WO1999060376A1

WO1999060376A1 - The method of microobjects' study

Info

Publication number: WO1999060376A1
Application number: PCT/RU1999/000152
Authority: WO
Inventors: Gennady Fedorovich Yaskevich
Original assignee: Individual
Current assignee: Individual
Priority date: 1998-05-20
Filing date: 1999-05-06
Publication date: 1999-11-25
Anticipated expiration: 2000-11-20
Also published as: AU3962999A; RU2154815C2

Abstract

The invention is assigned to a class of tools meant for study and analysis of particles and materials with the aid of optics. It may be applied in medical research, geophysics, mechanics, chemistry, powder metallurgy, control of environment etc. The aim of the invention is to raise measurement accuracy. Microobjects under study are irradiated by a radiation bundle whose maximum linear size of coherency volume in the area of irradiation does not exceed 30 % of an average distance between particles in space. Images of the studied microobjects are formed by an optical system and upon reading, their geometric parameters are read at the level of a signal depending on illumination coherency and aperture angle of the optical system.

Description

THE METHOD OF MICROOBJECTS ^Λ STUDY

Area of technology The invention is assigned to a class of tools meant for study and analysis of particles and materials through the use of optics or in more exact terms to a class of systems based on registration of a light flux scattered by individual particles. The invention may be applied for medical research, geophysics, mechanics, chemistry, powder metallurgy, control of environmental pollution etc.

Background of technology Methods and instruments for study of microobjects based on registration of a light flux scattered by individual particles are widely known [S.P. Belyaev at al. Optic-electronic methods of aerosol study. Moscow, Energy Publishers, 1981, p.p.12-98.] . In the course of measurement particles are passed through a measuring volume irradiated by a special light source and intensity of a light flux scattered by each of them is measured.

The indirect character of measurements is said to be the basic peculiarity of methods and instruments based on this approach. The ambiguity of relationship between a value measured directly (e.g. intensity of scattered radiation) and a parameter to be determined (e.g. a size of a microobject), makes it next to impossible to regulate an error of measurement by the abovementioned tools, when forms and optical properties of microobjects under study are not known in advance.

Methods of dispersion analysis of microobjects including illumination and image formation of microobjects under study with subsequent measurement of these images^' size are known [ The US patent No 3390229, cl. 176-8 , 1968; US patent No 4207001 , cl. G 01/15/02, 1980]. The true size of the objects as well as their concentration in a volume are judged by data being obtained.

However when the aforementioned methods are applied in reality, difficulties emerge since only a small share of the total amount of particles moving in a volume falls exactly into a focusing plane (the plane optically conjugated with the plane of an image register) of an optical system and is registered with true size and range of contrast. Images of most of particles appear to be defocused , therefore their size is determined with an error which may reach hundreds of percent. Since images of small objects are most of all distorted due to defocusing, a working volume size of an instrument appears to be dependent on a size of particles. Consequently the error related to the size of microobjects leads to the error related to their concentration, which also may reach hundreds of percent.

The troubles mentioned above are partly eliminated by dispersion analysis method [author^'s certificate of the USSR No 545174, cl. IPC G 01N 15/02, 1975]. When this method is applied, microobjects moving in a volume are partly irradiated by coherent radiation, their images are formed at a register display and the inner and outer diameters of the first light diffraction circles around defocused images of microobjects are measured. By data being obtained the true size of both particles and a working volume (i.e. a volume where microobjects are calculated with a view to determine their concentration) is calculated. However this method is fairly complicated, especially it is difficult to make accurate measurements when images are automatically processed.

Contrast of light diffraction circles and consequently an amplitude of a signal gained when they are scanned in the course of measurements is several times less than that gained from dark segments of microobjects' images. It hinders emission of signals from circles against a background of noises related to an optic-electronic path of an instrument being applied. Noises, caused, among other things, by comparatively high coherency of illumination, give rise to the situation when particles considerably removed from a focusing plane may be registered several times or not registered at all depending on a signal noise component value at the moment of scanning a given segment of an instrument's field of vision. As a consequence, errors related to both, size of microobjects and their concentration emerge.

Disclosure of invention The aim to raise reliability of studies through the increase of measurement accuracy constitutes the basis of this invention. This aim is attained by the fact that in the well-known method of microobjects^' study (it includes partial of microobjects under consideration by coherent, formation of their images with the help of an optical system, reading and subsequent analysis of signals coming from images) microobjects are irradiated by a radiation bundle, whose maximum linear size of coherency in the area of irradiation does not exceed 30 % of an average distance between particles in space. Geometric parameters of images are measured at the level of a signal coming from them. A value of the signal is set depending on irradiation coherency. In the course of measurements geometric parameters of images 95 are measured at the level of the signal U coming from them. A value of this signal is set by calibration or determined by the following relationship:

( 1 ) U = 0,28 + Uo , where

100

λ - average wave length in radiation spectrum;

Ik = λ / oc o _\ 1 ,22 ² + 16 / α o ² - maximum linear size of a radiation bundle coherency in the area of irradiation of microobjects; cc , α o - flat aperture angles of image formation system and irradiation

105 bundle accordingly;

Uo - amplitude of a signal gained when focused images of microobjects either studied or calibrated (reference) are read.

A further increase of measurement accuracy may be achieved by measuring image parameters of those microobjects only whose U signal no amplitude differs from the set level by not less than a root-mean-square value of photodetector^'s noises inherent in the aforementioned signal and/or by additional measurements of image parameters of microobjects at least at one more level of a signal coming from them. In case microobjects are studied in a flux, their images are projected on to us a lightsensetive layer of a photodetector (a ruler or a matrix of lightsensetive elements) in an impulse exposure mode, the obtained images are scanned and parameters of a videosignal coming from them are measured.

To increase statistical support of results a size of the space volume,

120 where microobjects are studied is modified by means of concurrent modification of illumination coherency and U measurement level of geometric parameters of images. To increase objectivity of studies a computerized analysis of

125 microobjects is performed. With this in view, in the course of scanning, images of microobjects are entered to computer storage and their parameters are measured. Thereafter images are displayed being grouped in one or several frames and results of the computerized analysis are visually assessed to reveal some other properties of

130 microobjects in addition to those determined by computer programme.

A comparative analysis of the proposed method with its prototype shows the following difference. First, microobjects are irradiated by a radiation bundle. The maximum linear size of coherency of this bundle in the area of irradiation does not exceed 30 % of an average distance

135 between particles in space. Second, geometric image parameters of microobjects are measured at the level of a signal coming from them. This signal depends upon illumination coherency. Hence, the proposed method meets requirement criterion of the novelty . The essence of the method is as follows.

140 It is based on the use of peculiarities of microobjects^' image structure and properties under conditions of partly coherent illumination, as well as peculiarities of microobjects^' distribution in space.

The ability of groups of microobjects illuminated by a high

145 coherency light bundle to form in space the so-called specie-structures i.e. a peculiar kind of intensity distributions of radiation scattered by microobjects, is widely known. Formation of species is caused by interference of radiation scattered by individual particles. Interference brings about formation of light and dark spots often similar in form with

150 the objects scattering radiation. When dispersion medium is studied using the prototype method, the abovementioned similarity is observed in an image plane even with comparatively low concentration of particles in a volume ~ 10³ ÷ 10⁴ cm -³. It causes formation of false

155 images, increase of a measurement error and restricts ability of the prototype method to study microobjects.

The proposed method prevents against this phenomena because here microobjects are irradiated by a radiation bundle. A coherency volume of the bundle is selected so that in the course of study at any

160 moment of time with high degree of probability no more than one particle may be found in it. Thus the conditions are created when the waves which interfere in an image plane are practically incoherent and do not generate species.

A coherency volume of a light bundle in the area of irradiation of

165 microobjects here and henceforward is considered to mean the volume of space [L.M. Soroko Fundamentals of holography. Moskow, «Nauka», 1971 , p. p.241-247; M.Born, E.Volf Fundamentals of optics. Moskow, «Nauka», 1970, p. p.541-545] around a focusing plane of an optical system forming images of microobjects, bounded by surface, on which a no complex degree module of a wave field coherency created by a radiation bundle, reaches the first zero. By analogy with [L.M. Soroko Fundamentals of holography. Moskow, «Nauka», 1971, p.247, and M. Franson Optics of species. Moskow, «Mir», 1980, p.12] a coherency volume of a light bundle Vk for an optical system with radial symmetry

175 (a pupil has a form of a circle) may be found from the following correlation:

( 2 ) Vk = π rk ^{2 •} δ = 1 ,49 π ^• λ ³/α ₀ ⁴, where r k = 0,61 λ / α ₀ - radius of coherency surface of a radiation bundle in section, perpendicular to its optical axis;

180 δ = 4 λ/α o ² - size of illuminator bundle coherency volume along the optical axis ; λ - an average wave length in a source radiation spectrum ; 2α o - an angle, at which a radiation source can be viewed from the point 185 of observation (In case under consideration it is a point of a focusing plane, which corresponds to a vision field center of a system forming images of microobjects). If a condenser or any other element increasing irradiation of microobjects under study is used for the purpose of source radiation concentration, 2cc₀ is an aperture angle of a condenser 190 on the side of microobjects being irradiated.

The maximum linear size Ik of this volume is as follows: n

( 3 ) lk = λ /ct o V 1 ,22 + 16 /ot o ²

195 When illumination coherency is selected it is taken in to consideration, that distribution of microobjects in space adheres to the Puasson law [L.V. Radushkevich Application of ultramicroscope for aerosol studies and fluctuation law of a number of article, weighted in the air. «JETPh», 1935, ch.5, uss.7, p.40.] and probability to find in the

200 volume particles with a distance between them less than 10÷20 % of a «modal» (average) distance at the given concentration of microobjects does not exceed several percent. Therefore if microobjects are irradiated by a radiation bundle with maximum linear size of coherency less than 10 ÷ 20 % of an average distance between particles in a volume,

205 radiation bundles scattered by particles will not generate in a plane specie-structure images, degrading quality of image analysis.

Numerous experiments showed that when microobjects moving in a volume are studied, species practically have no effect on image quality if Ik does not exceed 30 % of an average distance between

2io particles in space.

However, prevention of specie generation and elimination of their effect upon image quality is necessary but not sufficient to ensure high δ

accuracy measurements of microobjects^' geometrical parameters (e.g.

215 size, surface square, volume, form etc) and their concentration in a volume. As it was mentioned above when geometrical parameters and concentration of microobjects moving in a volume are measured the chief cause of errors has to do with distortion of images occurring because most of articles do not fall exactly into a focusing plane of an

220 optical system. At that a character and degree of distortions depend on illumination coherency and disfocusing size, and an error of geometrical parameters^' measurements depends on an image signal level, at which the analysis of this image is conducted.

An increase of measurement accuracy by the proposed method is

225 attained due to the image property found out experimentally. According to this property for any illumination coherency in a signal gained from reading images of microobjects, the signal level is available at which image size in a brood range of defocusing remains unchanged. A size of this level unambiguously depends on illumination coherency. Therefore

230 to raise realiabity of microobjects^' study by the proposed method it is stipulated that measurements of image geometrical parameters should depend on illumination coherency of particles.

Figures reveal the essence of the method, where ^'41 ^ gives typical empirical relationships between relative size of images and Δz distance

235 between a particle and a focusing plane of an optical system at various illumination coherency and various levels of measurements .

Curves designated by figures 1 ; 2; 3, snow relative variation of image size when images are defocused at signal levels equal to U=0,3Uo; U = 0,5 Uo ; U = 0,7 Uo accordingly.

240 Curves derived at illumination close to incoherent α o > α (r k <

0,8 mkm) are identified by dotted line. Curves derived at partially coherent illumination α o = 0,3 (r k = 245 15 mkm) are identified by dash-dot line.

Curves derived at illumination close to coherent α o = 0,015a (r k = 300 mkm) are identified by full line.

Areas, where at corresponding signal levels a size image measurement error exceed several percent, are identified by horizontal 250 and inclined hatching. d (Δz = 0) - diameter of a focused image. d (Δz) - diameter of a disfocused image with Δz distance between a microobject and a focusing plane of an optical system. r k value for radius of coherency surface of a radiation bundle in 255 section, perpendicular to its optical axis, is adopted as a quantative characteristic of illumination coherency with the fixed aperture of an optical ( see expression ( 2 ) :

2/5 presents an empirical relationship between optimal level U of 260 image geometrical parameters measurement and illumination coherency. The curve described by expression (1) is identified by dotted line. The area of dispersion U in size of microobjects ranging from several to hundreds microns is identified by hatching.

3/5 gives a block-scheme of the instrument required to realize the 265 proposed method of microobjects^' study.

1 - Impulse light source.

2 - Illuminator optical system.

3 - Device for modification of illuminator light bundle coherency.

4 - Optical system of microobjects^' image formation. 270 5 - Transmitting TV camera.

6 - Device for image processing.

7 - Videomonitor. 4/5 shows distribution in size of monodispersion latex with an

275 average certified diameter of articles to be d av. = 6,9 mkm.

5/5 gives distribution in size of fog particles in a stream of superheated vapor.

The curves given by 1/5 show that a maximum distance between a particle and a focusing plane, at which its image may be registered

280 considerably depends on illumination coherency. It is also shown that variation of image size with defocusing may have a tendency for decrease or increase, may be ambiguous or ever oscillating in character depending on radiation source coherency and a measurement level. So improper selection of a measurement level causes errors in measurement of

285 microobjects^' geometrical parameters.

From 1/5 it also follows that each value of coherency corresponds to it own (optimal) level of measurement U, at which a range of values Δz with permissible (e.g. 1 - 2 %) variation of image size caused by defocusing, reaches its maximum. So, for r k < 0,8 mkm (illumination

290 close to incoherent) optimal measurement level U equals ~ 0,3Uo (Uo - amplitude of signal coming from a focused image of a particle), for rk = 15 mkm U is close to 0,5 Uo, and for illumination close to coherent U ≤ 0,75Uo.

2/5 presents empirical relationship between optimal measurement

295 level and illumination coherency. The hatched area depicts experimental data dispersion in size of particles ranging from several to hundreds micron and variation of tg α from 0,1 to 0,6. The curve, described by the aforementioned expression ( 1 ) is identified at 2/5 by dotted line. When the proposed method is applied in reality it might be

300 reasonable to substitute curve ( 1 ) by the analogous curve: ( 5 ) U = 0,28 + Uo

2 ( 1 + 0,65 - cc o / α ) where optimal level is expressed by relationship between illuminator aperture angles α o and image formation system α .

It may also be recommended to develop calibration relationship between optimal measurement level and, say, ratio oc o / cc . The calibration curve derived from the specific device emplayed by the proposed method depicts U value more adequately, since it automatically considers all peculiarities of both the specific image formation optical system and the devices for image reading and processing. It may help to refine U value, derived from (1) or (5).

Further from the curves given at 1/5 it is evident that at any measurement level (including optimal) in the neighbourhood a working volume foundaries some areas (identified by hatching) are available where due to loss of contrast an amplitude of a signal coming from a defocused microobject image becomes close to a measurement level value. Due to image distortion and noises of an optic-electronic path of the instrument emplayed by the proposed method, size of particles falling within these areas may be measured with a considerable error.

To eliminate the aforementioned areas from a working volume, in the proposed method amplitude of a signal is reduced as images of microobjects are defocused. For this purpose selection of defocused images and isolation of working volume boundaries along the axis of an optical system are made by measuring image parameters of those microobjects only, whose signal amplitude differs from optimal U by no less than mean-root-square value of photodetector^'s noises, contained in a signal coming from images. Owing to this, subsequent analysis will consider only signals coming from images of those microobjects. which are measured at the set U level with sufficient accuracy (e.g. an error

335 does not exceed ± 1 ÷ 2 %) and the availability of signal noises practically have no effect on measurement results.

The similar result in accurate limitation an a working volume along the axis of an optical system may be attained also by measuring image size at the additional signal level Ui, which differs from level U,

340 derived from expressions (1) or (5) by no less than mean-root-square value of photodetector' s noises, contained in a signal coming from images. At that , if image size at Ui exceeds zero or differ from size at U by no more that some preassigned value (e.g. 30 %) a microobject is considered to be within the limits of a working volume and is measured

345 at basic level U with preassigned accuracy. A signal coming from this microobject is taken into account when volume concentration of particles is calculated and a function of their size distribution is constructed. Otherwise ( e.g. size at Ui is less than 30 % of U level size or equals zero, which is to say that defocused images signal amplitude

350 decreases to a value less than Ui ) a microobject is considered to be outside the limits of a working volume and is excluded from analysis. Hence defocused images, which could be measured with an error exceeding permissible value, are selected.

Measurement of image size additionally taken not at a single but

355 at several levels makes it possible to obtain information concerning the inner structure of microobjects (e.g. the inner areas, which absorb source radiation more intensively; insertions etc.).

The way discribed above is appropriate to measure parameters of microobjects moving in a volume relatively slow, when in a time of

360 particle registration its image is not noticeably «blurred» by travelling in a plane of a photodetector. A time of image registration is considered to mean here the period of time begining when a particle appears in a visual field of the image formation system and finishing either when

365 image reading is accomplished or when a particle escapes from the field of vision. To prevent image distortion caused by movement of microobjects under study, their images are projected on to a photodetector in the impulse exposure mode. The impulse exposure mode is realized, say, by the use of impulse radiation source and / or

370 by electric strob of a photodetector lightsensitivity. As a result the «set» images of moving particles are obtained with no distortion caused by their movement in space. Images are subsequently scanned to obtain a videosignal (videoimpulse), whose amplitude-time characteristics are determined by illumination and geometric parameters of images.

375 Measuring videoimpulse duration at U level, determined by expression (1) or (5) (where, Uo - amplitude of videoimpulse obtained as focused images of particles are scanned) geometric parameters of microobjects under study are defined.

Further more from relationships given by 1/5 and 2/5 it follows

380 that one more important image property is available with partly coherent illumination. It lies in the fact that modification of illumination coherency and selection of measurement level appropriate for this coherency ( according to expressions ( 1 ) or ( 5 ) allows a working level size of the instrument to be modified over wide limits. Owing to this it

385 appeared to be possible to provide realiable statistics of measurement as microobject are studied over a wide range of their concentrations without increase of time required to accumulate information. At the same time high accuracy of measurements taken to define geometric parameters of microobjects is retained.

390 The best way to realize the invention

The proposed method is realized with the help of the instrument ( 3/5 ), which incorporates impulse radiation source 1, illuminator opticalsystem 2 with device meant to set coherency of

395 illuminator light bundle, image formation optical system 4, transmitting TV camera 5, imageprocessing device 6, connected to TV camera outlet, videomonitor 7 meant to observe microobjects under study.

Illuminator optical system 2 includes lens system, capable to

400 realize, say, any one of methods of microobjects^' illuminator, which is currently known (Keller illumination, critical illumination etc.).

Device modifying illumination coherency 3 includes a band light filter with a pass band of Δ λ << λ av., where λ av. - average wave length in a filter pass band; and also an aperture diaphragm, which by

405 modification of diaphragm diameter ensure adjusting of coherency radius ( see above ) of illuminator light bundle within the following limits :

(6) 0,3 λ av. / A < r k < 100 λ av./ A , where

A - lens aperture of image formation system.

4io Image formation optical system 4 is a microprojection optical system generating images of particles at a lightsensitive element of transmitting TV camera 5, which converts images of microprojects to a videosignal.

Image processing device 6 includes a conversion block required to

415 convert a videosignal into digital form and computer required to process images of microprojects converted to digital form and to create an image archive. At that the archive is so organized that images of microobjects which fell in a visual field of the optical system over the whole period of information accumulation, are grouped within one or several frames in

420 horizontal (or vertical) series sequentially as they fell in the field of vision of the aforementioned system. This type of archive organization allows operational control of measurement results and quality of image processing by displaying images of all microobjects analyzed by the 425 instrument.

The proposed method should be used in the following sequence. First, the instrument should be calibrated.

Calibration has to fulfill the following tasks: 1. Determination of a working volume size of the instrument. 430 2. Determination of Uo - amplitude of a signal coming from focused images of microobjects. 3. Determination of U - optimal measurement level for image geometric parameters of microobjects.

Determination of a working volume size of the instrument,

435 includes measurement of visual field size x B of image formation

optical system and measurement of a working volume extension Z along its optical axis.

Measurement of visual field size is made by technique traditional for optical microscopy using standard object-micrometer. An object-

440 micrometer with a scale factor less than 10 mkm is placed in a focusing plane of the optical system. Image focusing is adjusted and monitor 7 displays a visual field size in two mutually perpendicular directions

(horizontal - a,vertical - B).

To measure a working volume size Z along the axis of the optical

445 system as well as Uo and U values, reference particles, for instance, latex particles are used. Particles are deposited on a microscopic slide. The slide is placed in the visual field of the instrument. With the help of the device, similar to a fine adjustment micrometer screw images of latex particles are adjusted while being displayed by the monitor 7 .

450 Thereafter Uo and U are determined. For this purpose an oscillograph is connected to TV camera 5 outlet to determine an

455 amplitude videoimpulse Uo coming from focused images of particles.

From expressions ( 1 ) or ( 5 ) U values for various ( I k - α ) or oc o / o values are calculated. Results of calculations are entered into computer storage and subsequently used when measurement are taken.

U measurement level values may be obtained by construction of

460 the calculation curve (e.g., U = f ( α o / o ) . For this purpose images of latex particles are adjusted again and results of measurement of their geometric parameters are fixed (e.g. size or particles). Then for various values of oc o / oc , a size of particles is measured at various levels of signal Ui and in this case both focused and defocused images are measured.

465 When all these measurements are accomplished, for each oc o / oc value U value is found, at which the result of measurement is less dependent on displacement of the microscopic slide with particles in relation to a focusing plane of the optical system. By data being obtained the required calibration curve is constructed.

470 To cut time for calibration of the instrument an optimal level U is determined in two steps. First, U value is calculated by relationships (1) or (5) and oscillograph measurement of Uo (see above)Then in sequence given earlier, U value is refined for a number of oc o / oc values. By data being obtained the required calibration relationship U = f (oc o / oc) is

475 constructed.

On accomplishment of the aforementioned operations a working level size Z along the axis of optical system 4 (3/5) is determined. With the help of the micrometer screw images of latex particles are adjusted again. Image processing device 6 is put into operation and geometric

480 parameters of latex particles are measured (e.g. diameter, perimeter, surface square etc.). With the help of the micrometer screw images are defocused while measurement of particles is being continued at regular 485 intervals. By limb of the micrometer screw Δ z distance is indicated, at which registration of particles ceases because image contrast is lost due to defocusing. Z = 2 Δ z is found. Similar measurements are taken for various oc o / oc values (see expression) (5), i.e. with various illumination coherency. Measurement data Z and x B are entered

490 into computer storage and subsequently used to calculate volume concentration of microobjects.

At this point calibration is finished and measurements are started. Microobjects are passed through irradiated volume, all blocks of the instrument are put into operation. Images of microobjects are viewed at

495 videomonitor 7 (3/5) display and measurement results at computer display. By oscillograph, amplitude of a signal coming from focused images is fixed and values of Uo and U are refined if required. By modification of aperture diaphragm of block 3 (3/5) and setting appropriate level of measurement (derived from calibration relationship

500 U = f (oc o / oc) ) mode of measurement is selected so that in permissible time of information accumulation τ the instrument could register, say, not less than 1000 microobjects. In this case an error of measurement stemming from statistical support of data will appear to be within 3 %. Of more reliable statistical support is required, ether time of information

505 accumulation τ is increased or diameter of aperture diaphragm of block 3 (3/5) is decreased, thereby illumination coherency of microobjects and hence a working volume extention along the axis of the optical system 4 (3/5) is increased.

When coherency is selected it is considered that maximum size of

5io light bundle coherency volume Ik (see expression ( 3 ) in the area of irradiation of microobjects should not exceed 30 % of an average distance between microobjects in space.

When required measurement level U is set, microobjects^' study is 515 continued. And here geometric parameters of those microobjects only are measured, whose signal amplitude does not exceed U l value:

(7) U l = U + U n , where

U n - mean-root-square value of noises of transmitting TV camera (rated value). 520 If image processing device 6 (3/5) has a noise suppression system with a noise suppression coefficient β, it is possible to measure geometric parameters of microobjects. whose signal amplitude exceeds U 2 value:

( 8 ) U 2 = U + U n / β .

Owing to this areas ( see 1/5), where results of measurement are

525 distorted by noises in a signal coming from microobjects (hatched areas) are eliminated from a working volume.

Similar aim is attained by additional measurement of microobjects^' geometrical parameters at levels U l or U 2 . But further, measurement results of these microobjects only are considered, whose

530 image size at U l (or U 2) exceeds zero.

In the course of measurement all images, which in time of information accumulation τ fell into field of vision of optical system 4 (3/5) , are converted to digital form and entered into computer storage. On accomplishment of information accumulation and signal processing

535 initial images (or processed images, e.g. images subjected to noise suppression or contrasting) are activated at a display of monitor 7 (or display of computer). Images of all microobjects are grouped within one or several frames for concurrent viewing and operational assessment of automatic processing quality. Within a frome images are grouped by

540 morphological properties (e.g. spherical and non-spherical) or located sequentially as they fell in field of vision of the system. In the latter case spatial characteristics of microobjects under study are judged by the data being obtained.

545 Owing to impulse radiation source the proposed instrument makes it possible to study both microobjects moving slowly in a volume, and microobjects moving fast.

Industrial applicability The proposed invention being used in actual practice will provide

550 the means of solving a broad range of fundamental and applied problems in various field of physics, chemistry and biology, will make it possible to exercise control over technological processes in many branches of industry employing or producing various types of microobjects, may find application in mobile diagnostic laboratories of

555 expeditious response and ecological condition control.

4/5 presents results of studies on quality of monodisperse latexes produced by one of American companies and meant for calibration of instruments. The studies have been conducted with the help of the device shown in 3/5.

560 From data, given in 4/5 it follows that an average diameter of latex particles measured by the device emplayed in the proposed method, equals 6,9 mkm, which corresponds to latex ratings. However in suspension along with the bulk of particles similar in size larger particles exist, which are actually conglomerates of basis type articles easily

565 observed at display of videomonitor. This fact should be taken into consideration in calibration of instruments based on indirect methods of measurement and having no way of interpreting results of such measurement with a fair degree of reliability.

5/5 presents results of investigations of fog microstructures arising

570 due to formation of microcracks in a high pressure steam boiler. It is obvious That under these conditions fog droplets are formed with size ranging from 0,5 to 7 mkm and distribution function, typical for fogs arising from turbulent gas mixing [A.G. Amelin Theoretical basis of fog

575 formation from gas condensation. Moscow , «Chemistry», 1972, p.p.86-110] .

The proposed method has the advantage over its prototype because it allows an error related to both measurement of size and concentration of microobjects in a volume, to be considerably (several

580 times) decreased. It is attained by solution of three basic and interconnected problems emerging when microobjects in a volume are studied through analysis of their images:

1. The problem of true size of microobjects, which fail to fall exactly into a focusing plane of image formation system (a focusing

585 plane is the plane in space of objects optically conjugated with the image registration plane). In this case a particle image is usually appeared to be blurred and actually present the Gabor microhologram ( see J. Goodman Introduction to Fourie-optics. Moscow, «Mir», 1979, p.270.), where illuminator light bundle is the reference bundle and

590 microscopic bundle is formed through light diffraction by particle.

2. The problem of accurate measurement of space volume size, where microobjects^' parameters are measured. Accurate measurement is required here to calculate microobjects' concentration. The difficult task is to define position of working volume boundaries along the axis of the

595 optical system, as when a particle removes from a focusing plane, images are blurred smoothly and methods currently known turn out to be unable to define when register device ceases to reproduce images due to lost of contrast. And the task of even greater difficulty is to define when measured image size of a microobject ceases to correspond it its true size.

600 3. The problem of regulated modification of working volume size, i.e. the volume of space where microobjects^' parameters are measured without increase of an error.

605 In the method currently known all attempts, say, to increase working volume size with the purpose to enhance statistical support of measurements lead to decrease of resolution or increase of errors associated with necessity of measuring geometric parameters of particles considerably removed from a focusing plane (i.e. measuring parameters

6io of highly focused images).

A side benefit of the proposed method is the extension of its functional potentialities in case microobjects studies are conducted automatically. The proposed method offers a way of operative assessing quality of automated image processing and makes it possible to reveal

615 properties of microobjects in addition to those provided by processing programme. It is achieved by accumulation in computer storage of images of various microobjects which fell into visual field of the optical system over the whole period of time of information accumulation and by grouping these images in one or several frames to be made available

620 for operative visual control. Within a frame images may be grouped either by morphological properties or sequentially as they fell in the field of vision of the optical system, by this a possibility for operative assessing morphological and spatial properties of microobjects under study is provided.

625

630

Claims

CLAIMS 635

1. The method of microobjects' study, including irradiation of the studied objects by partially coherent radiation, formation of their images, say, by an optical system, reading and subsequent analysis of signal coming from images of microobjects difftering by the fact that

640 microobjects are irradiated by a radiation bundle whose maximum linear size of coherency in the area of irradiation does not exceed 30 % of an average distance between particles in space, geometric parameters of images are measured at the level of a signal coming from them, and the value of the signal is set depending on

645 illumination coherency.

2. The method of microobjects^' study in item 1 difftering by the fact that geometric parameters of images are measured at the level a signal, whose value is set by calibration or determined by the following relationship :

650 1

U = 0,28 + Uo , where

2 ( 1 + 1,5 / l k - oc o - d o )

λ - average wave length in radiation spectrum;

655 Ik = λ/ oc o v⁷ 1,22 ² + 16/ oc o² - maximum linear size of a radiation bundle coherency in the area of irradiation of microobjects ; o , oc o - flat aperture angles of image formation system and irradiation bundle accordingly;

Uo - amplitude of a signal gained when focused images of microobjects 660 either studied or calibrated (reference) are read.

3. The method of microobjects^' study in item 1 difftering by the fact that image parameters of those microobjects only are measured

665 whose U signal amplitude differs from the set level of measurement by not less than a root-mean-square value of photodetector's noises, inherent in the aforementioned signal.

4. The method of microobjects^' study in item 1 difftering by the fact that image parameters of microobject are additionally measured

670 at least at one more level of a signal coming from them.

5. The method of microobjects' study in item 1 difftering by the fact that in case microobjects are studied in a flux, their images are projected on to a photodetector (a ruler or a matrix of lightsensetive elements) in an impulse exposure mode, the obtained images are scanned

675 and parameters of a videosignal coming from them are measured.

6. The method of microobjects^' study in item 1 difftering by the fact that a size of space volume, where microobjects are studied is modified by means of concurrent modification of illumination coherency and U measurement level of geometric parameters of images.

680 7. The method of microobjects^' study in items 1 and 5, difftering by the fact that computerized analysis of microobjects is automatically performed; with this in view in the course of scanning images of microobjects are entered to computer storage and their parameters are measured, thereafter images are displayed being grouped in one or

685 several frames, then results of computerized analysis are visually assessed to reveal some other properties of microobjects in addition to those determined by computer programme.

690