wave, from a single optical section. The images are registered within a range of wavelengths, which is limited by Your starting and ending points. The "step size \ lambda" is the magnitude (expressed in nm) between the lower and upper limits of the wavelength range in which a image.

The appropriate software supplied with the device specific CIS, controls the detector during spectral scanning and help in calculations, using definable algorithms, of the spectrum of emission after scanning an image. Thus, the spectrum of emission fluorescence of the selected area is obtained processing said spectral scan images.

In a more particular embodiment of the invention, the selected area corresponds substantially to a single cell. In another particular embodiment, the selected area comprises at least one pixel of phototrophic organisms. The term "pixel", based on the words " pic ture" and " el ement", 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 described as pixels. In a more particular embodiment of the invention, phototrophic organisms are selected from the group consisting of plants, algae, cyanobacteria and mixtures thereof. Plants and algae can be macroscopic or microscopic. In another particular embodiment, the sample has a thickness equal to or less than the detection thickness of the confocal scanning laser microscope used.

In another embodiment, the system settings for obtain the sample data of the emission fluorescence, from the selected area of the sample, are fixed with the objective of minimizing photobleaching. The photobleaching is the loss of fluorescence intensity of sample emission due to the destruction of fluorescent substances by a intense lighting In particular, the step size λ is fixed between 5 and 40 nm, and the bandwidth is set from 360 to 800 nm. In another particular embodiment, performance and adjustment are They keep constant. The performance value ("gain value") modifies the amplification of the detected signal and, consequently, The brightness and contrast of the image change. Setting value ("offset value") defines a threshold value and therefore only those signals that are above the threshold value are detected and represented in the image. Finally, the length of Laser excitation wave is set to one or more of the following values: 351 nm (UV laser Ar), 364 nm (UV laser Ar), 458 nm (laser Ar), 476 nm (Ar laser), 488 nm (Ar laser), 514 nm (Ar laser), 543 nm (HeNe laser) and 633 nm (HeNe laser).

In another embodiment, the sample data is processed according to definable algorithms that provide two-dimensional graphs of the average 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 an individual cell in situ , in thick 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. In summary, the main improvements achieved with the new method are: (i) the analysis of single or multiple fluorescent pixels; (ii) 3D location in vivo ; (iii) direct in situ analysis of single-cell fluorescent pigments in thick samples without prior isolation; (iv) establishing the relationship between fluorescent properties and the 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 the 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.

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. The summary of this application is incorporated here as reference. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The following particular embodiments and figures are provided to mode of illustration, and are not intended to be limiting of the present invention

Brief description of the drawings

Fig 1. shows the spectrophotometric analysis of the biofilm BF1 in vivo of the Roman catacomb of St. Callistus. Fig. 1A. shows the extended pseudocolor 3D projection in xy planes and the orthogonal view in the z-direction of the biofilm (49 optical sections). In Figs. 1B-D. in vivo spectral profiles derived from λ exc of 488, 514 and 543 nm, and the standard error (n = 5 cells) are represented.

Fig 2. shows the spectrophotometric analysis of the biofilm BF2 in vivo of the Roman catacomb Domitilla. Fig. 2A. shows the extended pseudocolor 3D projection of focus in xy planes and the orthogonal view in the z-direction of the biofilm (66 optical sections). In Figs. 2B-D. in vivo spectral profiles derived from λ exc of 488, 514 and 543 nm, and the standard error (n = 5 cells) are represented.

Detailed statement of embodiments CIS device operation

The CSLM analysis was carried out with a Leica TCS-SP2 microscope (Leica Microsystems Heidelberg GmbH, Manheim, Germany) using any of the Plan-Apochromatic 63x lenses (NA 1.32, oil) or 100x (NA 1.4, oil) (increase 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 of a laser of Ar; the 543 nm line of a green HeNe laser and the 633 nm line of a red HeNe laser. The Leica TCS-SP2 uses a spectrophotometric detection that allows the system to perform different sweeps from 360 to 800 nm of the spectrum using a motorized slit located in front of the photomultiplier. Although the CIS device could acquire spectral images of 5 nm in size step between 360-800 nm, each sequence of images (i.e. spectral scan or function "lambda-scan" system) was obtained by scanning the same x-y optical section using as 20 nm step size for detection (coordinate λ of a data set x-y- \ lambda) to avoid photobleaching The emission detection was placed 4-9 nm farther from the wavelength of excitation to avoid reflections of the laser beam.

The sweeps were performed using as a filter beam separator, substrate filter (for UV) or filter triple dichroic (488/543/633). The data series x, y, λ. it was acquired in the z position where the fluorescence was maximum Background noise was measured in areas without sample and then it was used to correct the primary spectra in the sections fine The laser impacted perpendicularly on the sample and, to avoid interference with background radiation (light in the laboratory or light sources of excitation), the images are They got in the dark. The performance and contrast were the same for each field in each excitation wavelength and not were altered throughout the scanning process.

Fluorescence analysis

From the above data, the mean fluorescence intensity ("MFI") of the data series x and -. It was obtained using Leica Confocal software, version 2.0. The region of interest (ROI) 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 presents the average intensity of all pixels within the ROI versus the wavelength. For pigment solutions, ROIs of 1000 µm 2 (n = 10 regions) were analyzed. For the analysis of the biofilms, ROIs of 1 [mu] 2, taken from the fluorescent region of the thylakoid within the cell, were fixed in each series of xy- [lambda] images. Spectral scans (lambdascans) of biofilms (n = 5 cells for each species present in the biofilm) were obtained for each excitation wavelength (λ exc) in at least three independent experiments. The numerical data was processed with Microsoft Excel® 97 or 2000. The mean and standard error were calculated for all regions or cells examined in each λ exc. The maximums of the pigments corresponded to their dispersion range in the different λ exc.

Calibrations using pure pigments

The pigments extracted show variations in the fluorescence spectra when compared with the pigments in vivo , so a control with pure pigments was performed to compare them with the published studies.

The fat-soluble pigments, chlorophylls (Chls) a and b , obtained from Spinacia oleracea and xanthophyll (Xant) from Medicago sativa (Sigma, St. Louis, MO, USA) were dissolved in pure ethanol. Water-soluble pigments such as R-phycoerythrin (R-PE) from Porphyra tenera and C-phycocyanin (C-PE) from Spirulina sp. they were dissolved in filtered distilled water. The allophycocyanin-XL (APC-XL) standard solution of Mastigocladus laminosus was dissolved in ammonium sulfate (60%) and potassium phosphate (pH = 7) (Sigma-Aldrich) with a final concentration of 38 mM. Four hundred µl of each pigment solution at a final concentration of 1 mg / ml was transferred to 8-well glass bottom chambers (Nunc Laboratorio-Tek ™, Nalge Nunc International, Roskilde, Denmark).

The pure pigment spectra (Table 1) correlated well with the published spectra of extracted pigments. The data are the mean ± standard error (n = 10 regions) from three independent experiments carried out under the same experimental conditions. The asterisk means presence of a shoulder. The maximum emission ranges of Chl a (672.4 ± 2.9 nm) and Chl b (662.4 ± 2.1 nm) partially overlapped. The maximum C-PE fluorescence was located around 577.2 ± 2.2 nm, with a shoulder around 660.1 ± 3 nm. This shoulder could correspond to another contaminant phycobiliprotein present in the standard. The peak C-PC peak fluorescence, located at 656.1 ± 4.3 nm, appeared slightly offset from published references. However, changes in the maximum fluorescence of the C-PC are reflected in the literature, depending on the extraction method used. The C-PC extracted from fresh biomass showed a great maximum at 615 nm while in the dry samples an additional maximum was observed at 652 nm.

TABLE 1 λ max and shoulders for different pigments pure by confocal imaging spectrophotometry in all \ lambda_ {exc}

one

Both the C-PC and the Xant They had a weak fluorescence as was known. While the weak fluorescence of the Xant was expected because the main de-excitation route occurs through the transition to triplet status, which is not fluorescent, could not explain the reason for this low fluorescence in pure C-PC. The maximum fluorescence for the allophycocyanin-crosslinked (APC-XL) It was at 676.2 ± 2.4 nm. The special structure of APC-XL, which prevented the dissociation of the molecule in diluted solutions probably affected the maximum emission and not could relate to the field material. On the other hand, this maximum matched a specific type of APC previously described. Other common low energy forms of APC have the maximum fluorescence near 680 nm, which is similar to the emission of intact phycobilisomas while the APC of Cyanobacteria had a maximum emission at 660 nm.

Preparation and analysis of biofilms

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 formed by Scytonema julianum and Leptolyngbya sp . The second biofilm (BF2), obtained from the Domitilla catacombs (Rome, Italy), was formed by the Bacillariophyta Diadesmis gallica and an unidentified cyanobacterium from the Chroococcales group. Both biofilms were obtained from artificially illuminated surfaces. The biofilm fragments were separated from their substrates (plaster, mortar or speleothems) or, rarely, taken together with small pieces of their support. The biofilms were maintained in a 2 mm layer of 10% agarized BG11 medium (1%, Merck), and processed during the first week. Biofilms and cultures were mounted in Nunc Lab-Tek ™ glass bottom chambers. The samples were processed at room temperature in the dark.

Extended focus images ("extended focus ", that is, the image is divided into three frames that represent the projection of maximum fluorescence intensity for the x-y, x-z e planes and -z) of the two stratified biofilms showed a differential distribution of microorganisms in depth in the biofilm (Fig. 1A and 2A). The emission spectra are shown for 488, 514 and 543 nm of λ exc for each biofilm (Fig. 1 B-D and 2B-D).

Fig. 1A shows a 3D projection pseudocolor of extended focus in xy and orthogonal planes in the z direction of the biofilm (49 optical sections). The image represents the maximum autofluorescence emitted in the range of 590-775 nm when excited at 543 nm. The volume under observation was 465.03 x 465.03 x 398.73 µm3. Z step: 0.4 µm. Magnification factor: 1. Thickness: 19.54 µm. Figs. 1B-D show the in vivo spectral profiles derived from 488, 514 and 543 nm λ exc and the standard error (n = 5 cells). MFI means average fluorescence intensity. The differences obtained in the emission fluorescence profiles obtained in the biofilm indicated the presence of different species of cyanobacteria. Leptolyngbya sp. It is represented with a continuous line and Scytonema julianum with a discontinuous line. Magnification factor: 2.

According to the data obtained, in the biofilm BF1, the thin filaments of Leptolyngya sp. they were oriented horizontally above the width Scytonema julianum (Fig. 1A). Both cyanobacteria had a broad λ max at 658.4 ± 3 nm, overlapping Chl a and phycobiliproteins (Fig. 1B-D). In addition, Leptolyngybya sp., But not S. julianum , presented an emission peak (579.7 ± 3.8 nm) attributable to the presence of C-PE (Fig. 2B-D).

Fig. 2A shows 66 xy optical sections of the stratified biofilm, which consists of two strata, the upper layer composed of colonies of Diadesmis gallica and the lower layer formed by colonies of Chroococcales. The volume under observation was 75.82 x 75.82 x 98.51 µm3. Z step: 0.1 \ mum. Magnification factor: 3.86. Thickness: 6.6 µm. The image represents the maximum autofluorescence emitted in the range of 590-775 nm when excited at 543 nm. Figs. 2B-D show the in vivo spectral profiles derived from λ exc of 488, 514 and 543 nm, and the standard error (n = 5 cells). The difference in the emission profiles obtained in the biofilm indicates the presence of different groups of algae and cyanobacteria. Chroococcal is represented with a continuous line and Galician Diadesmis with a discontinuous line. In this biofilm a large decrease in fluorescence was observed in the λ exc 544 nm, attributable to photobleaching after successive spectral scans. Magnification factor: 2.

The CSLM analysis revealed two layers in the biofilm BF2: Diadesmis gallica (Bacillariophyta) was mainly concentrated in the upper part of the biofilm while the unidentified Chroococcal formed a discontinuous layer in the lower part (Fig. 2A). D. gallica , 3 µm in diameter, showed less fluorescence than cyanobacteria. Its λ max, at 676.2 ± 5 nm (Fig. 2B-D), did not match the λ max of the other groups due to the presence of Chl c . To avoid photobleaching, caused when these cells were consecutively excited with different λ exc, different optical fields were used to obtain the emission spectra in all λ exc. Unidentified Chroococcal presented the same spectral form as Leptolyngbya sp.

Table 2 shows the emission results of fluorescence acquired from biofilms after excitation at four wavelengths (λ exc)) (351, 488, 514 and 543 nm). Each value \ lambda_ {max} was obtained from 5 cells.

TABLE 2 Comparison of \ lambda_ {max} for each λ exc of the two aerophytic biofilms

2

Differences in mean fluorescence intensity (MFI)

In the biofilms, the cyanobacteria presented the highest MFI in the range 640-740 nm at any of the \ lambda_ {exc} when compared to Bacillariophyta (Fig. 1B-D and 2B-D). The cyanobacteria also presented a high MFI at 577-580 nm λ max, due to the C-PE.

Each photosynthetic pigment present in the species absorbs light of a certain wavelength, but in In general, pigments capture photons from a wide range of wavelengths when excited at the wavelengths of 351-633 nm. Only at 364 nm (UV) and 458 nm (blue) \ lambda_ {exc} one more emission response was observed Small in all microorganisms. In the case of cyanobacteria, this indicates that the minimum efficiency of photoreceptors is at 430-460 nm.

Claims (11)

1. Non-invasive method for the analysis of a sample using a scanning confocal laser microscope coupled to a spectrophotometer detector comprising the steps from: (i) select a particular area of the sample; (ii) obtain spectral scan images of the emission fluorescence from the sample area selected at selected system settings; Y (iii) process said scan images spectral according to definable algorithms to obtain a spectrum of emission fluorescence of said area; where the sample emits fluorescence under a Laser excitation, without any previous marking. 2. Method according to claim 1, wherein spectral scan images are images x-y- \ lambda. 3. Method according to any of the claims 1-2, wherein the selected area It corresponds substantially to a single cell. 4. Method according to any of the claims 1-2, wherein the selected area It comprises at least one pixel of phototrophic organisms. 5. Method according to claim 4, wherein the phototrophic organisms are selected from the group consisting of plants, algae, cyanobacteria and their mixtures. 6. Method according to any of the claims 1-2, wherein the system settings are selected to minimize photobleaching. 7. Method according to claim 6, wherein the passage size λ is set between 5 and 40 nm, and the width of band is set from 360 to 800 nm. 8. Method according to any of the claims 1-2, wherein the performance and the Contrast remain constant. 9. Method according to any of the claims 1-2, wherein the wavelength of Laser excitation is set to one or more of the following values: 351  nm (UV laser Ar), 364 nm (UV laser Ar), 458 nm (Ar laser), 476 nm (Ar laser), 488 nm (Ar laser), 514 nm (Ar laser), 543 nm (laser HeNe) and 633 nm (HeNe laser). 10. Method according to any of claims 1-2, wherein the algorithms of step (iii) provide two-dimensional graphs of the average fluorescence intensity versus the emission fluorescence wavelengths. 11. Method according to any of the claims 1-2, wherein the sample has a thickness equal to or less than the detection thickness of the Scanning confocal laser microscope used.