DE4019929A1 - Enclosing main stream of suspended particles by surrounding stream - introducing non-rotating, wide, pure, transparent fluid pref. electrolyte around input channel in nozzle chamber - Google Patents

Abstract

The liq. of the core stream (4) is introduced into a first input channel with an outlet nozzle (6) extending in the direction of an output channel (3) of a nozzle chamber (9) with a smooth, hydrophile or aerophobe streamlined inner wall. The liq. of the surrounding stream (26) is introduced into a second channel opening in the inner wall in the region of the circumference of the first channel. The surrounding stream is non-rotating and wide to cover at least the cross-section of the nozzle chamber. An obstacle in the first input channel and a nozzle (6) setting a small angle w.r.t. the length direction of the channel form a partial stream in parallel and subsidiary streams (30',30''). USE/ADVANTAGE - Trhoughflow particle counter, the particles being organic or inorganic but pref. living cells. Particle sorter can be coupled to the appts. Fully stable so that air bubbles are not formed either in the starting phase or while operating. Allows epi- or direct lighting of flowing particles with laser in same appts.

Description

The invention relates to a method for including a thread-like consisting of a fluid particle suspension thin core flow with an enveloping flow of a pure, transparent fluid, in particular an electrolyte. they also relates to a device for performing the method and the use of the device in an analyzer and in an analysis system.

In the following, if transported by the core flow, to be analyzed particles, it can be inorganic or organic, inanimate or animate matter act. The term "particle" is also intended in particular to be living Cells include. Furthermore, in the course of the following text alternatively related to the meaning of the particles from epi or reflected light mode on the one hand and from direct light mode on the other hand - e.g. B. from an epilaser or from a direct laser - spoken. These modes differ themselves primarily by the way they are captured  Light that already interacts with the respective particle kicked: In the epimode there is an area the direct (original) beam, e.g. B. scattered light, measured.

A nozzle chamber for creating a hydrodynamically focused Core flow in combination with a core flow enclosing flow is first described by P. J. Crosland-Taylor, Nature, Jan. 3, 1953, Volume 171, No. 4340, pages 37/38. In this device the enveloping liquid quasi radial through two entry holes at the top, that is, at the upstream end, a cylindrical cavity injected with a vertical axis whose diameter is larger than the one that is entry holes. This cavity becomes today referred to as "nozzle chamber". Under the term "focus" becomes a kind of radial constriction in the present context understood or bundling the core flow.

According to Crosland-Taylor (op. Cit.), A suspension of blood cells - as core flow - axially through an injection needle 0.125 mm in diameter injected into the nozzle chamber, and the envelope flow is around the - now called "nozzle" - needle and thus also formed around the core flow, so that the above-mentioned flow combination results. In the known, the combined flow becomes vertical directed downwards. The hydrodynamically focused Blood cell suspension, i.e. the core flow, is included observed under a microscope; the cells can be optically counted because they scatter a beam of light directed at them. After counting, the cells and the surrounding cells leave  Envelope flow the nozzle chamber radially through a hole that at Downstream evenings of the chamber is provided.

The core flow containing the blood cells had according to information von Crosland-Taylor (op. cit.) a diameter of about 10 µm, the diameter around ± 50% Average fluctuated. The article also reports that the core flow fluctuated even more if only one Entry hole was used to introduce the envelope liquid. (Here and below there is a linguistic difference between the envelope liquid itself and through it in the nozzle chamber formed flow shape.) This instability (the core flow) can be explained by the fact that the flowing into the (filled) nozzle chamber Sheath liquid their moment on the one already in the chamber Liquid transfers and therefore a slow rotation of the enveloping flow around the nozzle chamber axis. There the needle (or nozzle) was probably not exact at the time in the nozzle chamber axis, caused the rotation of the Envelope flow a corresponding fluctuation of the blood cells transporting core flow.

There are modern, so-called "jet analyzers" that work in a similar way Wise like the aforementioned Crosland-Taylor analyzer be constructed so that those also occurring here Stability problems at least in part on the characteristic instabilities of the core flow are caused by the early version of the Nozzle chamber is recognizable.  

Such fluctuations in the central flow are of course only acceptable if the analysis volume is large enough. This includes Lenses with a long focal length, that means great depth of field. Because such lenses are only a small fraction of the scattered or fluorescent emitted on the particle Collecting light was the modified nozzle chamber, that is that of the jet type, put into practice. However, that was only possible after the development of high-power lasers.

In the jet analyzer, the nozzle chamber tapers at its Stream evenings in a very thin capillary tube with an inner diameter of about 0.1 mm. If this part of the capillary tube Made UV transparent (down to about 200 nm) the capillary tube itself is suitable as an analysis volume. The focal plane of the lens can be on the center of the capillary tube be served. Alternatively, the focal plane the lens to a position outside the capillary tubes, where the jet flow in free space in a downward direction flows, to be aligned. In the analysis volume are the core flow, the optical axis of the lens and the laser beam is essentially perpendicular to each other, such that the core flow is approximately in the focal plane of the lens; see. see M.A. van Dilla et al., IEEE Transactions in Nuclear Science, 1977, NS-21, page 716.

If the named capillary tube - as in the case of the drop deflector after M. J. Fulwyler, Science, 1965, volume 150, page 910 - is not transparent, the analysis volume placed in the open jet flow. The instability of the  Current causes problems not only in the analysis, but also in the sorting operation because of the instabilities generated disturbance of the jet flow the drop formation according to Fulwyler (loc. cit.) severely impaired and thus the sorting quality also deteriorated.

An additional disadvantage of jet analyzers is that Requires high power lasers. These devices are right costly and space consuming, and requires the full system an optical one for the necessary mechanical stability Table. Finally, the operation can be used because of the High power lasers in some cases only under strict Security precautions take place. It is therefore right for an easier to represent alternative with lighting lower performance and with simplified operating conditions has been searched with the aim of the particle analyzer train as a tabletop device.

It is clear that a high-power laser can only be Low intensity UV light source can be replaced if you have the large focal length and small lens Light-gathering power used in the jet system through a UV object with a higher numerical aperture (approx 1.3) can replace, whose light collecting ability accordingly, preferably larger by more than an order of magnitude than that to be used in the prior art Is objective. In such a case the particles would have to the core flow in a fairly flat channel of about Move 0.05 to 0.1 mm depth so that the focal plane of the mentioned strong lens to adjust to the core flow  is. A duct cover must be taken into account e.g. B. with a cover glass of 0.17 mm thickness, at a Focal length of the strong lens of only about 0.24 mm.

Such a system is described in German Patent P 19 19 628.2 described. However, the corresponding analysis results were pretty weak because of the core flow - as a result of the above described simple nozzle chamber - was not stable enough around each particle to be analyzed exactly through the focal plane to lead. W. Dittrich and W. Göhde therefore have one in the US-PS 37 38 759 described flow system developed in which the particles are not in the focal plane of the strong UV lens, but be moved perpendicular to it.

According to Dittrich and Göhde, the particles with a inexpensive and easy to operate High pressure mercury lamp lit by 100 W, whose UV component has about 10 mW. there has been a Koehler lighting optics used in incident light mode. After crossing the analysis volume, the particles quite abruptly by a so-called perpendicular to the optical Axis of the lens and thus perpendicular to the Particle stream flowing cleaning flow into another Channel washed away, which is the continuation of the channel, from which the cleaning flow comes. This channel system will in the known covered with a 0.17 mm thick cover glass, whereby the branch channel mentioned also as an output channel for the Particle / electrolyte mixture, which is under the effect a suction pump flows into a collection bottle as waste.  

Obviously, the channels and the vertical hole form in the aforementioned flow system in which the particles in Flow towards the focal plane, a flow system with the Form a vertical "T". In addition to the simplified lighting conditions there is a clear advantage to this system in that the small fluctuations in the core currents are none cause big mistakes in particle analysis because that Field of view of the lens fairly close to the optical axis is homogeneous. Because of this, you would get pretty good results expect this type of analyzer. Indeed it will however, this expectation is not always met.

Because of the 90 ° collision of the core / shell flow combination with the cleaning flow, the T flow system tends to turbulence in the vicinity of the analysis volume, which can affect the analysis itself. That some users still get good results with this type of analyzer is probably due to the fact that it there is a speed range in which the currents can collide without significant turbulence in the analysis volume to create.

Another disadvantage of the 90 ° deflection of the core / envelope flow combination is noticed when a small thread in the Flow is carried along. If this thread of distraction the Currents do not follow and therefore in the union volume of the channels, i.e. around the analysis volume should, it can clog up this volume and possible complete stoppage of the analyzer operation have as a consequence. That such blockages occur quite often  is both of this and direct observations other flow systems known. Cleaning such devices is often tedious and time-consuming, not counting the stress, who is working with such unsatisfactory equipment sets. Another disadvantage of the flow system von Dittrich and Göhde (op. cit.) is that as a result the turbulence flow described and as a result of the space requirement for the attached nozzle chamber, this analyzer not easily with currently known particle sorting can be combined. Particle sorting, in particular Sorting cells is becoming more and more necessary.

There are two other papers on cell analyzers, which should be mentioned for the sake of completeness. The one of these works concerns the two-parameter analyzer after E. Severin et al., Cytometry, 1983, Volume 3, No. 4, pages 308-310, in which an argon laser beam is applied to the flow system by Dittrich and Göhde (op. cit.). This laser beam reaches the core flow and thus the core analyzing particles through a in the main body of the Device cut perpendicular to the cleaning exit channel Channel. The authors do not report experimental Results. There is a major disadvantage of this analyzer but definitely in the non-combinability with one of the known sorting devices.

The other analyzer still to be discussed is by H. B. Steen, Cytometry, 1980, Volume 1, No. 1, pages 26-31. The particles, here cells of the latex spheres, become with a core / envelope flow combination  Jet stream to the top of an illuminated in reflected light mode Sprayed microscope glass. The fluorescent light is collected with a relatively low magnification UV lens. The UV lens is placed under the cover glass at the point where the jet stream and thus the particles hit the coverslip fall, arranged. For 1.5 µm beads and cells from about 10 µm in diameter gives good results, however this type of device cannot be combined with a sorter either will. The jet stream is also very likely just as unstable as that of the jet analyzer described above, because in both cases the same nozzle chamber type is used.

There is currently only one electrical analyzer or sensor of practical value, its original version by W. H. Coulter was invented in the mid-1950s (cf. van Dilla et al., op. cit.). This analyzer was originally used to count particles that come from a large Container through an aperture in another large container reach. The cross section of the aperture was only slightly larger than that of the particles to be analyzed. Both containers were filled with electrolyte. Contained in the current tank the electrolyte also contains the particles still to be analyzed, while there are already analyzed particles in the other container found. By applying electrodes a through the Leading electrical circuit opening closed. A was an essential part of the resistance of this circuit focused on the aperture. So if a (non-conductive or much worse than the electrolyte conductive) Particles slid through the aperture became a sudden  Resistance increase in the fluid system as electrical Voltage or current pulse registered.

In a more modern version of the particle counter explained last (see U. Zimmermann et al., Bioelectrochemistry and Bioenergetics, 1976, volume 3, pages 58-83) are cells in including a hydrodynamically focused core flow, which in turn consists of a particle-free envelope electrolyte is surrounded. This ensures that those to be examined Particles flow through the center of the opening, where that electric field is homogeneous to a high degree. On this enables precise classification of the cells, because, as has been found experimentally, the amplitude of the generated when the particle passed through the opening electrical impulse is proportional to the particle volume. In both versions, however, the particles go after passing the aperture is lost, so they represent another Handling, e.g. B. for sorting, not available.

Such further handling, in particular sorting, is possible in the flow system developed by the inventor according to US-PS 42 37 416. There the particles move with the Core flow through a very narrow part of the closed Channel system, which is referred to here as an aperture. The particles can therefore be analyzed according to their volume and in the same Core flow is passed on to a sorting system; see. the US PS also attributed to the applicant 41 75 662. In the known, the sorter can be in the same Basic body like the analyzer can be integrated, so that the Analyze and sort based on the same amount of particles  becomes possible. Experiments with these systems led for excellent results even when combined with optical lasers, the UV lenses of large numerical aperture and magnification and where reflected light after the Koehler lighting system for uniform illumination the field of view of the UV lens was used.

According to US-PS 41 75 662, the hydrodynamic focusing takes place and thus the generation of the constricted core flow in a nozzle chamber into which the core flow through a very flows in a narrow capillary tube ending in a nozzle. At this The liquid comes with the position of the core flow Liquid of the enveloping flow in contact. The the enveloping flow forming enveloping liquid is known in the nozzle chamber fed through a hole a few mm long, which is almost is perpendicular to the capillary tube or its nozzle. The The entrance hole of the enveloping liquid is on Current end of the nozzle chamber, at a point where the thin capillary tube also enters the nozzle chamber.

Because of the described geometry of the nozzle chamber and in it sufficient thin capillary tube according to US-PS 41 75 662 the capillary tube itself forms an obstacle in the flow path the enveloping liquid. The thin capillary tube actually splits the enveloping liquid in three flow parts. A first one Partial flow experiences an approximately 90 ° turn and flows under the capillary tube towards the tip of the nozzle, the others both parts of the flow flow half as a sub-flow around the capillary tube and meet in frontal collision at the top of the capillary. The two lower flows  repel each other by about 90 ° and flow towards the nozzle, where the three flow parts reunite and the one coming out of the nozzle Form core flow enclosing envelope flow. This core / shell flow combination then happens the above Cover.

There are several flow systems built according to US-PS 41 75 662 been. They all worked the same way. The nozzle chamber was free of air bubbles after a break-in period, it there was a very stable core flow of high quality. By incorporating hydrodynamic resistance into the flow the core fluid could be core flow diameters of adjust down to 1 µm, even when observing not with a stereomicroscope with 50x magnification swayed. The central flow was included in these cases colored black ink to make them distinguishable from the surrounding To make enveloping flow.

In the known system, however, the nozzle chamber could be operated free of air bubbles often only after many switch-ons, that is replenishments, because in general Air bubbles are formed in the part of the nozzle chamber where the thin capillary tube enters the nozzle chamber and generally in the starting phase, that is, if the empty duct system under the suction effect of one at the system outlet, for example over buffer bottles and silicone tubes, connected pump is filled. It was found, that these air bubbles have different sizes and through and during the frontal collision of the two  Partial flows of the enveloping liquid arise when the latter enters the empty nozzle chamber and these flow parts in Direction towards the chamber exit, that is towards the described aperture, be distracted. Because such air bubbles change the position and shape of the core flow and the this flow system can even make the latter unstable not very reliable, and because of this relative imperfection it has only marginal application in clinical Found practice because there are stable and reliable devices needed for quick and reliable diagnoses will.

The following results from the prior art described above: Large nozzle chambers, especially with rotating ones Liquid are undesirable because of such flow systems cause the core flow to fluctuate or flicker. Sharp (90 °) collisions of canals and currents must because of the resulting turbulence and air bubbles in such systems can also be avoided. Although one in the US-PS 41 75 662 described nozzle chamber relatively close the solution to the underlying task, a reliable one and to ensure stable operation Reliability of the known nozzle still to be desired, that is, a new nozzle chamber should be developed, which are practically 100% reliable in the sense of the above is to the stress that the operator with less experienced reliable facilities.

The object underlying the invention is therefore mainly in entering a flow particle counter  to create the type mentioned, to which a particle sorter can be connected and practically 100% in the above sense is stable and reliable. Education in particular of air bubbles in the nozzle chamber both in the switch-on phase as well as practically excluded in continuous operation. Furthermore, the analyzer itself should concern Maintenance and operations are to be improved, and it should alternative and simultaneous use of epi and Direct lighting of the particles flowing in the core flow with the help of lasers in one and the same device will.

The solution according to the invention is used in process and device and main usage claims. Improvements and further developments are in the subclaims described.

The invention makes it highly stable and reliable Multiparameter flow analyzer for examining Particles, especially cells. To the invention also include a process and means for complete containment a stable flow called core flow thin stream of current with another fluid flow, the Envelope flow. The core flow is said to pass through the center of a small tube or a channel of capillary size. The Union of the two flows is said to be in a nozzle chamber take place whose inner walls have such a geometry, that the enveloping flow at all times the capillary tube Operating without any turbulence and without generating air bubbles surrounds it before it comes out of the capillary tube  Core flow reached and embraced.

In the flow combination, the ones to be analyzed Particles carried into specified analysis rooms. Kicks there each particle depends on its physicochemical properties interacting with at least one sensor. The The result of such an interaction is an electrical one Voltage signal and / or a current pulse in the respective Sensor associated electronic system. The voltage signal or the current pulse can continue in others on the relevant Sensor switched electronic devices to be analyzed.

A typical facility to be used in the present context is a multi-channel analyzer that is suitable for this is the number of particles depending on one by the To represent sensor-determined physical quantity and / or to register. For example, if the physical size is the particle volume (e.g. the cell volume), the Multichannel analyzer a volume distribution of the suspended Deliver particles.

Normally optical in the context of the invention Sensors used. The particles to be examined are in the flowing suspension with light from the UV spectrum illuminated. With the help of a UV microscope lens the scattered or by fluorescence will match the previously colored particles collected emitted light. Currently is only a single electrical sensor for determining the Known particle size.  

The analysis volume of an optical sensor is by definition equal to the product of field of view (area) and depth of field (Depth of focus). This volume is often quite small and / or inhomogeneous. Therefore, the core flow during the Operating the analyzer when particles - one by one - pass the analysis volume, very stable at all times be. The invention therefore also relates to an improved one Nozzle chamber, in which the union of core and envelope flow, without creating turbulence and air bubbles is, so that an extremely stable and reliable core flow is produced in the nozzle chamber and a downstream Multi-parameter analysis device correspondingly reliable is working.

The invention primarily improves the operational quality of the Analyzer improved. In addition, however, the maintenance the device compared to the prior art simplified. It follows from the foregoing that the flow analyzer mainly in cytology and in this Area used in cancer research. The volume analysis is also very important in aerosol research.

The following improvements, among others, are achieved by the invention reached: The flows flowing in the device are made as short as possible to the probability the emergence of turbulence according to the Rayleigh principle to be kept to a minimum. The currents hit yourself at the smallest possible angle. Sharp (90 °) Flow deflections are avoided everywhere  Blockages in the channels causing such distractions to avoid. The core flow diameter is not very much larger than the particle diameter in order for each particle regardless of its size, the same analysis condition create. Nevertheless, the diameter of the core flow, if the analysis volume is large enough, even larger than that Minimum amount. There are no sharp corners or edges allowed in the flow system because of such edges tend to anchor air bubbles firmly. These would namely how solid pieces of material act on the flow and act accordingly distracting.

The nozzle chamber according to the invention should at least partially have an approximately rectangular cross-section around a rotation to prevent the enveloping liquid. In addition, the nozzle chamber tapered at its exit end so that its cross section finally in the cross section of the output channel transforms. The walls of the nozzle chamber as well as the walls of the other channels are made with a non-reflective, non-fluorescent Black, either because of the walls be painted accordingly or because the whole device or whose base body is made of such a material.

The output channel of the analysis device according to the invention serves also to the particles with the core flow from the analysis part to carry to a sorting part of the device if these parts are housed in the same body will; in the latter case the output channel is called Denoted "main channel".  

In general, any "necessary" obstacle in the way of Flow so streamlined that the split Currents after passing the obstacle without any noticeable turbulence, that is, without causing instability unrest leading to analyzer operation, reunited will. Each input channel and output channel should be made as short as possible to have a look at this Channels with a simple microscope at low magnification to enable and to be able to remove these holes from to clear any dirt that has occasionally penetrated. The mentioned input and output channels should be flexible (e.g. made of silicone) cables with the corresponding Liquid containers are connected. These lines are supposed to be fitted with a sieve filter at their entrance. The smallest dimension of the associated filter openings should not be larger than the smallest dimension of the cross section of the flow system. Take these precautions sure that the flow system of the analyzer is permanently connected can be sealed with a microscope cover glass, whereby here - like everywhere - non-reflecting, non-fluorescent black epoxy should be used as an adhesive.

In the multi-parameter particle analyzer according to the invention there is a clear distinction between three units. The first Unit is the sensor that is one of the physicochemical Sizes (parameters) of a particle in an electrical impulse converts whose amplitude is proportional to the particle property is. The analysis volume assigned to the sensor lies in the second unit, that is, in the output channel  of the analyzer. The second unit, in turn, is inside arranged in the third unit, i.e. in the analysis device, which follows that the latter two units are functional through the first unit, that is the sensor for one Multiparameter analyzer can be coupled.

The base body of the device preferably has - if also not necessary - the shape of a fairly flat cylinder, the conditions of short input and output channels to fulfill. These channels occur on the floor and / or on the Sides of the device on or off where the funds are arranged with which the device on the analysis device is attached. For the above reasons, the Base body diameter of the device is also so small be as possible. The device is said to consist of a non-conductive and non-reflective, non-fluorescent black material, or at least all active channel walls should be covered with such a material.

The analysis device, that is the third unit, takes also the various optical, mechanical and electro-optical Devices for operating the analysis device are necessary.

The various active channels and the nozzle chamber are either cut, molded, milled etc. or on other way into the smooth, polished top of the body introduced the device. The channels or cavities are generally called active spaces because of the combination of the different currents or the particle analysis or  particle sorting if a sorter with core flow deflection according to US-PS 41 75 622 provided in the analyzer will take place in these channels, etc.

The different analysis volumes are in the output channel, located near the downstream of the nozzle chamber. There should the output channel should be so wide that a UV object is larger numerical aperture and correspondingly large opening angle (about 120 °) "see" a particle movement in the core flow can. The core flow is said to be approximately in the center of the channel run freely through its side walls to ensure that that of the one to be analyzed Fluorescent light emitted by the particle Wide-angle UV lens is collected.

Taking into account the teaching of the invention, the Core flow can be easily adjusted so that it is exactly in the Focal plane flows. The associated duct system runs namely parallel to the cover slip and thus to the focal plane of the UV lens. According to the invention there are also Possibility to shape the nozzle chamber in such a way that the Core flow essentially perpendicular to the focal plane flows through this plane. But there is obviously no reason for the particles, said focal plane perpendicular to traverse.

A necessary condition for a correct analysis of the particles is that their diameters are always smaller than the focal depth or depth of field defined above. Since the Depth of field of lenses of large numerical aperture and  Magnification is very small, that is about 10 to 20 µm for a lens with a numerical aperture of A = 1.30, it is necessary for normal operation of the analyzer, that the particles are parallel or quasi parallel to the Focal plane flow. This may be an extreme one high core flow stability required. To these conditions to meet, the nozzle chamber according to the invention created.

The nozzle chamber according to the invention should be in the same way as the output channel is placed in the top of the analyzer, e.g. B. incised, and with the same cover slip as the output channel will be covered. The nozzle chamber can be seen from above about like an elongated drop of oil that Tapered towards its transition to the exit channel. The cross section of the nozzle chamber should be rectangular rather than be circular. The nozzle is said to be in a nozzle holder that they can be easily removed from the analyzer means from the nozzle chamber, can be removed.

The input channel of the enveloping liquid (electrolyte) should be in the nozzle chamber at the bottom from its upstream end a considerably smaller one with respect to the nozzle or the chamber axis Enter angles as 90 ° so that the envelope liquid already when entering the nozzle chamber at an angle of flows about 30 ° to the nozzle. Indeed, the nozzle conceives considerable and necessary obstacle in the way of the current the enveloping liquid, this is especially true in the Start phase when the chamber is still empty. Therefore, by this nozzle in the start-up phase caused severe turbulence  and a possibly related development of Eliminating air bubbles will make the transition of the Input channel of the enveloping liquid in the wall of the nozzle chamber rounded off so that the nozzle chamber like a light curved continuation of the entrance hole looks. The input channel the shell liquid can be at its beginning and any cross-section outside the device have. However, its cross section should gradually (in the longitudinal direction of the channel) so change that at the transition to the nozzle chamber wall quasi a rectangular cross section is created, which in turn should go smoothly into the wall.

To the possibility of the formation of turbulence and thus the formation of air bubbles at the entrance of the entrance channel to reduce in the nozzle chamber, according to others Invention not only this meeting point in the aforementioned Way, it should also be smoothed Special shape lining between the top of the nozzle and the cover slip. The feeding should however not to the nozzle, but precisely delimited to that Current end of the nozzle chamber wall and possibly if that Cover glass yourself on top of the analyzer device is also glued to the cover slip.

The last-mentioned lining according to the invention is intended to be such have curved sides that look like the prow of a Aircraft carrier looks. With the help of such a surface system, that in relation to that through the nozzle axis and the symmetry plane defined by the output channel be symmetrical should, the two are split up by the nozzle, beforehand  defined sub-streams merged smoothly again. A another partial flow of this sheath liquid flows - as I said - Immediately approximately parallel to the nozzle in the direction of the Output channel. However, the said feeding is said to be one have such a form that the split and now united Sub-streams and the last-mentioned lower sub-stream even the nozzle itself after a certain flow path wrap completely and without turbulence before touching it with the liquid coming from the tip of the nozzle flowing core flow come.

As can be seen by comparison with the prior art according to the US-PS 41 76 662 results, a stable from the outset Core flow can only be generated in such a nozzle chamber if there are no air bubbles in the areas in the starting phase develop in which, according to the invention, the special feeding is attached. The use of this lining is therefore well founded.

After it is created, the core flow flows into the outlet channel and carries the particles to be analyzed into the respective one Analysis volume. After passing through these analysis volumes the flow either flows directly into a waste collection bottle or in another part of the device, e.g. B. in a sorter in which the core flow is deflected for sorting becomes. With such a sorter, which is in the US-PS 41 75 662 is described, the analyzer according to the invention easy to combine.

The analyzer according to the invention can of course also in combination  with the UV microscope system with reflected light, as it was in connection with the flow system according to Dittrich and Göhde (op. cit.) is used will. The analysis volume of the UV microscope should also be used easily in the straight output channel of the device downstream of the meeting point of the nozzle chamber and Output channel can be positioned. The considerable additional The advantage of this combination is that for Analysis volume essentially nothing more than a straight line Canal is needed. The device combination according to the invention is therefore incomparably easier than that of Dittrich and Göhde (op. Cit.) Device described. Nevertheless, the device according to the invention all good properties, in particular the optical properties of the known known Systems. If the said simple device according to the invention with that described in US Pat. No. 4,237,416 and further combined in US-PS 41 75 662 explained electrical sensor other parameters, namely that Particle volume to be analyzed. Such a device will therefore be called a combined analyzer in the future will.

Furthermore, an additional light source, namely a Laser comparable with the energy of the mercury lamp mentioned UV energy, with the mercury lamp in one "and / or" combination over a number of mirrors and lenses can be combined in the usual way. The advantage of such a "switchable" Lasers, hereinafter referred to as Epilaser is that its spatially filtered and homogenized beam into a very thin and narrow band-like  Beam can be reshaped. Then if the beam much thinner than the longitudinal dimension of each to be analyzed Is only a small part, one that Slit corresponding strip of the particle through which Beam illuminated, and when the particle is suitably colored (e.g. with a fluorescent substance), the distribution the colored unit (DNA) or protein) within the Particle determined when the particle glides past the UV lens. This way additional information can be found obtained on the particle, in particular cell structure will. Of course, a laser device with one Epilaser as simple as well as combined Laser device can be used.

For exciting light energy in the order of 1 W or the epilaser mode cannot do more or more to be used. It is known from experiments that heat sensitive Material covering the space between the lenses of the UV lens fills, burns out at such energies and thereby the lens is unusable for further use makes. In some cases it is still so high Light energy required. That is the case with the analysis many chromosomes and bacteria, their DNA amount pretty much is small. It will therefore have a higher light excitation energy needed if one of the particles is sufficient for analysis strong fluorescent light is to be delivered.

Another variant of the system described - both in the simple as well as in the combined version - will obtained when using a high power laser that the  Core flow and thus the particles to be analyzed directly illuminated or illuminated. If necessary, the spatial filtered and equalized laser beam in a channel system be directed from two half cones with one common axis exists. The common axis should be vertical to the optical axis of the UV lens and to that Core flow, that is to be directed to the exit channel. The two half cones can be in the top of the device in the same way as the nozzle chamber and the outlet channel be incised. The pointed ends of the two half-cones, that are on both sides of the exit channel are sufficient then on or in the vertical according to UV transparent Sidewalls of the canal.

It follows from the above description that the said direct laser beam, the optical axis of the UV lens and the core flow is an approximately orthogonal axis system form. In this regard, the analyzer according to the invention similar to that of the known jet analyzer system. However, the system according to the invention is much more stable and more reliable because of its fluctuating core flow and because of the ability to use a UV lens that Is 10 times as strong as that in the known analyzer.

The half-cones, also known as light channels, do not have to be have a circular cross-section, their cross-section can be the same be good rectangular; your opening angle should be so big be as possible to make that too under appropriately large Angles on the particles as they pass through the beam to be able to collect scattered light from the direct laser. The  Cross section of the two light channels at the output channel should be in of the order of 0.1 × 0.1 mm² so that the beam of the direct laser can easily pass through the exit channel can. The walls of the light channels are said to be non-reflective and be non-fluorescent black. These cone channels themselves are said to have a UV-transparent material, e.g. B. one Good quality epoxy that is transparent down to 200 nm is with a piece of quartz of suitable shape or with light-guiding Fibers of the right quality can be filled. The outer ends of the light channels may become flat ground and polished. After all, a little piece High quality epoxy cover slip on the polished Surface to be glued to an easy to clean window to get these channels. In case of application Direct lasers of very high energy use quartz as a filling the channels preferred, and the base body of the device is made of metal for better cooling guarantee.

From the foregoing it follows that one is from a high power direct laser laser beam coming through one of the half cones (Light channels) goes into the exit channel, the core flow traverses and if necessary with a particle interacts. The result of one Interaction is a partial scattering of the laser beam, the small angle scattering component from the continuous or persistent beam when leaving the other half cone (Light channel) can be separated.

The component of the small-angle scatter of direct laser light  is used to classify the respective particle. If the particle is colored, the wide-angle UV lens collects the fluorescent light coming from the particle. The associated analysis volume is around the intersection of Direct laser beam and core flow. Because this again usable wide angle lens about 10 times more Can collect fluorescent light from a colored particle than the lenses that can be used in the known jet system large focal length is the direct lighting system according to the invention correspondingly more sensitive than the jet system. However, this higher sensitivity is important for analysis and sorting small particles such as cells or chromosomes. If necessary, the direct laser is used together with the other parts of the analysis device to an optical Table mounted. Of course, the same safety precautions are then also like the jet system because of the use of high-power lasers.

Since the rays of the Epilaser and the direct laser in the invention Device almost perpendicular to each other, can be a so-called two-dimensional Slot scan analysis can be performed. Prerequisite for this is that both types of jets take the form of a very thin ribbon imposed and both jet types used at the same time will. This way, more information about the internal structure of the analyzed particles can be obtained. With the help of such a double slit scanning can be Cell swellings often from deformed cells, the latter are cancerous in some cases; see. L. L. Wheeles et al., J. Histochem. Cytochem., 1979, volume 27, page  596.

As I said, the flow system of the invention Device to be constructed so that every part of the interior either directly or through the coverslip that the channels open on covering the top, is clearly visible. The visibility should exist regardless of whether the system is empty, or whether electrolyte flows in it. In addition, each part of the System interior by a mechanical means, e.g. B. by a thin wire, a thread or a jet of water be such that any unwanted particle is easily removed from the Flow system is to be removed. In particular the inside of the Nozzle chamber can be easily reached in this way because the nozzle is releasably inserted into the nozzle holder and therefore the two parts are easily separable.

Because of the good accessibility through the canals, this can Flow system at the end of the assembly with a cover slip underneath Use a non-reflective and non-fluorescent black epoxy are permanently sealed. Since further the entire interior of the duct system also the aforementioned Backlight is said to have optical properties reduced accordingly and thus the signal-to-noise ratio considerable compared to previous ones Devices in which the cover slip is detachable with a ring of springs on the top of the device, possibly under Inserted vacuum grease for better coverage was enlarged. Experiments have shown that this earlier Procedure for covering the duct system fairly It is impractical, because it seems absurd, to be careful  Clean coverslip on the greased top of the device to put. Through the permanent provided according to the invention Glue the cover slip onto the top of the device the correspondingly arduous work becomes complete superfluous.

Routine cleaning of the device inside and outside is easy to do: the inside can be Rinse the canal system with a lukewarm detergent that allowed to flow through the system for some time, cleaned will. The surface can be covered with a cotton cushion (Q-Tip), that is immersed in white spirit, cleaned to the remove used immersion liquid and other dirt. The overall maintenance of the new device is therefore considerable compared to the case of known institutions, such as they are given, for example, by Dittrich and Göhde (op. cit.) will.

An important advantage of the "top-transparent" according to the invention Channel system is that one with black ink colored, threadlike thin core flow using of a stereo microscope clearly from the one constricting it Envelope flow is distinguishable. It follows that form and Size of the core flow from its starting point at the top the nozzle to its end at the exit hole of the device can be closely observed.

The shape and size of the core flow can be matched to the appropriate one Cross section either by setting the cross section the nozzle tip or by reducing the flow of the  Core flow forming liquid outside the device to be brought. The reduction can be done either by Increasing the hydrodynamic resistance of the Connection line of the core flow liquid or through Decrease their level relative to the level of Enveloping liquid. Very small cross sections of the Lines are said to be blocked because of the risk of clogging be avoided.

The above description of the analyzer with three Light sources shows that this system is step by step can be built, initially with a simple To start the analysis device with only one mercury lamp and later either one or both of the above Laser types to continue. This "domino" construction it also makes research groups with little Budget possible, over time the most complicated To set up analysis facility.

For mass production of the device according to the invention becomes a press molding process for manufacturing the flow system suggested because of the depth of the different parts of the system is different. So once a device high quality has been developed and mass production is only required of the device a negative to be molded, with the help of which a multitude Devices according to the invention always of the same quality is to be produced.

Based on the schematic representation of exemplary embodiments details of the invention are explained. It shows

Figure 1a is a plan view of an analyzer.

Fig. 1b shows a section along the line Ib-Ib of Fig. 1;

Figure 2a is a plan view of an analysis system with three primary light sources. and

Fig. 2b is an elevation of the system of Fig. 2a.

The section plane Ib-Ib marked in FIG. 1b represents a plane of symmetry of a practically executable, highly stable and reliable analysis device according to FIG. 1a. The plane of symmetry is defined by the axis 1 of a cylindrical base body 2 of the device and by the center line of an output channel 3 . Through the outlet channel 3 is a core stream flows. 4 This is a hydrodynamically focused continuation of the liquid 5 of the core flow coming from a nozzle 6 .

In the exemplary embodiment according to the drawing, the base body 2 of the device according to the invention consists of a double-jacketed cylinder, that is to say of two concentric cylinders, of which the outer cylinder has a diameter of approximately 30 mm, while the diameter of the inner cylinder can be approximately 18 mm. The latter limits the top 7 of the device. By choosing an inner cylinder with a diameter of 18 mm, the use of a standardized microscope cover glass 8 is made possible, which also has a diameter of 18 mm.

The cover slip 8 not only covers the outlet channel 3 , but also a nozzle chamber 9 ( FIG. 1b). Normally, the cover slip 8 should be permanently glued to the top 7 of the device. At the beginning of the device assembly, however, the cover glass 8 can be pressed onto the upper side 7 with the aid of a ring (not shown) of finger-like springs for testing purposes. For this purpose, three holes 10 , one of which is shown in FIG. 1a, and three associated screws - preferably outside the diameter range of the inner cylinder - can be provided.

The device according to the invention has in the embodiment Shape of a flat cylinder, its help with a diameter of about 30 mm should only be about 8 mm. Out of this follows that the different input and output channels can be quite short, as is also explained above Construction principles is required.

The material of the base body 2 should preferably be electrically non-conductive and optically non-reflective and non-fluorescent black. If this condition cannot be easily met, an appropriate hard plastic can be used to produce the base body 2 . The walls of the active channels, which are also referred to as cavities and in which either the different flows are combined or combined or the particle analysis takes place, should be covered with a thin layer of a material with the aforementioned properties. Black epoxy has already proven itself for this. In the final phase of assembly, the cover slip 8 is to be glued to the top 7 with the same type of epoxy.

An approximately V-shaped groove 11 is preferably cut into the lower surface of the part with a larger diameter of the base body 2 . With the help of this groove 11 and three screws provided in a (not shown) bayonet socket, the device can be fixed in the socket. The bayonet mount is in turn arranged in an adjusting part of the analysis device, in which it and with it the analysis device is to be displaced in a preferably horizontal XY plane in such a way that the device axis 1 is at least in a parallel position to the optical axis of a UV lens (not shown) arrives, but preferably coincides with the optical axis.

In FIGS. 1a and 1b, an ideal case is assumed in which the axis of the instrument 1 and the optical axis of the ultraviolet objective lens coincide, so that they can both be referred to by the numeral 1. Under this condition, which simplifies the description, not only the core flow 4 , but also the optical axis 1 and the beam 13 of a direct laser pass through the area of an aperture 12 of the device according to the invention. In the illustrated embodiment, the direct laser beam 13 enters the device from the right side of the core flow 4 . Three units or determinations define a quasi-orthogonal axis system on the aperture 12 .

For the direct laser beam 13 , the output channel 3 on the diaphragm 12 should not only be UV-transparent on the top 7 (through the cover glass 8 ) but also on its vertical sides. This is achieved through two windows 14 a and 14 b, which can be produced from the same material as the filling of the optical channels 15 a and 15 b assigned to the laser beam 13 . The opposite end of each laser of the optical channel 15b is designed with a small piece of glass 16 (Fig. 1a) are closed. If necessary, the glass piece 16 should be glued to the respective channel end with the aid of the material of the filler, which also fills the channels. An epoxy of high optical quality is known to be transparent down to 200 nm and is therefore well suited for the present purpose. A suitably shaped quartz wedge can also be inserted into the channels. However, before the channels are filled, their walls should be coated with a non-reflective and non-fluorescent black material, preferably with the aforementioned black epoxy. The filling should be sanded and polished together with the top 7 of the device. Then the cover slip 8 is to be glued to the top 7 .

In the arrangement shown, the UV lens is located vertically above the top 7 of the device and the focal plane of the lens runs parallel to the top 7 . The UV lens can therefore be positioned with the help of a fine thread screw so that the core flow 4 passes through the focal plane diametrically.

According to the design principles of the invention and the general description above are high for analysis Quality two criteria of great importance, namely

• - The depth of focus of the UV lens should be greater than  the diameter of the particle to be examined; and
• - The core flow 4 should run in the focal plane at the beginning of each operation and each time the device is operated.

For particles (e.g. chromosomes) with diameters up to 20 µm the second criterion (the stability criterion) is met well, when using the ZEISS NEOFLUAR 100/1.30 UV lens becomes; the latter, incidentally, was also used in US Pat. No. 3,738,750 (W. Dittrich and W. Göhde) known system used. With larger diameters, other lenses, e.g. B. the lens NEOFLUAR 63 / 1.25 or lenses with magnification even smaller than 63 can be used. Latter Lenses have a greater depth of field than the former. However, it should be noted that lenses with lower magnification and lower numerical Aperture are quite undesirable because they are either less Collect light or because your field of vision is quite large and therefore the excitation by incident light is less. That in turn has a lower signal-to-noise ratio and therefore one Decrease in quality of the analysis device. Therefore, whenever possible, a high performance lens of the type of NEOFLUAR 100 / 1.30 can be used.

According to the invention, the flow system is to be constructed so that the core flow 4 is extremely well stabilized. For this purpose, linings 17 and 18 are provided in the nozzle chamber 9 . Without these linings 17 and 18 , the nozzle chamber 9 has the shape of the bow of a relatively simple ship. The front part 19 of the nozzle chamber 9 is tapered and aligned with the tip on the outlet channel 3 . In general, the cross section of the front part 19 is approximately rectangular. The important role of the liner 17 and 18 is only described below.

The nozzle 6 is generally made of a flexible plastic tube in order to be able to position it precisely aligned. If necessary, the core flow 4 is also precisely aligned within a sheath flow with the aid of the nozzle 6 . The nozzle 6 is inserted into the upstream end of the nozzle chamber 9 with the aid of a nozzle holder 20 . The nozzle holder 20 is placed in an elongated hole 21 . For this purpose, a tube 22 made of rubber or soft plastic (silicone) is provided, which fixes the nozzle holder 20 in an airtight but detachable manner. A small piece 23 of hard plastic is glued to the base body 2 of the device in order to enable the drilling of a suitable elongated hole 21 for the nozzle holder 20 .

According to FIG. 1a and 1b, the inlet flow system of the suspension or liquid 24 is the core flow quite simple: The liquid 24 is filled open container in a (not shown), the liquid passes, and a short from there through a screen filter, preferably made of silicone existing pipe (not shown) into an inlet channel 25 and flows to the tip of the nozzle 6 . There, the core flow 4 formed here first assumes a conical shape, that is to say the liquid 5 is focused directly by the envelope flow 26 indicated in FIG. 1 a with the two flow lines and indirectly by the tapered front part 19 of the nozzle chamber 9 . Through this transition and as a result of the inclusion in the enveloping flow 26 , the liquid 5 is focused in the form of the core flow 4 and as such is fed to the analysis volume in the area of the orifice 12 . The core flow 4 then either reaches an exit hole or a sorting device, preferably a deflection sorting device according to US Pat. No. 4,175,662.

While the flow system of the core flow 4 transporting the suspension to be examined is relatively simple, the same is in no way true for the flow system for producing the envelope flow 26 represented by the flow lines of FIG. 1a. In fact, as previous studies show, all difficulties, that is, the unreliability and unstability of other analysis devices, result from the fact that the core flow 4 is unsuitably surrounded by the liquid 27 of an envelope flow 26 .

With the earlier device design, precise adjustments were made of the flow system with the aim of reliable and stable particle analysis uses the following principles: The channels and currents are said to be at a very small angle collide to create turbulence and hence of air bubbles, especially at the start of operation (i.e. with an empty flow system) to practically zero to reduce. There should also be no abrupt transitions Changes in the channel cross-section occur. Others too sharp edges or corners within the flow system that Any air bubbles that may be generated should be avoided will. The cross section of the nozzle chamber is said to be quasi be rectangular or when using additional ones Linings have a shape that merges  previously split partial flows under one turbulence-free small angle allowed.

In the following it is shown that the flow system according to FIGS. 1a and 1b fulfills the aforementioned conditions for the flow of the sheath liquid and for the sheath flow itself: According to FIG. 1b, a flow of the sheath liquid 27 occurs (generally a pure physiological saline solution or distilled water). through an input channel 28 at an angle of less than 90 ° with respect to the axis of the nozzle 6 into the base body of the analyzer. The liquid 27 is filtered with a very fine sieve (not shown) and fed to the inlet channel 28 from a flexible tube (preferably not made of silicone).

The inlet channel 28 of the enveloping liquid 27 preferably has an initially circular cross section with a decreasing diameter in the direction of the nozzle 6 . A consequent increase in the flow velocity (in the direction of flow) is generally desirable because in this way small air bubbles of often unknown origin can be washed out of the flow system better. These bubbles are known to adhere fairly firmly to every corner or sharp edge in the wall and act like solid pieces of material, so that they often produce such substantial changes in the flow of liquid that the operation of the device as a whole becomes impossible. If necessary, the situation can only be managed by renewing the electrolyte filling of the flow system.

After a short distance, the cross section of the input channel 28 changes from a circular to a rectangular shape. In the exemplary embodiment according to FIG. 1b, this transition begins at line 29 , which represents the projection of a semicircle onto the paper plane. The gradual reduction in the cross-section of the remaining part of the input channel 28 is partly made by removing, in particular cutting or milling, the upper part of the circular channel cross-section to achieve the approximately rectangular shape and partly by filling the lower part with the lower liner 18 .

For the further description of the flow of the enveloping liquid 27 , this is considered to be theoretically divided into a lower and an upper partial flow 31 or 30 . According to FIG. 1 b, the lower partial flow 31 of the enveloping liquid 27 flows smoothly under the nozzle 6 or its inlet channel 25 . In contrast, the upper partial flow 30 is split again into two parts by the nozzle. These two sub-streams 30 'and 30 ''flow smoothly together again above the nozzle 6 . In this combination, the upper lining 17 according to the invention, which is provided in relation to the upper side of the nozzle 6 , plays an essential role. Since this upper lining 17 is to be shaped in the manner of an aircraft carrier bow, it forces the two lower part streams 30 ', 30 ''to unite at such a small angle that they cannot collide head-on, but rather merge into one another tangentially.

In earlier devices of this type, in which an upper liner 17 is missing, a fairly large air bubble develops in its place. The bubble in the earlier devices in particular almost always occurs in the initial phase of operation, i.e. when the flow system is filled with electrolyte under the action of a suction pump connected to the outlet. Obviously, these bubbles result from the frontal collision of the two lower part flows. If a bubble was observed at the location mentioned, the operation of the flow system was not acceptable, but if such an air bubble did not form, core flow diameters down to 1 µm could be observed under a stereomicroscope with a 50-fold magnification; a flow contrasted with black ink did not fluctuate at all. This means that with the aid of a flow system according to FIGS. 1a and 1b, an analyzer of high reliability and stability can be constructed if one only prevents the formation of air bubbles in the initial phase of the flow operation.

The different partial flows and lower part flows 31 or 30 'and 30 ''should continue to unite with each other well before reaching the nozzle tip and surround the nozzle 6 . Only in this way can it be achieved that the actual envelope flow 26 encloses the conical part 5 of the core flow correctly and then focuses hydrodynamically.

Furthermore, it is desirable, if not absolutely necessary, to adjust the cross-sectional dimensions of the outlet channel 3 - in terms of width and depth - in such a way that these dimensions become proportional to those of the tapered front part 19 of the nozzle chamber 9 , because only in this case, even if the outlet opening of the Nozzle 6 has a circular shape, a circular cross section of the core flow 4 can be achieved. This fact is the result of the laminar flow in the tapered front part 19 of the nozzle chamber 9 and at the same time the result of the entire rest of the flow system.

Core flows with other cross sections can also be adjusted by adjusting the geometry of the outlet of the nozzle 6 . For example, while flows with a circular cross section are well suited for round or elongated particles, band-like core flows are preferred if flat particles are to be oriented horizontally or vertically, i.e. if the cross section of the core flow 4 is comparable to that of the particles or cells to be examined. The cross-section of the core flow can also be adjusted or specified from the outside by selecting the hydrodynamic resistance in the connecting pipe of the sheath flow liquid.

In the latter cases, band-shaped laser beams that are directed perpendicular to the particle stream can be very suitable for one- or two-dimensional slot scanning or for multiplexing (see the article by Severin et al. Given above) if both lasers have the same part of the core flow 4 or illuminate different parts of the latter. Since the stable core flow 4 always flows in the focal plane of the UV objective, the particle to be examined also moves in the plane. In this way, an optimal condition can be achieved both for the lighting and for the alignment of the UV lens, so that there is a considerable improvement compared to earlier flow systems of the type described.

It follows from the foregoing that the optical axis 1 of the UV objective, which is regarded here as coinciding with the axis of the analysis device, is perpendicular to the upper side 7 of the device and thus perpendicular to the core flow 4 . The direct laser beam 13 thus runs approximately perpendicular to the axis 1 . As already mentioned, the three axes accordingly form an orthogonal coordinate system. Of course, more than one epilaser and / or direct laser can be provided in the same analysis device. In the case of two direct lasers, one of them should be placed on one side and the other on the other side of a simple analysis device. The beams should then be inclined equally with respect to one another in order to avoid an undesired combination of the persistent and the scattered parts of the beams, which are designated 32 and 33 in FIG. 1a. As already mentioned, the beam from a direct laser can be used as an excitation source for fluorescence analysis if the particles are stained with a fluorescent substance. In addition, or instead, the portion 33 of the original laser beam 13 scattered forward at a small angle (up to about 2 °) can be used to measure particles.

From FIGS. 1a and 1b It is also apparent that the maintenance of the device according to the invention is very simple. The various cavities, e.g. B. the nozzle chamber, the input and output channels (the latter is not shown) are easily observable, partly because of the transparent top of the device and partly because the cavities are generally larger than 1 mm in diameter. By pulling out of the nozzle holder 20 , the interior of the nozzle chamber 9 is not only more visible, but each part of the chamber is easily accessible either with a thread or with a water jet for cleaning any dirt. However, dirt is very rarely found in the interior of the cavities, because the filters mentioned are placed in front of the inputs of the tubes or channels supplying the two liquids 24 and 27 . Incidentally, during such maintenance, the nozzle 6 can also be observed, aligned or even replaced by another nozzle; in this way, the cross-section of the core flow can also be easily changed.

If the device is a pure analyzer, the output channel 3 should be as short as possible to reduce the likelihood of clogging. On the other hand, when the analyzer is to be combined with a deflection type sorter, the output channel is made about 1 to 3 mm long and is called "main channel". This part of the device can also be viewed and reached in the aforementioned manner, and dirt can also be easily removed.

The nozzle chamber 9 is also easy to clean. The same applies to the outside of the device because its surfaces are all smooth and can even be cleaned with a Q-Tip (cushion). The outside of the lid should occasionally be cleaned with benzine to wash off unwanted immersion liquid, while the interior of the flow system can be successfully cleaned with a detergent by allowing it to flow through the entire system for a period of time. Since there is no need to remove the cover slip and all maintenance work can be carried out easily from the outside, the risk of clogging is considerably reduced. This fact also represents a great advantage over comparable known systems, because there the removal of the cover slip is a rather time-consuming and dangerous work for the device.

FIGS. 2a and 2b show a plan view and a front view of an analysis system, which is on an optical table 41. The main parts of this system are the analysis device 42 (double-framed rectangle), an Epilaser 43 and a direct laser 44 . The XYZ coordinate system indicated in the drawing helps to define the different directions. The X and Y coordinate axes should lie in the plane of the optical table 41 and therefore also in the plane of FIG. 2a. The axes X and Z lie in the plane of Fig. 2b. The direction of the Z axis is identical to the vertical direction.

The analysis device 42 is thought to be divided into five main parts. These include a high-pressure mercury lamp 45 with a power of 100 W ( FIG. 2a), which delivers about 10 mW of power in the UV range via a collector lens (not shown) arranged in the lamp housing and after filtering by an optical filter system 46 .

If a mirror slide 47 symbolized in FIG. 2a is brought into such a position that the dashed line 48 of the slide lies at the beam crossing 49 , then the filtered UV beam 50 enters the first closed housing 51 . There are two lenses and at least one photomultiplier described in more detail below. One of the lenses from the housing 51 is an alignment lens for Koehler optics and is used to project at least part of the light from the mercury arc of lamp 45 in such a way that the Koehler optics are correctly illuminated. The Koehler lens comprises the other lens and the UV lens.

The UV lens (not shown) is placed within a second closed housing 52 , it can be adjusted with a fine thread along the vertical (Z-axis) to align its focal plane so that the core flow described above flows in the focal plane. The basic device body 2 , in which the core flow flows with the particles to be analyzed, is fastened in a bayonet mount 53 ( FIG. 2b) together with the inlet tubes for the suspension and sheath flow liquids 55 a and 55 b, respectively. The bayonet mount 53 indicated by dashed lines in FIG. 2b within the base body 2 is fixed in a small microscope stage in the form of a disk, which is arranged in a light-tight cylinder 54 . In Fig. 2a, the base body 2 , the bayonet mount 53 and the light-tight cylinder 54 are shown as concentric dashed circles. The small microscope stage and thus also the basic device body 2 can be adjusted horizontally, that is to say in the XY plane, with the aid of fine-thread screws 56 a and 56 b, which are assigned to the Y and X axes. With these two screws, the diaphragm 12 ( FIGS. 1a and 1b), which can be moved vertically with the help of a fine thread for optimal adjustment, can be exactly positioned under the UV microscope.

If the device described is a sorting device according to US Pat. No. 4,175,662, the fine length screws 56 a and 56 b can be used to conveniently observe the entire length between the screen 12 and the branching of the actual sorter because the light source is at the illumination (epilight) is moved together with the field of vision of the UV lens. In fact, lenses can be seen in the field of view of the UV lens either because they reflect parts of the UV spectrum or because they are colored with a substance that only fluoresces when their molecules are mixed with some very special molecules, e.g. B. DNA or cell protein come together.

Part of the reflected or fluorescent light is collected by the UV lens and can be seen through the eyepiece 57 ( FIG. 2b) when the tilting mirror 58 is 45 ° relative to the optical axis of the microscope, to which the UV Objectively heard, is pivoted. In the other position of the tilting mirror 58 , the mirror is outside the path of the collected light, so that it is collected by the cathode of a photomultiplier, not shown, which is arranged in the first housing 51 , and converted into electrical energy. If a suitably colored cell moves under the UV microscope, a fluorescent light is generated which is converted into an electrical pulse in the photomultiplier and further processed in the electrical devices connected downstream in the photomultiplier.

In the following, the term "analysis device" 42 is used to distinguish the device according to the invention with the mirror slide 47 from conventional analyzers which do not have such parts ( 47 ). Conventional analyzers are also called pulse cytophotometers or ICPs.

The mirror slide 47 is integrated into the analysis device 42 in order to enable a combination of the beam 59 a of the epilaser 43 with the UV light 50 of the mercury lamp 45 . This is a new type of beam union. The Epilaser 43 with its various beam parts is only shown in Fig. 2a. The "raw" epilaser beam 59 a first enters a spatial filter 10517 00070 552 001000280000000200012000285911040600040 0002004019929 00004 1039860 in order to eliminate undesired modes and to obtain a uniform light distribution over the beam cross section. The spatial filter 60 can also be a beam consumer or a beam diameter reducer, if this should be necessary in the special case. It is known from general experience that such a filter 60 is necessary in any case when a high quality laser beam is used as the end product.

In the beam path, the space filter 60 is followed by a slit diaphragm 61 with a gap arranged in broken lines. This is followed by a biconvex lens 62 and two cylindrical plano-convex lenses 63 and 64 . These lenses are advantageous for generating a laser incident light beam of the desired cross section. In the conventional case, where only a horizontal beam, that is to say in the X direction, with a circular cross section is used, only the biconvex lens 62 is required in addition to the spatial filter and beam expander 60 (beam expander). This provides a beam 59 b of z. B. 15 mm diameter, which can be moved in the Y and Z directions with the jet valve 65 to generate a beam 59 c, which the beam 50 of the mercury lamp 45 crosses. The latter two beams 50, 59 c can be combined and guided together into the first closed housing 51 if the (first) dichroic mirror 66 of the mirror slide 47 is moved into the beam crossing 49 .

With a second dichroic mirror 67 , two epilaser beams 59 c and 59 d with different UV spectral bands can be combined. If the epilaser beam 59 d is to be coupled into the analyzer device 42 alone, the mirror 67 should be totally reflective. Here, the device for generating the epilaser beam 59 d is preferably arranged on a common fixed frame with the additional beam shaping means described. In this way, a particle analysis system can be obtained as a table device, so that a complex optical table is not needed.

After entering the first closed housing 51 , the epilaser beam follows the same path that was previously described for the beam of the mercury lamp. Of course, the beam intensity must generally not be higher than the order of 10 mW, otherwise the material filling the space between the lenses of the UV lens would be destroyed. The two above-mentioned beams reach the objective after they have been reflected by a dichroic mirror arranged in the second closed housing 52 . This latter mirror is said to have a cutoff wavelength such that it reflects the UV rays into the UV objective, but transmits the fluorescent light spectrum emanating from the particles illuminated by the UV rays.

Apparently, the UV lens in this system represents both the condenser lens for the Koehler illumination optics and the lens of a microscope. The latter collects the fluorescent light emanating from the particles. This light can either be seen through the eyepiece 57 or collected through the cathode by one or more photomultipliers connected to the microscope system.

If the totally reflecting mirror 68 is located at the beam crossing 49 instead of the first dichroic mirror 66 , only the beam 59 c and / or 59 d of the epilaser enters the analysis part of the device. If this beam is used, it is in any case desirable to adjust the laser beam 59 c having a uniform circular cross section onto the lens 62 such that the beam is still substantially uniform when it illuminates the respective particle. The lens 62 is very desirable because otherwise there are only three lenses in two closed housings 51 and 52 (including the UV lens) and because simple considerations of the geometrical optics mean that a beam of uniform cross section passes through a certain number of Lenses is guided, maintains its uniform cross section only when an even number of lenses are used. Lens 62 is the fourth lens in the path of the epilaser beam, so that there is then an even number of lenses.

Very small beam cross sections can be obtained with the highly cleaned laser beams. As previously described, a ribbon-like beam is advantageous for slit or slit scanning, the slit being said to be thinner than the length of the particle to be analyzed. Such a beam can be formed from the laser beam 59 a if a horizontal slit 51 is placed in the beam path of the filtered and homogenized beam. The lenses 63 and 64 according to FIG. 2a are only required if the beam is also to be compressed in the Z or Y direction (cf. LL Wheeles et al., Loc. Cit.).

The horizontal laser beam 59 b or 59 c is projected into the analysis part with a flat surface also horizontally aligned. After reflection by the dichroic mirror 66 arranged directly in front of the objective, the beam 59 b or 59 c runs vertically or in the Z direction and with its upper surface perpendicular to the core flow flowing in the positive Y direction. If this beam ( 59 b / c) is thin enough in relation to the longitudinal extension of the particle to be analyzed, only a thin cross section of the particle is illuminated and analyzed. Under the conditions mentioned, the UV lens can collect fluorescent light only from the area of the particle that is illuminated by the UV excitation beam ( 59 b / c) with the band shape of the epilaser. Because of this "epi" or incident light nature, it is very easy to work with such a system, because the illumination of the particles and the collector of the fluorescent light are aligned by the same means, namely by the fine-thread screws 56 a and 56 b together with the not drawn fine-thread screws for vertical adjustment of the UV lens.

If the particle to be analyzed is so small that the illumination of neither the mercury lamp 45 nor the epilaser is strong enough, then a direct laser 44 can be used as the light source ( FIG. 2a). The beam 69 a of the direct laser 44 is spatially filtered by a filter 70 in the same way as in the Epilaser and formed by a biconvex lens 71 or a vertical slit diaphragm 72 and / or by cylindrical plano-convex lenses 73 and 74 . In this way, a beam is obtained b 69, which is to bring by a beam shifter 75 in such a position that the resultant at the end of direct laser beam 13 (identical to the beam 13 of Fig. 1a and b) through a small hole in the Bayonet mount 53 and possibly enters the base body 2 of the analysis device or sorting device, as is shown in detail in FIG. 1a.

When the direct laser beam 13 thus produced strikes a particle moving in the core flow 4 ( FIG. 1a) in the positive Y direction, it is split into a substantially persistent main beam 32 and a scattering beam 33 , as shown in FIG. 1a. If the beam 13 has a circular cross section, its diameter should vary between 0.02 and 0.2 mm depending on the size of the particle to be analyzed. Correspondingly, the cross section of the outlet channel 3 ( FIG. 1a) should be selected so that it can barely hold the largest particle carried with the core flow.

Slit scanning can also be performed with this beam if it is in the form of a horizontal band with vertically (z) aligned flat surfaces. As with the use of an epilaser, the beam should also be much thinner than the longitudinal dimension of the particle to be analyzed. A direct laser beam of such a shape can be produced with the aid of a slit diaphragm 72 and / or the plano-convex lenses 73 and 74 . The parts 72, 73 and 74 focus namely the originally cylindrical beam along the Y and Z axes and form a band-like, vertically oriented beam, which is also vertical to the core flow.

Of course, the epi and direct laser beams can be band-shaped can be used simultaneously when a two-dimensional Slot scanning to be performed. That way you can for example cell swellings from deformed cells, which are often cancerous, can be distinguished.

Since both laser beams are UV-type and therefore dangerous for the human eye, all rays should be covered with tubes that are either only semi-transparent or impermeable at all. The small tubes 78 and 79 , which are attached to both sides of the bayonet mount 53 according to FIGS. 2a and b, are particularly advantageous for this purpose because the different parts of the analyzer device, such as. B. the eyepiece 57 , the fine-thread screws 56 a and b and the inlet tubes 55 a and b etc., which are in the field of view of the respective operator, are arranged around the bayonet mount 53 .

Claims (30)

1. A method for enclosing a thin core flow ( 4 ) consisting of a fluid particle suspension with a sheath flow ( 26 ) of a pure, transparent fluid, in particular an electrolyte, characterized in that the liquid ( 24 ) of the core flow ( 4 ) by a in a nozzle chamber ( 9 ) extending into a first inlet channel ( 25 ) with an outlet nozzle ( 6 ) in the direction of an outlet channel ( 3 ) into a smooth, hydrophilic or aerophobic and streamlined inner wall having a nozzle chamber ( 9 ) that the liquid ( 27 ) the enveloping flow ( 26 ) through a in the peripheral region of the first input channel ( 25 ) into the inner wall opening second input channel ( 28 ) in the form of a rotation-free, wide stream with at least the cross section of the nozzle chamber ( 9 ) is also introduced into the latter that the wide Current on the body of the first entrance, which is a flow obstacle for him Angle channel ( 25 ) with nozzle ( 6 ) by choosing a small angle relative to the longitudinal direction of the first input channel ( 25 ) in a first partial stream ( 31 ) parallel to this in front of the first input channel ( 25 ) and two against each other half around the first input channel ( 25 ) flowing lower part streams ( 30 ', 30 '') is split up so that these three flow parts ( 30 ', 30 '', 31 ) through the shape of the wall of the nozzle chamber ( 9 ) and the surface of the first input channel ( 25 ) with nozzle ( 6 ) at a small angle with respect to each other and on the first input channel ( 25 ) in front of its outlet nozzle ( 6 ) as a complete covering of the nozzle and as a turbulence and air bubble-free, complete filling of the space between the first input channel ( 25 ) and the surrounding inner wall of the nozzle chamber (9) are recombined, so that the ready-formed and calmed envelope flow (26) at the downstream end of the Austrittsd se (6) is combined nearly tangential with the core flow (4) and that the core flow (4) indirectly by the envelope flow (26) wall surrounding the nozzle chamber (9) hydrodynamically constricted and then focused directly through the envelope flow (26) and becomes. 2. The method according to claim 1, characterized in that the core flow ( 4 ) coming from the nozzle ( 6 ) is constricted radially by the hydrodynamically concentrating effect of the surrounding sheath flow and the surrounding, conically tapering nozzle chamber wall and is thereby accelerated in the flow direction . 3. The method according to claim 1 or 2, characterized in that a rotation of the sheath flow liquid ( 27 ) introduced into the nozzle chamber ( 9 ) is prevented in that a partial flow ( 30 ) of the incoming sheath liquid ( 27 ) in two against each other around the second input channel ( 25 ) with the nozzle ( 6 ) flowing lower part streams ( 30 ', 30 '') is split. 4. The method according to at least one of claims 1 to 3, characterized in that the nozzle chamber ( 9 ) is kept completely free of air bubbles at each start of the process by - with streamlined nozzle chamber inner wall without sharp corners or edges - all the air present in the still liquid-free flow system is washed out with the front wave of the incoming liquid by choosing a correspondingly high flow rate. 5. Apparatus for enclosing a filamentary thin core flow ( 4 ) consisting of a fluid particle suspension with a sheath flow ( 26 ) of a pure, transparent fluid, preferably an electrolyte, in particular for carrying out the method according to at least one of claims 1 to 4, characterized that a first inlet channel ( 25 ) equipped with a nozzle ( 6 ) for axially introducing the liquid ( 24 ) of the core flow ( 4 ) along an axis of a smooth, hydrophilic, streamlined inner wall in the direction of its outlet channel ( 9 ) 3 ) it is provided that a relatively large part of the space in the nozzle chamber ( 9 ) is filled by the nozzle ( 6 ) with the inlet channel ( 25 ), that opens into the wall of the nozzle chamber ( 9 ) at a relatively small angle with respect to the nozzle second input channel ( 28 ) for the liquid ( 27 ) of the envelope flow ( 26 ) is provided that de r first input channel ( 25 ) of the nozzle ( 6 ) a from the second input channel ( 28 ) during operation enveloping liquid ( 27 ) in three streams ( 30 ', 30 '', 31 ) - of which a first partial stream ( 31 ) from second inlet channel ( 28 ) flows parallel to the axis directly along the nozzle ( 6 ) and the other half against the first input channel ( 25 ) with nozzle ( 6 ) flowing around sub-streams ( 30 ', 30 '') form - represents a splitting obstacle That the inner wall of the nozzle chamber ( 9 ) around the nozzle ( 6 ) is conically tapered in the direction of the nozzle chamber outlet ( 3 ) lying in the axis so that the three split streams ( 30 ', 30 '', 31 ) are already in front the contact with the core flow ( 4 ) form a united, hollow, turbulence-free envelope flow which completely fills the space between the nozzle ( 6 ) and the inner wall of the nozzle chamber ( 9 ) and screams in the direction of flow the tapering of the surrounding nozzle chamber wall exerts a hydrodynamically focussing or constricting effect on the core flow ( 4 ) which is already included directly at the outlet of the nozzle ( 6 ). 6. The device according to claim 5, characterized in that above the nozzle ( 6 ), the second input channel ( 28 ) opposite, on the inner wall of the nozzle chamber ( 9 ) a shaped as the bow of an aircraft carrier lining ( 17 ) is provided, which Sub-streams ( 30 ', 30 '') forces to unite almost parallel to the first partial stream ( 31 ) without turbulence. 7. The device according to claim 6, characterized in that the lining ( 17 ) of the nozzle chamber wall having the shape of an aircraft carrier bow does not touch the nozzle ( 6 ), but is so close to the nozzle ( 6 ) that the two lower part flows ( 30 '30 '') Flow almost tangentially into each other in the direction parallel to the axis of the nozzle chamber ( 9 ). 8. The device according to at least one of claims 5 to 7, characterized in that the second inlet channel ( 28 ) provided for supplying the enveloping liquid ( 27 ) has a decreasing cross section in the direction of the transition into the nozzle chamber wall and that the cross section at least at the transition in the nozzle chamber wall is approximately rectangular. 9. The device according to claim 8, characterized in that the second input channel ( 28 ) on its inlet side facing away from the nozzle chamber ( 9 ) has a cross-section deviating from the rectangular shape and that at the transition ( 29 ) to the rectangular cross-sectional area of the channel a second lining ( 18 ) is provided which, in particular, forces the first partial flow ( 31 ) of the enveloping liquid ( 27 ) flowing directly along the nozzle ( 6 ) without turbulence in the direction almost parallel to the axis of the nozzle chamber ( 9 ). 10. The device according to claim 8 or 9, characterized in that the cavity of the nozzle chamber ( 9 ) is a slightly curved continuation of the second input channel ( 28 ), which prevents the incoming enveloping liquid ( 27 ) from rotating in shape and cross section. 11. The device according to at least one of claims 5 to 10, characterized in that the cross-sectional shape of the core flow ( 4 ) by the size and shape of the cross section of the nozzle ( 6 ) is substantially predetermined and that the outer part of the flow of the core flow means for fine adjustment of the core flow cross section are assigned by adapting the hydrodynamic resistance. 12. The device according to at least one of claims 5 to 11, characterized in that the angle between the first input channel ( 25 ) and the direction with which the flow of the enveloping liquid ( 27 ) at the transition of the second input channel ( 28 ) to the nozzle chamber ( 9 ) enters this, is less than 90 °. 13. The device according to at least one of claims 5 to 12, characterized in that the outer cross section (circumference) of the nozzle ( 6 ) tapers in the direction of its outlet end where a corresponding cross-sectional reduction or bundling of the combined envelope flow ( 26 ) surrounding and bundling nozzle chamber wall is provided. 14. The device according to at least one of claims 5 to 13, characterized in that a except for the inputs and outputs ( 25, 28, 3 ) completely closed nozzle chamber ( 9 ) is provided, which on at least one of its sides with a UV -Permeable cover glass ( 8 ) is sealed. 15. The apparatus according to claim 14, characterized in that the cavity of the nozzle chamber ( 9 ) and the associated feed and discharge channels are embedded in a flat and smooth surface of a base body ( 2 ) and with a microscope cover glass ( 8 ) which is also transparent to UV light. is covered, so that the nozzle chamber ( 9 ) and the adjoining channels form an air-tight flow system, except for the channel entrances and exits. 16. The apparatus according to claim 14 or 15, characterized in that each part of the nozzle chamber ( 9 ) including the input and output channels is constructed with the aid of a conventional stereomicroscope. 17. The apparatus according to claim 16, characterized in that the material or at least one of the surfaces of the cavities of the base body ( 2 ) either a non-reflective and non-fluorescent black plastic or, if considerable heat is to be dissipated through the base body ( 2 ), consists of a metal , which has the same optical properties as the black plastic, and that the cover glass ( 8 ) on the top ( 7 ) is glued using the same black plastic. 18. The apparatus according to claim 16 or 17, characterized in that the base body ( 2 ) has a minimum diameter or a minimum extent perpendicular to the axis of the cylinder, that in the base body ( 2 ) drilled inlet or outlet channels ( 24, 28, 3 ) the nozzle chamber ( 9 ) are so short that their interior can be viewed through a standard microscope of relatively small magnification and that these channels at a very small angle, for. B. less than 30 °, meet the cavity of the nozzle chamber ( 9 ). 19. The device according to at least one of claims 5 to 18, characterized in that the nozzle ( 6 ) is mounted on a hollow nozzle holder ( 20 ) which is to be pulled out of the supporting base body ( 2 ). 20. The apparatus according to claim 9, characterized in that the nozzle holder ( 20 ) consists of a hollow cylindrical body, at the outer end or upstream end of a flexible tube is connected, and that the tube is in turn equipped at its upstream end with a filter system, the Micro-openings are not smaller than the size of the largest particle of the suspension to be analyzed or treated. 21. Use of the device according to at least one of claims 5 to 20 in an analysis device for counting particles suspended in a liquid electrolyte taking into account predetermined properties, for. B. particle size, DNA and / or protein content, the particles flowing in the thin core flow ( 4 ), which is enclosed by the clear, particle-free envelope flow ( 26 ), the core flow together with the envelope flow flowing through different analysis volumes in which the Particles interact with a sensor depending on their physicochemical properties and the size of the quantity to be analyzed is transformed into a proportional electrical pulse, with the following further features:

• a) means for installing the analysis device in an analysis system where other optical and electro-optical parts of the sensors are arranged,
• b) an output channel ( 3 ) which is UV-transparent not only on its top ( 7 ), which is closed with the cover glass ( 8 ), but also on part of its two vertical walls, this analysis volume having at least one additional illumination of the core flow ( 4 ) is associated with moving particles by appropriate lasers; and
• c) a double-cone laser guidance system with two half-cones ( 15 a, 15 b) of any cross-sectional geometry, which have a common axis and points towards each other and with their tips reach the output channel ( 3 ) from both sides where the UV-transparent side walls are provided, the two half-cones ( 15 a, 6 ) being filled with a UV-transparent material and sealed at least on one base with a thin piece of glass ( 16 ).

22. Analysis device according to claim 21, characterized in that at very high energy of the laser beam used, the double cone laser guide system is filled with a heat-resistant and UV-permeable material, preferably with an appropriately shaped quartz piece, and that the base body ( 2 ) or the coating of the inner surfaces the main body ( 2 ) and the double cone laser guidance system consists of a metal with such good optical qualities as non-reflecting and non-fluorescent black. 23. Analysis device according to claim 21 or 22, characterized in that the basic body ( 2 ) containing the various channels and cavities has the shape of a flat cylinder, which consists either of plastic or metal, which is neither reflective nor fluorescent for any part of the measurement The electromagnetic wave spectrum to be used is that one of the flat sides of the base body ( 2 ) is finely polished, that various channels and cavities are cut, cut or otherwise introduced into the body ( 2 ) in this side that the walls of the channels and cavities have the required optical quality, that their top side, which is open during manufacture, is covered during normal operation with a UV-transparent microscope cover glass ( 8 ) or quartz glass and that even the adhesive material with which the cover glass ( 8 ) may be permanently on the polished top side the main body is attached, the required op table quality. 24. An analyzer according to any one of claims 21 to 23, characterized in that the one outlet in the flow compartment of the nozzle chamber (9) defining output channel (3) houses the various analytical volume. 25. Analysis device according to claim 24, characterized in that a UV microscope with a lens of high magnification and with a maximum numerical aperture, but with a small volume sharpness is directed towards the output channel ( 3 ) such that the core flow ( 4 ) and thus the Analyzing particles in the focal plane flow through the field of view of the lens, and that a lens is provided with a depth of field exceeding the amount of the diameter of the largest particle to be analyzed from the suspension. 26. Analysis device according to claim 25, characterized by means for combining at least one laser beam with the beam of a conventional high-pressure mercury lamp ( 45 ) which operates in the reflected light mode. 27. Analysis device according to at least one of claims 21 to 26, characterized, that three light sources are assigned to an analysis volume at the same time are. 28. Analysis device according to at least one of claims 25 to 27, characterized in that the nozzle chamber ( 9 ) is cut out on its front part facing the cover glass ( 8 ) such that the particles to be analyzed refer to the field of view at any angle less than 90 ° cross on the field. 29. Analysis device according to claim 15 or 21, characterized in that the dimensions of the base body ( 2 ) are so small and the channels, cavities and holes combine with each other at such a small angle that the entire flow system can be easily observed through a standard microscope and Even if the cover glass ( 8 ) is permanently glued to the polished surface of the base body, every part of the flow system can be reached with a very thin wire or plastic thread or with a water jet. 30. Use of the device according to at least one of claims 5 to 20 and the analysis device according to at least one of claims 21 to 29 in an analysis system for analyzing and sorting particles suspended in a liquid electrolyte with the following features:

• a) a conventional UV microscope equipped with a conventional high-pressure mercury lamp ( 45 ), Koehler optics for homogeneously illuminating the object after the reflected light mode and using at least one photomultiplier for converting the emitted by the particle to be analyzed and by the UV lens of the Microscope collected fluorescent light into electrical impulses;
• b) the interaction of the mercury lamp ( 45 ) with at least one laser ( 43, 44 ) using mirrors and lenses, also after the reflected light mode; and
• c) the additional or alternative switching on of at least one laser ( 44 ) of very high power in direct mode.