ITRM20070371A1 - LASER CITOMETER IN FLOW FOR THE SIMULTANEOUS SIZE OF SIZE REFERENCE INDEX ASPHERICITY AND FLUORESCENCE OF MICROSCOPIC PARTICLES IN LIQUID OR GASEOUS SUSPENSIONS - Google Patents

Description

Description of the industrial invention entitled: FLOW LASER CYTOMETER FOR THE SIMULTANEOUS MEASUREMENT OF SIZE, REFRACTION INDEX, ASPHERICITY AND FLUORESCENCE OF MICROSCOPIC PARTICLES IN LIQUID OR GASEOUS SUSPENSIONS;

The present invention substantially relates to an apparatus for the simultaneous measurement of the physical characteristics of microscopic particles present in liquid or gaseous suspensions.

More specifically, the invention relates to a laser flow cytometer comprising a source of circularly polarized light and a cell with a capillary. The source irradiates the capillary in which microscopic particles are made to flow in liquid or gaseous suspensions. Ó each particle, due to the interaction with the coherent light radiation, emits by diffusion and fluorescence.

According to the invention, this emission is collected - simultaneously - by the detection channels of the non-polarized light diffused forward, of the linearly polarized light diffused forward, of the non-polarized light diffused laterally in different spectral bands.

On the basis of the emission measurements carried out in these channels, a special acquisition software allows to determine simultaneously and in real time: size, refractive index, depolarization (asphericity) and fluorescence of each particle.

Tests conducted with aqueous samples of polystyrene spheres, fluorescent or non-fluorescent, and phytoplankton cells show that the invention is capable of reconstructing size and refractive index with an accuracy of 1% and that the measurements of depolarization and fluorescence allow to classify otherwise indistinguishable particles.

The simultaneous measurement of size and refractive index of microscopic particles with laser systems is based on Mie theory which describes the diffusion of light by dielectric spheres [1] In general, instruments based on this technique are called laser counters of particles [2] and laser flow cytometers [3], depending on whether the particles are suspended in air and water, respectively. The great interest in the rapid classification of microscopic particles in liquid suspensions (marine phytoplankton, blood cells, etc.) has favored the widespread diffusion of commercial systems based on laser flow cytometry (LFC).

The most established companies in this field are:

■ Becton, Dickinson and Company [4],

• Beckman Coulter [5],

• Partec [6].

In LFC, the particle flux and laser beam are orthogonal. As the particle passes through the beam, the radiation is scattered with an angular distribution that depends on size and refractive index. In general, commercial systems are limited to measuring forward diffusion (FSC) and lateral diffusion (SSC), without obtaining the size and refractive index. Often, the fluorescence of the particles is revealed in some spectral bands. Typically, forward scattering is associated with the size of the particle [7], while lateral-scattering is dominated by refractive effects [8]. Although simplifications, this approach allows rapid classification of cells in the clinical field, especially if the cells are labeled with monoclonal antibodies conjugated to fluorescent dyes. As for the applications of LFC to marine phytoplankton, we point out the products of CytoBuoy which manufactures laboratory systems, for buoys and submarines [9].

It is important to note that, up to now, the measurement of size and refractive index has always been carried out separately and distinctly from any measurement of polarization (asphericity) and fluorescence. This means that currently it is not possible to detect all these data for the same particle because the aforementioned measurements, made at different times, do not allow to detect the data relating to the same particle.

In other words, by detecting the diffusion separately from the fluorescence, it is not possible to associate - a posteriori - these measurements to the particle to which they refer.

For example, in the context of the characterization of phytoplankton cells, it should be noted that:

• phytoplankton cells can be discriminated not only by size and refractive index, but also by shape and pigmentation,

and shape and pigmentation can be observed, respectively, due to diffusion depolarization and fluorescence emission.

The main purpose of the present invention is to overcome the limitations of conventional products by providing a device capable of combining LIF, LFC and polarizing optics in order to simultaneously measure the size, refractive index, depolarization or asphericity and fluorescence of microscopic particles in liquid or gaseous suspensions.

This has been achieved, according to the invention, by providing to combine together the laser flow scanning cytometry (LSFC) [15], capable of reconstructing the size and refractive index, with suitable means for measuring the depolarization or asphericity of microscopic particles.

A better understanding of the invention will be obtained with the following description and with reference to the figures which illustrate, purely by way of non-limiting example, a preferred embodiment.

In the drawings:

Figure 1 shows the principle of operation of a laser flow cytometer.

Figure 2 is an example of data obtained with a laser flow cytometer: note how the fluorescence of chlorophyll allows to distinguish phytoplankton (point clouds along the 45 ° line) from other particles (horizontal point cloud). By observing known cultures, it is possible to attribute the point clouds to types of phytoplankton.

Figure 3 is a schematic of FACScan (Becton, Dickinson and Company).

Figure 4 is a photo of the invention: the main elements are indicated with the nomenclature followed in Table 1 below.

Figure 5A is an optical diagram of the invention: the three deflection mirrors (inserted between D and Q) and the further deflection mirror (inserted between M2 and 11) are irrelevant and have not been indicated.

Figure 5B schematically shows the optical path of the laser beam and of the light scattered by the particles reflecting on the spherical mirror at the bottom of the cuvette.

Figure 6 shows the interface of the acquisition and analysis program. Tracks 1, 2 and 3 represent the signals relating to "indicatrix", "trigger" and fluorescence (the second has been inverted for viewing convenience).

Figures 7A and 7B are, respectively, images of Chlamydomonas reinhardtii obtained under the scanning electron microscope and under the optical microscope obtained.

Figures 8A-8D are histograms respectively relating to: size, refractive index, fluoresceinza and depolarization obtained by analyzing a sample of Chlamydomonas reinhardtii mixed with fluorescent and non-fluorescent polystyrene spheres, having a diameter of 6 pm.

Figure 9 is a two-dimensional "Scatter plot" (size and depolarization) analyzing a sample of Chlamydomonas reinhardtii mixed with fluorescent polystyrene beads, having a diameter of 6 μπι.

Figure 10 is a "Scatter plot"; three-dimensional (size, depolarization and fluorescence) obtained by analyzing a sample of Chlamydomonas reinhardtii mixed with fluorescent and non-fluorescent polystyrene spheres, having a diameter of 6 pm (the smallest points are the projections on the planes).

Figure 11 is a three-dimensional "Scatter plot" (size, fluorescence at 530 nm and fluorescence at 680 nm) obtained by analyzing a sample prepared by mixing green fluorescent and non-fluorescent polystyrene spheres, having a diameter of 2 μπι, with polystyrene spheres fluorescent in red and non-fluorescent, having a diameter of 6 μιη.

The principle of operation of the LFC is illustrated in Fig. 1, an example of data is provided in Fig. 2 and a popular commercial tool is shown in Fig. 3.

Typically, LFC allows to simultaneously acquire 5 parameters: forward diffusion, lateral diffusion and fluorescence in 3 spectral bands. The counting rate can exceed 10,000 particles per minute. The advantages of LFC include the ease of sample preparation, the absence of chemical treatment and the possibility of carrying out measurements in situ.

According to the invention, an apparatus is provided comprising:

- means for illuminating the particles to be analyzed - with a circularly polarized laser light; - a forward scattering detector adapted to simultaneously measure non-polarized and polarized light. linearly diffused by the particles in a wide range of the polar angle, typically between 5 ° and 100 °, where the non-polarized and polarized beams are perpendicular;

- a lateral scattering detector adapted to simultaneously measure the non-polarized light scattered by the particles in the lateral direction and the fluorescence of the particles in different spectral bands;

- a specific acquisition and processing software able to - provide in real time the size, refractive index and a particle depolarization parameter, on the basis of said measurements of non-polarized and polarized light detected by the forward diffusion detector;

- a suitable acquisition and processing software able to provide in real time the instant of passage of the particle in a known point and the fluorescence parameters of the particles, on the basis of the measurements of non-polarized light and fluorescence detected by the lateral diffusion detector;

said measurements and / or calculations of size, refractive index, depolarization or asphericity and fluorescence being carried out simultaneously. In the experiments, a D diode laser with a high polarization ratio (100: 1) was used. Moreover, this source is characterized by large power and short wavelength, allowing the system to have good sensitivity and to detect sub-micrometric particles (up to about half of the wavelength).

Optionally, it is possible to increase the polarization ratio by inserting a polarizer such as a Glan-Taylor Thorlabs GT10 into the beam ("clear aperture" diameter: 10 mm, extinction ratio: 100,000: 1). The linearly polarized beam 1, after being reflected by three mirrors to compact the system, passes through a quarter-wave plate Q, exiting circularly polarized. Subsequently, after being focused by a first Li lens and passing through a perforated mirror M2, the beam 1 coaxially affects a flow of particles 2 in liquid suspension that crosses the capillary (diameter: 254 pm) of a cuvette C. In the zone of interaction (length less than 5 mm) the beam has a small cross section (diameter around 30 p FWHM) and therefore the flow of radiant energy is high. The light 3 diffused by the particle 2 is reflected towards the perforated mirror M2 by a spherical mirror 4 which forms the bottom of the cuvette C. The radiation reflected by the perforated mirror M2, after having been returned by a special mirror (not shown) to compact the system , is divided into two parts by a non-polarizing beam splitter B. The transmitted part is detected by a first photomultiplier PMi, after passing through a polarizer P, the reflected part is revealed as it is by a second photomultiplier PM2. The "beam splitter" B, the polarizer P and said photomultipliers PMi and PM2 are mounted on an aluminum block that allows only the light coming from the perforated mirror M2 to pass through. A first adjustable diaphragm carries out the spatial filtering of the incoming beam.

With reference to Figure 5B, it is interesting to note that since the radiation collimated by the spherical mirror 4 is observed, for each position of the particle 2 along the capillary, only the light 3 coming from a well-defined polar angle of diffusion is detected. Therefore, by reconstructing the position based on the acquisition time of the signal, thanks to the measurement of the speed of particle 2 and the instant of its passage in a known point, it is possible to determine the intensity of the diffusion as a function of the polar angle ( "indicatrix") in a wide range (as mentioned above: typically between 5 ° and 100 °). Finally, the size and refractive index of the particle are reconstructed starting from the "indicatrix" by means of an inversion method [20] of the Mie theory. The instant of passage of the particle 2 in a known point is measured, as we will see, by observing the lateral diffusion. The velocity of the particle 2 is a function, previously calibrated, of the pressures in the hydrodynamic circuit, selected by the operator.

If the particles 2 are spherical, the signals and I∑r respectively in output from the aforementioned photomultipliers PMi and PM2, are equal (except for a constant factor, linked to the different efficiency of the respective detection channels, which can be made equal to 1 by adjusting the gain of photomultipliers).

-i3e, on the other hand, the particles are not spherical, the depolarization:

it is not nothing.

Therefore, the measurement of depolarization allows to discriminate spherical particles: this is particularly useful for classifying phytoplankton cells. .

With reference to the optical diagram of Figure 5A, the part of the invention: relating to the determination of the size and the refractive index of the particle ("indicatrix"), as well as of the shape of the particle (polarization) comprises:

- a diode laser (405 nm) D.

- a quarter-wave plate Q

- a lènte of: focus Li

- a cuvette C

- a perforated M2 mirror

- an iris diaphragm li

- a non-polarizing "beam-splitter" B - a polarizer P

- two photomultipliers PMi and PM2

According to a peculiar characteristic of the invention, the device is able to simultaneously detect also the lateral radiation by means of a microscopic objective.

With reference to the optical scheme of Figure 5A, the part of the invention relating to determining the pigments of the particle (fluorescence), as well as detecting the passage of the particle in a known point ("trigger") comprises:

- L2 collimating lens

- dichroic mirror M4

- iris diaphragm I2

- dichroic mirror M5

- interierential filter (680 nm) Fi

- PM3 photomultiplier

- interference filter (530 nm) F2

- PM4 photomultiplier

- iris diaphragm I3

- interference filter (405 nm) F3

- PM5 photomultiplier

The light is separated into three spectral zones by two dichroic mirrors. The reflection of a first dichroic mirror M4 directs the lateral diffusion to a fifth photomultiplier PM5 which measures the instant of passage of the particle in a known point and provides the "trigger" of the measurement. The fifth photomultiplier PM5 is mounted on an aluminum block which allows only the light coming from particle 2 to pass. A third adjustable diaphragm I3 and a third interference filter F3 carry out, respectively, the spatial and spectral filtering of the incoming beam.

A second dichroic mirror M5:

and reflects the green radiation to a fourth photomultiplier PM4 which measures the fluorescence of the particle around 530 nm, after the spectral filtering carried out by a second interference filter F2,

• transmits the red radiation to a third photomultiplier PM3 which measures the fluorescence of particle 2 around 680 nm, after the spectral filtering carried out by a first interferential filter Fi.

The photomultipliers and interference filters just described are mounted on an aluminum block which allows only the light coming from particle 2 to pass. A second adjustable diaphragm I2 performs spatial filtering of the incoming beam. A photo of the invention is shown in Fig. 4. The optical elements are described in Table 1.

The signals detected by the photomultipliers are amplified by the "transìmpedance" amplifiers indicated in Tab. 1, digitized by an ADLINK DAQ-2010 analog digital converter (4 channels, 14 bit, 2 MS / s) and, finally, stored and analyzed on a personal industrial computer. The graphic interface of the acquisition and analysis program developed by the inventors (FastSFC) is shown in Fig. 6. Once the sample has been inserted (typically a few thousand particles), the system analyzes it in a few minutes providing - for each particle - the data relating to: size, refractive index, depolarization (or asphericity) and fluorescence.

The operation of the invention will now be illustrated by describing the measurement of a sample of Chlamydomonas reinhardtii, a unicellular green eukaryotic alga, about 10 pm long, which moves using two flagella, about 10 pm long (Fig. 7A and 7B). This cell offers an ideal test for the invention having an elliptical shape and containing chlorophyll-a which imply, respectively, non-zero depolarization and fluorescence around 680 nm. The test results, illustrated in Figs. 8A-8D and Tab. 2, were obtained with a sample of Chlamydomonas reinhardtii (which we will simply call "chlamydomonas") mixed with fluorescent and non-fluorescent polystyrene spheres, having a diameter of 6 pm, (indicated as "6 pm F" and " 6 pm NF ", respectively).

First of all, we observe that the invention correctly measures the size and refractive index of spheres. In fact, the expected values: 6.0 μπι and 1.61 - 1.62. [19], respectively, are compatible with the measured values: 6.07 ± 0.27 pm. and 1.62910.022, respectively (obtained by averaging the results of 6 pm F and 6 pm NF), corresponding to relative errors of 1% in both cases. Also, as we would expect, all spheres have zero depolarization and the fluorescence is nonzero only for 6 pm F.

As far as the classification of the particles is concerned, we note that, due to the great natural variability, in the size of the cells (visible in the microscope), the relative histogram is quite large and it is impossible to distinguish them from the spheres. The same applies to the refractive index: although the values for cells and spheres are significantly different, there is a non-negligible overlap in the histograms: a cell can be mistaken for a sphere and vice versa. The situation improves if fluorescence is observed: the non-fluorescent spheres are completely resolved. Unfortunately, there can be some confusion between fluorescent spheres and cells. Finally, if depolarization is taken into account, it is practically impossible to exchange cells and spheres.

These results are even more evident in the two and three dimensional "scatter plots" of Fig. 9 and 10, respectively, and demonstrate how useful the measurement of depolarization is for the accurate classification of microscopic particles: without it, it would have been impossible. totally distinguish cells and spheres.

To exemplify the usefulness of observing fluorescence in two spectral bands, a sample prepared by mixing green fluorescent and non-fluorescent polystyrene spheres, having a diameter of 2 pm (indicated as "2 pm F" and "2 pm NF", was measured. , respectively), with red fluorescent and non-fluorescent polystyrene spheres, having a diameter of 6 pm (indicated as "6 pm F" and "6 pm NF", respectively). The three-dimensional "scatter plot" of Fig. 11 demonstrates that CLASS resolves the four types of particles into distinct point clouds.

BIBLIOGRAPHY

[1] Tsang L., Kong J.A., Ding K.-H /, "Scattering of.

Electromagnetic Waves: Theories and Applications, "John Niley & Sons, New York, US (2001).

[2] Provder T., Texter J., eds., "Particle Sizing and Characterization," American Chemical Society, Washington, US (2004).

[3] Shapiro H.M., "Practical Flow Cytometry," John Wiley & Sons, New York, US (2003).

[4] http://bd.com

[5] http://www.beckmancoulter.com

[6] http://www.partec.de

[7] Burger D.E., Jett J.H., Mullaney P.F., "Extraction of morphological features from biological models and cells by Fourier analysis of static light scatter measurements," Cytometry 2, 327-336 (1982).

[8] Salzman G.C., Mullaney P.F., Price B.J., "Lightscattering approaches to celi characterization," in "Flow Cytometry and Sorting," Melamed M.R., JUindmo T., Mendelsohn M., eds., John Wiley & Sons, New York, US (1979).

[9] http://www.cytobuoy.com

[10] http://wwwrob.brindisi.enea.it/miao

[11] http: //www.miur. it

[12] http: // www. aeneas. it / com / Frascati / laser applications. html [13] http: // www. kinetics. nsc. ru

[14] Fiorani L., Paiucci A., "Locai and remote laser sensing of bio-optical parameters in naturai waters," Journal of Computational Technologies 11, .39-45 (2006).

[15] Maltsev V.P., "Scanning flow cytometry for individai particle analysis," Review of Scientific Instruments 11, 243-255 (2000).

[16] Aristipini P., Fiorani L., Menicucci I., Paiucci A., "Portable laser spectrometer for the analysis of non-opaque liquids in yes," Italian Patent Number: RM2005A000269 (2005).

[17] Maltsev V.P., Chernyshev A.V., Method and device for determination of parameters of individai microparticles, "United States Patent Number: 5650847 (1997).

[18] Fiorani L., "Une première .mesure lidar combinée d <7> ozone et de vent, à partir d'une instrumentation et d <7> une méthodologie coup par.coup," Swiss Federal Institute of Technology, Lausanne, Switzerland (1996).

[19] Barnaba F., Fiorani L., Paiucci A., Tarasov P., "First characterization of marine particles by laser scanning flow cytometry," Journal of Quantitative Spectroscopy and Radiation Transfer 102, 11-17 (2006).

[20] Semyanov K.A., Tarasov P.A., Zharinov A.E., Chernyshev A.V., Hoekstra A.G., Maltsev V.P., "Single-particle sizing from light scattering by spectral decomposition Applied Optics 43, 5110-5115 (2004)

Claims (17)

CLAIMS :. 1. Apparatus for the simultaneous measurement of size, refractive index, asphericity and fluorescence of microscopic particles in liquid or gaseous suspensions, characterized in that it comprises, in combination: means for flow scanning laser cytometry (LSFC) suitable for reconstructing size and refractive index of the particles, and suitable means for measuring the depolarization or asphericity of microscopic particles, as well as their fluorescence and / or pigmentation. 2. Apparatus according to the preceding claim, characterized in that it comprises: - means for illuminating the particles, to be analyzed with a circularly polarized laser light; - a forward scattering detector capable of simultaneously measuring the non-polarized and linearly polarized light scattered by the particles in a wide range of the polar angle, typically between 5 ° and 100 °, where the non-polarized and polarized beams are perpendicular; - a lateral scattering detector able to simultaneously measure the non-polarized light scattered by the particles in the lateral direction and the fluorescence of the particles in different spectral bands; - a specific acquisition and processing software able to provide in real time the size, refractive index and a depolarization parameter of the particles, on the basis of said measurements of non-polarized and polarized light detected by the forward scattering detector; - a suitable acquisition and processing software able to provide in real time the instant of passage of the particle in a known point and the fluorescence parameters of the particles, on the basis of the measurements of non-polarized light and fluorescence detected by the lateral diffusion detector; said measurements and / or calculations of size, refractive index, depolarization or asphericity and fluorescence being carried out simultaneously. 3. Apparatus according to the preceding claim, characterized in that said means for illuminating the particles (2) comprise a diode laser (D) with a high polarization ratio (for example 100: 1). 4. Apparatus according to claim 1, characterized in that said means for determining the size and refractive index of the particle ("indicatrix"), as well as the shape of the particle (polarization), comprise: - a diode laser (405 rati) (D); - a quarter-wave sheet (Q); - a focusing lens (Li); - a cuvette (C); - a perforated mirror (M2); - an iris diaphragm (li); - a non-polarizing "beam-splitter" (B); - a polarizer (P); - two photomultipliers (PMi and PM2). 5. Apparatus according to the preceding claim, characterized in that the "beam splitter" (B), the polarizer (P) and said photomultipliers (PM <'> i and PM2) are mounted on an aluminum block which allows the passage only to the light coming from the perforated mirror (M2). 6. Apparatus according to claim 1, characterized in that said means for determining the pigmentation of the particle (fluorescence) and for detecting the passage of the particle in a known point ("trigger") comprise: - collimation lens (L2); - dichroic mirror (M4); - iris diaphragm (I2); - dichroic mirror (M5); - interference filter (680 nm) (Fi); - photomultiplier (PM3); - interference filter (530 nm) (F2); - photomultiplier (PM4); - iris diaphragm (I3); - interference filter (405 nm) (F3); - photomultiplier (PM5). 7. Second apparatus; the preceding claim, characterized in that said photomultipliers and said interference filters are mounted on an aluminum block which allows only the light coming from the particle (2) to pass through. 8. Apparatus according to claim 2, characterized in that said lateral diffusion detector comprises a microscopic objective. 9. Apparatus according to claim 6, characterized in that it is adapted to separate the light into three spectral zones by means of two dichroic mirrors (M «and M5). 10. Apparatus according to claim 6, characterized in that a first dichroic mirror (M4) is able to reflect the lateral diffusion towards a fifth photomultiplier (PM5) able to measure the instant of passage of the particle in a known point and to provide the "trigger" of the measure; said fifth photomultiplier (PM5) being mounted on an aluminum block which allows only the light coming from the particle (2) to pass. 11. Apparatus according to claim 6, characterized by the fact that a third adjustable diaphragm (I3) and a third interference filter (F3) are adapted to carry out, respectively, the spatial and spectral filtering of the incoming beam 12. Apparatus according to claim 6, characterized in that a second dichroic mirror (M5) is adapted to: • reflect the green radiation to a fourth photomultiplier (PM4) able to measure the fluorescence of the particle around 530 nm, after the spectral filtering carried out by a second interierential filter (F2), • transmit the red radiation to a third photomultiplier (PM3) suitable for measuring the fluorescence of the particle (2) around 680 rati, after the spectral filtering carried out by a first interferential filter (Fi). 13. Apparatus according to claim 6, characterized in that a second adjustable diaphragm. (I2) is adapted to effect the spatial filtering of the incoming beam. 14. Apparatus according to claim 4, characterized in that a laser polarizer is provided between the laser (D) and the focusing lens (Li); said laser polarizer being adapted to increase the polarization ratio of the laser beam. 15. Apparatus according to claim 1, characterized in that it provides a quarter-wave sheet for rotating the polarization of the laser beam. 16. Apparatus according to claim 4, characterized in that said polarizer (P) is a Glan-Thompson polarizer. 17. Apparatus according to claim 14, characterized in that said laser polarizer is a Glan-Taylor polarizer.