WO2000063673A1

WO2000063673A1 - Apparatus to detect shape, size and fluorescence of fluidborne particles

Info

Publication number: WO2000063673A1
Application number: PCT/GB2000/001379
Authority: WO
Inventors: James Mcdonald Clark
Original assignee: The Secretary Of State For Defence
Priority date: 1999-04-20
Filing date: 2000-04-19
Publication date: 2000-10-26
Also published as: AU4577900A

Abstract

Apparatus to determine the shape, size and fluorescence of a fluidborne particle is described. The apparatus comprises a light source directed to impinge on a particle under analysis; a first detector means arranged to detect the resulting fluorescent radiation from the particle; a second detector means to detect light elastically scattered from the particle; and analyser means connected to said detectors. The apparatus may further comprise a second light source. At least one light source is preferably in the ultraviolet region. In a preferred embodiment the second detector means is capable of recording the spatial distribution of the elastically scattered light in both radial and azimuthal directions.

Description

Apparatus to Detect Shape. Size and Fluorescence of Fluidborne Particles

This invention relates to a method and apparatus for the analysis of fluidborne particles.

More particularly the present invention relates to the simultaneous measurement of shape,

size, and fluorescent properties of individual particles entrained within a fluid.

In a wide variety of environmental, occupational, military and industrial scenarios, fine

particles, typically within the size range from a few tenths of a micron to a few hundred microns, play an important role. Environmental fluidborne particles, usually comprising

mineral dusts, combustion products and biological particles, carried by winds and other air

movement, can result in breathing difficulties, allergic reactions and possible degradation of the body's immune system. Particulate material arising from industrial activity can contaminate industrial products and processes and can also present a respirable health hazard, such as in the case of asbestos fibres or fugitive pharmaceutical powder particles. In the military field, the deliberate generation of hazardous aerosols poses a major threat

since a wide variety of biological and chemical weapons are believed to be possessed by both national governments and terrorist organisations. Similarly water-borne particles

may cause disease or allergic reactions or act as pollutants in a water-course. For example, it is desirable to be able to detect cryptosporidium in water.

The in-situ characterisation of fluidborne particles has therefore become important in both

civilian and military fields, and considerable effort has gone into developing techniques

which can analyse certain particle parameters and provide some degree of identification or

classification. Moreover, since even brief exposure to some of the aforementioned

aerosols can damage health and may even prove fatal, the speed of response of the measurement technique has been an important consideration.

A potentially powerful technique of gas-borne particle analysis involves the introduction

of individual particles into a near vacuum where they are fragmented using an intense laser

light pulse. The resulting atomic and molecular fragments are then measured using a time- of-flight mass spectrometer or similar, yielding a detailed assessment of the material

content of the particle. (See for example, Marijnlssen J et al, Proposed on-line aerosol

analysis combining size determination, laser induced fragmentation, and time-of-flight

mass spectrometry', Journal of Aerosol Science, volume 19, pages 1307-1310, 1988).

Such methods offer a high degree of particle discrimination but remain expensive and

cumbersome to implement and, because they are comparatively slow in terms of the rate at which individual particles can be analysed; they do not offer the real-time aerosol analysis capability (i.e. response to a change in aerosol composition within a few seconds desired

in monitoring applications). _*

Of other possible particle characterisation techniques, those based on elastic optical scattering have become popular because they offer genuine real-time non-destructive particle analysis. Here, the term elastic denotes that the scattered light is at the same wavelength as the illuminating light. In their simplest form, optical scattering instruments

are designed to draw ambient fluidborne particles through a measurement space in single file. A light source, usually a laser, illuminates the measurement space and each particle

thus scatters some radiation to an appropriately positioned detector. The magnitude of the

scattered radiation may, to a first order, be used to determine a particle size, whilst the rate

of generation of the light pulses can be related to particle concentration within the sampled atmosphere. Whilst comparatively straightforward to implement, simple light scattering techniques

such as these do not yield sufficient information about the particles to provide anything

other than a very superficial overview of the ambient aerosol. They do not, for example,

provide any indication of the material nature of the particles; whether the particles are of

solid or liquid form; or whether the particles are biological or non-biological.

In order to discriminate more effectively between fluidborne particles of different types, a number of methods have been developed which measure multiple parameters from

individual particles in addition to their (optical scattering) size. For example, analysis of

the spatial distribution of light scattered by each individual fluidborne particle passing through the measurement space of an optical scattering instrument has proved to be an

effective method of improving particle discrimination. This is because the spatial pattern of scattered light contains information relating to the shape of the scattering particle. Examples of instrument geometries which embody this approach to spatial scattering analysis are described in: Portable Particle Analysers', Ludlow, I. K. and Kaye P H. European Patent EP 0 316 172, July 1992; Particle Asymmetry Analyser', Ludlow, I. K.

and Kaye, P. H. European Patent EP 0 316 171, Sept. 1992; 'Apparatus and Method for the Analysis of Particle Characteristics using Monotonically Scattered Light', Kaye, P.H. and Hirst, E. US Patent 5,471,299, Nov. 28, 1995; and Hazardous Fluidborne Fibre Detector'. Hirst, E. and Kaye, P.H. UK Patent Application No: 9619242.2; filed 14th September 1996.

US Patent 5,471,299 above describes an instrument which employs an imaging system

capable of recording both the radial and azimuthal (about the illuminating beam axis) variations in the pattern of scattered light from individual particles carried in a sample

airflow. Using this type of instrument, particles may be effectively classified on the basis

of their shape (whether, for example, spherical, cuboidal, flake-like, or fibrous) as well as

on their size, the latter being derived from an assessment of the total scattered intensity.

However, spatial light scattering analysis instruments of the type described above cannot

discriminate particles on the basis of their material structure. For example, a non-

biological silicate based particle may yield an essentially identical spatial scattering pattern

to a biological cell of similar size and shape. In order to discriminate particles on the basis

of their material structure it is necessary to employ other techniques such as an analysis of light which is scattered inelastically by the particle. Such light is manifest as either a fluorescence emission or, far more weakly, a Raman emission. Since useful Raman

signals from individual microscopic particles in flow have, to date, proved unattainable, they will not be discussed further here. In contrast, several workers have demonstrated

successful measurement of fluorescent spectra from single particles and have used this technique to attempt particle discrimination on the basis of fluorescence. For example,

Pinnick et al (Fluorescent Particle Counter for Detecting Fluidborne Bacteria and Other

Biological Particles' Pinnick R G et al., Aerosol Science and Technology, volume 23, pages 653-664, 1995) developed an instrument in which a stream of airborne particles passes through a measurement space and is illuminated with light at 488nm wavelength from an Argon-ion laser. The light excites some naturally occurring fluorophores within

the particles and the fluorescence emission spectrum between 500nm and 800nm

wavelength is recorded and analysed. Based on the fact that biological particles such as

bacteria or spores produce measurable fluorescence, the authors proposed the technique as

a possible means of discriminating biological from other non-biological particles that may be present in an environment.

However, on many occasions, environments will contain non-biological particles which

also fluoresce, such as hydrocarbon-based fuels and combustion products, and the

technique fails to discriminate these from biological particles present. Other workers, (for example see Hairston P P et al, Design of an instrument for realtime detection of

bioaerosols using simultaneous measurement of particle aerodynamic size and intrinsic

fluorescence', Journal of Aerosol Science, vol. 28, no. 3, pages 471-482, 1997), have combined a measurement of the magnitude of fluorescence from a particle with a measure

of its size, in this case the aerodynamic size of the particle. This dual-parameter

measurement approach provides a greater degree of particle discrimination than

measurement of particle fluorescence alone. However, this type of technique again fails to discriminate between particles which may be of very different nature (such as a biological particle and a droplet of hydrocarbon-based fuel) but are of similar aerodynamic size and produce similar levels of fluorescence.

The invention comprises an apparatus to determine the shape, size and fluorescence of a fluidborne particle, the apparatus comprising; a) a light source directed to impinge light on a particle under analysis; b) first detector means arranged to detect the resulting fluorescence from the particle;

c) second detector means to detect light elastically scattered from the particle; and

d) analyser means connected to said detectors to analyse the fluorescence and

light scattering of said particle. Advantageously, the apparatus can be used to detect particulate matter in a liquid, for example water, or a gas, for example air.

The apparatus may further comprise a second light source, but preferably the first and

second light sources are one and the same i.e. a single light source is used.

Where it is advantageous to use two separate light sources, it is preferable to use two sources, each of which emit light of mutually different wavelengths. This reduces the

mutual interference between light from the two sources. Where two light sources are used,

it is further preferable for the first light source to emit light which stimulates the

fluorescence detected by the first detector means and the second light source emits light that is detected by the second detector means. The first light source should emit ultraviolet

light to achieve efficient excitation of fluorescence from particles. The first and second light sources may respectively comprise a source of ultraviolet light, for example a neodynium:YAG laser (emitting at 266nm), and a source of visible light, such as a

continuous wave laser. The neodynium-YAG laser provides a high intensity beam of light

which is preferable for the probing of particles which only give low levels of fluorescence. It is often convenient to locate the second light source orthogonal with respect to the first light source.

Where a single light source is used, means for optically splitting the resulting fluorescent and elastically scattered light may be required prior to detection. Where it is advantageous to utilise two separate light sources operating at different wavelengths such means for splitting the fluorescent and elastically scattered light may not be required and the two detector means can lie along the same or similar axes. Switched

filtering may also mean that only one detector is required, however this is not preferred as

the detectors are usually specially adapted for their different function.

Where a single light source is used, an illuminating wavelength in the ultraviolet region is

desirable to achieve efficient excitation of fluorescence from particles. Such a preferred

source is a continuous wave laser. Such a laser provides a high intensity beam of light

which is preferable for the probing of particles which only give low levels of fluorescence.

Where an ultraviolet light source is used to stimulate fluorescence, it is advantageous to locate an ultraviolet filter between the first detector and particle for allowing only fluorescent radiation to be received by the first detector. This prevents the first detector

from being exposed to light other than fluorescent light emitted by the particle.

It is preferable that the or each light source comprises a laser. Lasers provide

monochromatic, powerful and well-collimated beams of light.

It is further preferable that the second detector means is capable of recording the spatial distribution of the elastically scattered light in both radial and azimuthal directions. For example, the second detector means comprises a DEP high gain spatial scattering detector.

The second detector means may comprise at least one detector to detect shape data, and at

least one second detector to detect size and/or count data of the particle. The first detector means preferably comprises a spectrometer. This is capable of measuring

the fluorescence spectrum of the particle (i.e. intensity of fluorescence as a function of wavelength). Such a spectrum facilitates the identification of the particle. Alternatively, the first detector means may comprise a detector capable of measuring the intensity of

fluorescent radiation emitted by said particle. Such a detector would not measure a spectrum, but merely record the intensity of fluorescence at a particular wavelength or

over a particular waveband.

It is preferred that the fluidborne particles are drawn through the apparatus by a pump

means. This provides an easy and efficient way of transporting the particles into the

measurement space of the instrument.

The invention further provides a method of determining the shape, size and fluorescence of a fluidborne particle, the method comprising; a) directing light from a first light source to impinge on a particle under analysis;

b) detecting the resulting fluorescence from the particle; c) directing light from a second light source to impinge on said particle;

d) detecting light elastically scattered from the particle, and e) analysing the fluorescence and elastic light scattering detected by said detectors. The invention also provides a method for determining the shape, size and fluorescence of a fluidborne particle comprising;

a) directing light from a light source to impinge on a particle under analysis;

b) detecting the resulting fluorescence by first detector means;

c) detecting elastically scattered light by second detector means;

d) analysing the fluorescence and elastic light scattering detected by said detectors.

The assessment of particle shape and size is preferably achieved through the capture and

analysis of the spatial distribution of radiation scattered by the particle when traversing the

beam from a continuous wave laser. The fluorescence data are recorded by illuminating the particle at a suitable wavelength, normally in the ultraviolet. A single continuous wave ultraviolet laser may be used to produce both the spatial scattering data and the fluorescence data. Alternatively, separate lasers may be used providing their beams are spatially coincident at the measurement space through which the particles flow. In the latter case a practical arrangement would incorporate a continuous wave visible laser to

generate spatial scattering data and a pulsed ultraviolet laser, triggered by passage of the particle through the visible beam, to generate fluorescence data. The determination of

parameters relating to the shape, size, and fluorescent properties of the scattering particle affords an effective means of discriminating particle classes such as biological and non- biological particles.

The present invention will now be described in more detail, by way of example only and

with reference to the following drawings of which Figure 1 shows one embodiment of a multi-parameter particle analysis system according to the invention,

Figure 2 shows an embodiment of a multi-parameter particle analyser having two light sources,

Figure 3 shows a schematic representation of the number density of particles as a function

of particle shape measured using an apparatus and method in accordance with the present invention,

Figure 4 shows a schematic representation of the intensity of fluorescence from particles

as a function of particle shape, and

Figure 5 shows a schematic representation of the intensity of fluorescence from particles

as a function of particle size and shape.

Fluidborne particles are drawn by a pump arrangement into the instrument through an aerodynamic focusing nozzle 1 which delivers the particles in single file through the measurement space 2. Coincident with the measurement space is the beam from a continuous wave ultraviolet laser 3. In the embodiment this is a helium-cadmium laser emitting at 325nm wavelength. The beam from the laser 3 passes through beam-shaping

optics 4 before being turned through 90° by a front-silvered prism 5 mounted on a thin

mechanical support 6. A second front-silvered mirror 7 finally reflects the unscattered beam to a beam stop 8.

A particle traversing the measurement space 2 is illuminated by the beam and generates

both elastically scattered radiation and fluorescent radiation in all directions. Some of this radiation falls on an ellipsoidal reflector 9 whose focus is coincident with the measurement

space and is reflected towards an iris 10. The radiation passing through the iris then passes through a suitable optical assembly incorporating an ultraviolet blocking filter 11

such that only the fluorescence wavelengths (which are greater than 325nm) are

transmitted. This transmitted radiation is directed onto a spectrometer 12 such that the

spectral description of the fluorescence emission may be recorded. If only the total

magnitude of fluorescence emission is required, this spectrometer may be replaced by a single optical radiation detector.

Elastically scattered radiation and fluorescence emission from the particle which is emitted

in the forward direction (close to the direction of propagation of the illuminating beam) passes through an aperture in the ellipsoidal reflector and is imaged onto a detector array 13. Since the magnitude of the fluorescence signal at the detector array will be several

orders of magnitude less than the elastically scattered light signal, the former may be ignored. The detector array is capable of recording the spatial distribution of the elastically scattered light in both radial and azimuthal directions, and this contains information relating to both the shape and size of the scattering particle (see, for example, Spatial Light Scattering as a Means of Characterising and Classifying Non-spherical

Particles', Kaye, P.H. Measurement Science and Technology, vol. 9, no.2, pages 141-149. 1998). Finally, the fluorescence data from the spectrometer and the spatial scattering data from the array detector are directed to a Particle Discrimination Data Processor 14. This electronic processor analyses the incoming particle data and classifies or identifies the

particle on the basis of particle size, shape, and fluorescence properties. A preferred embodiment of this processor would be an artificial neural network which have been

shown to provide good discrimination capabilities. An alternative embodiment of a multiparameter particle analysis instrument incorporates two lasers of differing wavelengths and is illustrated in Figure 2. In this embodiment a

continuous wave visible laser 15, such as a diode laser operating at 635nm wavelength,

illuminates the flow of particles through the measurement space within the instrument.

When a particle passes through the beam elastically scattered light is collected in the

forward direction and directed onto an optical detector such as a photomultiplier tube

(PMT) 16. The signal from the photomuitiplier is in the form of a pulse whose duration is

equivalent to the time of flight of the particle through the beam and whose magnitude may

be related to the size of the particle. Light scattered by the particle at higher scattering angles falls on an ellipsoidal reflector 17 which redirects the light to an optical assembly at

the opposite end of the instrument. This assembly comprises suitable collimating optics 18 together with a dichromatic filter 19. The filter is chosen to allow the transmission of

the 635 ran radiation but the reflection of all shorter wavelengths. The 635 nm scattered light therefore passes through the filter and is imaged by lenses 20 onto a detector array 21 which is capable of recording the radial and azimuthal variations in the pattern of

scattered light. This information is used to determine the shape of the scattering particle.

The pulse signal from the photomultiplier tube 16 may be used to activate the second laser of the system. This second laser is an ultraviolet laser giving a pulsed output, such as a

frequency-quadrupled neodynium-YAG laser operating at 266nm wavelength. The laser (not shown) is arranged such that the beam 25 passes through the measurement space of the instrument in a direction orthogonal to the plane of the Figure. The spatial

arrangement of the two laser beams would be with the ultraviolet beam typically a fraction

of a millimetre below the visible beam in the direction of travel of the particle flow. The

timing of the firing of the pulsed ultraviolet laser is controlled electronically to coincide with the arrival of the particle at the trajectory path of the ultraviolet beam. In this way, the beam excites any fluorophores within the particle and fluorescence emission is produced.

A large proportion of the fluorescence emission, together with elastically scattered ultraviolet radiation, is reflected from the ellipsoidal reflector 17 and directed through

collimating optics 18 onto an ultraviolet blocking filter 22. Only the fluorescence

wavelengths, typically between 300 and 500nm, pass through this filter. The fluorescent

light is then reflected by the dichroic filter 19 to a spectrometer 23 for spectral separation. Alternatively, the spectrometer can be replaced by a single optical detector if a single

measure of magnitude of fluorescence is required.

Finally, the particle size data derived from the pulse magnitude of the continuous wave scattering recorded by the photomultiplier 16, together with the particle shape data from the detector array 21 and the fluorescence data from the spectrometer 23 is fed to the

electronic Particle Discrimination Data Processor 24 where the particle is classified or identified on the basis of these parameter values.

The apparatus of example 2 was used to measure the characteristics of a stream of particles in air, providing the results shown schematically in Figures 3-5. Figure 3 shows a schematic representation of the number density of particles as a function of particle shape. In Figures 3 and 4, the position of each data point reflects a certain particle shape, with

perfect spheres being at the centre of the triangular plot and more asymmetric particles

further towards the periphery. In practice, the data points are of a particular colour which

indicates the number of particles of a certain shape. Figure 4 shows a schematic representation of the intensity of fluorescence from particles as a function of particle shape. The colour _.of each data point reflects the intensity of fluorescence. Such

fluorescence/shape data permit the identification of individual particles. The data shown in

Figure 4 can be represented in an alternative manner as shown in Figure 5. The position of

each data point reflects particle size and shape. AF is the asymmetry factor, with 0 being a

perfect sphere and 100 being an infinitely long fibre. The colour of each data point reflects

the fluorescent intensity. These data displayed in Figures 3-5 were obtained using a

fluorescence detector that records only intensity. This could be replaced with a

spectrometer which could measure the spectral response as a function of particle size and shape.

Although the data presented were accumulated from gas-borne particles, it will be

appreciated that such data could be obtained from liquid-borne particles. For the measurement of liquid-borne particles, the measurement space 2 would be filled with liquid. This demonstrates that an apparatus and method in accordamx with the present invention can measure the shape, size and fluorescence characteristics of individual

fluidborne particles.

The unique combination of essentially simultaneous shape and fluorescence measurements

from the same particle overcomes the problems associated with instrument systems which record only one of these parameters. In the aforementioned example, a fluorescent fuel

droplet may be discriminated from a fluorescent biological particle because the accurate

spherical shape of the droplet will be identifiable in contrast to the aspherical or rod-

shaped shape normally associated with a biological particle. Equally, a biological particle

may be discriminated from a similar shaped and sized inorganic particle on the basis of the fluorescent signatures of each particle type. Furthermore, the apparatus and method in

accordance with the present invention provide a very important improvement over the state of the art. Either of the shape/size data or the fluorescence spectrum alone may

permit identification of the analysed particle. However, a combination of the two

techniques increases the likelihood of a positive identification manifold. Furthermore, the

recordal of the two characteristics provides a vast increase in the confidence with which a particle can be identified.

Claims

1. Apparatus to determine the shape, size and fluorescence of a fluidborne particle,

the apparatus comprising;

a) a light source directed to impinge light on a particle under analysis; b) first detector means arranged to detect the resulting fluorescence from the

particle; c) second detector means to detect light elastically scattered from the particle; and

d) analyser means connected to said detectors to analyse the fluorescence and

light scattering of said particle .

2. Apparatus as claimed in claim 1 wherein the apparatus further comprises a second light

source.

3. Apparatus as claimed in claim 2 wherein the first and second light sources each emit light of mutually different wavelengths.

4. Apparatus as claimed in any one of claims 2 or 3 wherein the first light source emits light which stimulates the fluorescence detected by the first detector means and the

second light source emits light that is detected by the second detector means.

5. Apparatus as claimed in any one preceding claim wherein the first light source has a

wavelength in the ultraviolet region.

6. Apparatus as claimed in claim 5 including an ultraviolet filter located between said first detector and particle for allowing only fluorescent radiation to be received by the first detector.

7. Apparatus as claimed in any one preceding claim wherein the or each light source

comprises a laser.

8. Apparatus as claimed in any one preceding claim wherein said second detector means is

capable of recording the spatial distribution of the elastically scattered light in both radial

and azimuthal directions

9. Apparatus as claimed in claim 8 wherein said second detector means comprises at least one detector to detect shape data, and at least one second detector to detect size and/or count data of the particle.

10. An apparatus as claimed in any one of claims 2 to 9 wherein the second light source is

orthogonally located with respect to the first light source.

11. An apparatus as claimed in any one preceding claim wherein the first detector means comprises a spectrometer.

12. An apparatus as claimed in any one of claims 1 to 10 wherein the first detector means

comprises a detector capable of recording the intensity of fluorescent radiation emitted

by said particle.

13. An apparatus as claimed in any one preceding claim wherein fluidborne particles are

drawn through the apparatus by a pump means.

14. A method of determining the shape, size and fluorescence of a fluidborne particle, the

method comprising; a) directing light from a first light source to impinge on a particle under analysis;

b) detecting the resulting fluorescence from the particle;

c) directing light from a second light source to impinge on said particle;

d) detecting light elastically scattered from the particle, and

e) analysing the fluorescence and elastic light scattering detected by said detectors.

15. A method for determining the shape, size and fluorescence of a fluidborne particle comprising;

a) directing light from a light source to impinge on a particle under nalysis;

b) detecting the resulting fluorescence by first detector means; c) detecting elastically scattered light by second detector means; d) analysing the fluorescence and elastic light scattering detected by said detectors.

16. A method as claimed in claims 14 or 15 wherein the or each light source has a

wavelength in the ultraviolet region.

17. A method as claimed in any one of claims 14 to 16 including an ultraviolet filter

located between said first detector means and particle under analysis for allowing only fluorescent radiation to be received by the first detector.

18. A method as claimed in any one of claims 14 to 17 wherein the or each light source comprises a laser.

19. A method a claimed in any one of claims 14 to 18 wherein the first detector means

comprises a spectrometer.

20. A method a claimed in any one of claims 14 to 18 wherein the first detector means comprises a detector capable of recording the intensity of fluorescent radiation emitted by said particle.