WO2000036396A2 - Axial pattern analysis and sorting instrument for multicellular organisms employing improved light scatter trigger - Google Patents

Abstract

An improved instrument that consists of an optical analyser and a fluid switch using light scatter and fluorescence means to optically identify and activate fluidic sorting of multicellular organisms from live populations of organisms such as various life cycle stages of Caenorhabditis elegans, the larval stages of Drosophila melanogaster, and the embryonic stages of Danio rero. In the case where fluorescence from these organisms is very weak, comparatively high levels of electronic noise accompany the electronic signals that are generated by the fluorescence detector and its associated circuitry. Because these weak signals cannot be used to mark the presence of an organism, another, less noisy, signal must be used to gate fluorescence detection. A gate derived from the low-noise light scatter signal from the organism collected over an acceptance angle of at least 20 degrees. Such a light scatter signal unambiguously gates even weak fluorescence signals. These signals can then be correlated with position along the major axis of elongate, multicellular organisms and used as enhanced analysis and sorting parameters.

Description

AXIAL PATTERN ANALYSIS AND

SORTING INSTRUMENT FOR

MULTICELLULAR ORGANISMS

EMPLOYING IMPROVED

LIGHT SCATTER TRIGGER

BACKGROUND OF THE INVENTION

The present application is based on and claims priority from U.S. Provisional

Application No. 60/112,280, filed December 15, 1998 which is incorporated herein by

reference.

1. Field of the Invention

The present application concerns instruments to analyze and separate objects

suspended in a fluid— specifically such instruments optimized to analyze and separate

elongated multicellular organisms.

2. Description of Related Art

The present invention pertains to high-speed mechanisms for automatically

identifying and physically selecting multicellular organisms with certain spatially

distinct, optically detectable, phenotypic characteristics from mixed populations.

Examples of applicable multicellular organisms are all stages of Caenorhabditis

elegans, Drosophila melanogaster (fruit fly) larvae, or Danio rero (zebrafish)

embryos. These are useful as model organisms for human disease and functional genomics studies. Examples of spatially distinct, optical characteristics are; the

localized expression of DNA encoded fluorescent protein molecules, localized

variations of the index of refraction or granularity, or localized variations in specific

binding sites (receptors) for optically labeled antibodies, lectins, or other specific

ligands.

Intact multicellular organisms, such as C. elegans, D. melanogaster larvae, or

D. rero embryos are frequently used as model systems to help understand the function

of human genes that have been implicated in disease. Human gene homologues have

been identified in these model organisms and mutations have been induced specifically

in those gene homologues. Such mutations frequently result in an easily observable

phenotypic change in the model organism and it has been shown that certain mutants

respond to pharmacological compounds and these responses collaterally produce

optically detectable changes in the organism.

Mutants of intact organisms are now used as a new class of in vivo drug

screens for libraries of potential pharmacological compound produced through use of

combinatorial chemical methods. With these organisms, one can identify targets for

drug intervention without the need to completely understand complex biochemical

pathways that relate the genome to the phenotype. This allows rapid and economical

screenings of the compound libraries for new and useful human drugs while limiting

politically controversial testing on mammals. The exposure of model organism mutants to diverse drug compound libraries,

even when the specific mutations involved have not yet been linked to human gene

homologues also helps define gene function. The addition of such functional genomic

techniques to the repertoire of molecular biology and biochemistry methods can greatly

accelerate the drug discovery process. Investigators can annotate drug libraries for

toxicity, non-specific activity, or cell membrane permeability by observing their

behavior in intact organisms. This way, toxic or ineffective libraries and/or library

members can be discarded at an early stage without wasting valuable resources.

While model organisms such as the nematode C. elegans, the fruit fly D.

melanogaster, and the zebrafish D. rero have been proven useful in the study of

human disease, they have not yet been successfully used in the field of high speed,

high throughput drug discovery. Until now high-speed preparation and analysis

techniques have been missing for these large organisms. This presents a roadblock to

investigators that need to search through thousands of multicellular organisms for a

new mutation or for response to a given sample drug. For example, with today's

molecular biology techniques, a large laboratory can produce deletion mutations in a

multicellular test organism at a rate of 20 to 30 per month. Then, in order to evaluate

the effect of a chemical compound library (that frequently contains 100,000 discrete

compounds) on a class of mutated organisms, one must first manipulate and deposit a

precise number of organisms of the mutant strain and the same development stage into

various containers such as wells of a microtiter plate array. Wild type or deviants from the desired mutant strain, or organisms at a different development stage must be

eliminated. Using slow, manual methods, the selection and deposition of organisms of

the proper type is a bottleneck in time to the entire process of drug discovery.

Additionally, manual methods rely on pipettes that dispense accurate volumes of fluid

but not accurate numbers of organisms. In many studies where reproduction rate is

altered by the mutation, it is necessary to begin the study of the effect of a compound

from the combinatorial library with an exact, and known number of multicellular

organisms in each well. This is, at best, a daunting requirement.

In addition to the need for rapid preparative methods there is a need for rapid

analysis methods. For example, if a mutant strain or expression system can be

characterized by a spatial pattern of fluorescence or staining, then the effect of

therapeutic compounds or toxic environments on these strains might be determined by

changes in these patterns. For example, green fluorescent protein (GFP) is used as

reporter gene to indicate that an inserted gene has been expressed. The expression of

the fluorescent protein usually occurs in a specific spatial pattern within a multicellular

organism. Discrimination of one pattern from another is currently carried out manually

with the fluorescent microscope. This is an extremely tedious task requiring a

significant number of workers that are trained at very high academic levels.

Co-pending United States Patent Application Serial No. 09/378,634, filed 20-

Auguat-1999, (which is incorporated herein by reference) describes an instrumentation

system for the rapid analysis and sorting of multicellular organisms using optical characteristics such as light scatter and fluorescence to classify each organism in a

flowing stream. A single value of fluorescence intensity at a given emission

wavelength is detected and assigned to each organism. The present invention is an

improvement that enables a flow analyzer and sorter to localize and report not only the

intensity but also the position of fluorescence along the major (long) axis of the

organism and use this new spatial information to sort the organisms.

If a mutant strain or transgenic organism is characterized by a stable, spatial

pattern of fluorescence, staining or other optically detectable characteristics, then the

effect of therapeutic compounds or toxic environments on these strains can potentially

be determined by monitoring changes in these spatial patterns. Discrimination of one

pattern from another is currently carried out manually with the fluorescent microscope.

This is an extremely tedious task requiring a significant number of workers that are

trained at very high academic levels. Automating the detection of spatial patterns of

fluorescence will improve the objectivity and the speed of measurement.

Flow instruments have been used before to count the number of nematodes in a

fluid volume. Such a device was described by Byerly et al (L. Byerly, R.C. Cassada,

and R.L. Russell, "Machine for Rapidly Counting and Measuring the Size of Small

Nematodes" , Rev. Sci. Instrum. Vol. 46, No. 5, May 1975) where the flow cytometer

employed sheath flow to orient the nematodes along the direction of flow so that their

size could be measured and organism-by-organism counts could be made by an

electrical impedance method. The device was similar to a commercial Coulter counter. The present invention differs from the Byerly device in that it can provide a device that

selects and deposits (sorts) specific organisms. The present invention is also not limited

to using an impedance sensor, which can only estimate overall size, but instead uses

optical sensing to spatially resolve localized features along the major axis of the

organism and use these to analyze and sort.

An optical flow instrument for analyzing elongate organisms such as plankton

with widths of 500 μm and lengths over 1000 μm has been described with sheath flow

to achieve orientation of the plankton. (J.C. Peeters, G.B. Dubelaar, J. Ringelberg,

and J.W. Vissεr, "Optical Plankton Analyser: a Flow Cytometer for Plankton

Analysis, I: Design Considerations" Cytometry 1989 Sept 10 (5): 522-528; and G.B.

Dubelaar, A.C. Groenwegen, W. Stokdijk, G.J. van den Engh, and J.W. Visser,

"Optical Plankton Analyser: a Flow Cytometer for Plankton Analysis, II :

Specifications" , Cytometry 1989 Sept 10 (5): 529-539). The size range of the plankton

used in these optical flow cytometers is similar to that encountered with C. elegans

nematodes, fruit fly larvae, and zebrafish embryos; however there is no provision that

avoids the ambiguous light scatter signals that are eliminated by the present invention.

SUMMARY OF THE INVENTION

The present invention uses a fluid flow stream to orient elongate, multicellular,

organisms and a narrowly focused, stationary, optical beam to scan them along their

major axis as they flow. Features such as cell density, refracfility, granularity, and fluorescence can be detected and recorded as a function of position along the length of

the oriented organism (i.e., an axial pattern scan). The invention is an improvement in

speed and statistical precision over current manual techniques for analyzing

multicellular organisms one by one under the microscope. The information from the

scan can be used to characterize gene expression and enable physical selection and

deposition of phenotypes with desired characteristics, or it can be used to determine

alterations in gene expression caused by toxic or therapeutic compounds.

In the case where fluorescence from these organisms is very weak,

comparatively high levels of electronic noise accompany the electronic signals that are

generated by the fluorescence detector and its associated circuitry. These weak signals

cannot be used to mark the presence of an organism, and another, less noisy, signal

must be used to gate fluorescence detection. Axial light loss might be used as such a

gate. Another preferred gate can be derived from the low-noise light scatter signal

from the organism. Conventional light scatter gating, such as is practiced in flow

cytometry of single cells, creates ambiguous signals when used on multicellular

organisms and thus leads to false gating of fluorescence. A light scatter detection

means is herein described which unambiguously gates these fluorescence signals.

These signals can then be correlated with position along the major axis of elongate,

multicellular organisms and used as enhanced analysis and sorting parameters.

Traditional optical flow cytometers analyze and sort small particles and single

cells in liquid suspension by detecting light scatter within (over) narrow cone or solid angles at various angles to the incident optical beam and fluorescence emission at

various wavelengths. Information about cell size and structure can be derived from

light scatter collected at different angles. For example, information about size can be

derived from light scatter detected at low angles relative to the incident optical beam

while information about internal cellular granularity can be derived from light scatter

detected at a wide angle (near a right angle) relative to the optical beam. Further, the

prior art shows that size of the granular structures to be detected determines the angle

and acceptance cone for optimal wide-angle detection.

Light scatter signals collected at specific angles and over narrow cone angles

are also used to gate detectors of weak fluorescence from single cells. Weak

fluorescence signals cannot be effectively used to mark the presence of a cell in the

optical beam because high levels of electronic noise accompany these signals. Noise

spikes frequently exceed the threshold level for fluorescence detection and produce

false readings that are confused as weakly fluorescing cells. To avoid this, flow

cytometers generally use signals from one or more detectors situated to detect light

scatter at one or more angles relative to the beam to produce relatively noise free

signals that can effectively discriminate against false fluorescence from electronic

noise, and gate true fluorescence from cells (The reader attention is drawn to U.S.

Patent 4,284,412).

It is key to the use of these light scatter detectors as fluorescence gates that

their solid angle of detection be narrow. For example, so-called "low angle forward scatter" (LAFS) detectors are frequently placed as close as 0.5 degrees to the optical

axis and collect light only within a one degree cone. Wide-angle light scatter detectors

are frequently placed at positions ranging from approximately 10 degrees to 90 degrees

off axis and also collect light within small cone angles of less than five degrees. If the

cone angle of collection is not kept as small as possible, then information about

granularity and size can become merged. Under these conditions for example, large

cells become indistinguishable from small cells and granular cells become

indistinguishable from non-granular cells of the same size.

When narrow acceptance cone light scatter (NACLS) detectors are used to

monitor the passage of multicellular organism such as C. elegans, three problems arise

that do not occur with single cells such as blood cells. First, it is found that the light

scatter signal does not necessarily rise above baseline (zero) at the beginning of the

passage of the organism through the optical beam, but instead rises at an unpredictably

later time. Second, it is found that the light scatter signal does not necessarily return to

baseline (zero) at the end of the passage of the organism through the optical beam, but

instead returns at an unpredictably early time. Third, it is also found that the light

scatter signal frequently returns to baseline (zero) at one or more unpredictable times

while the organism is in the beam.

Therefore, the most basic effort to size multicellular organisms based on their

"time of flight" through the analysis light beam is thwarted by this unpredictable

behavior of light scatter signals that are collected over narrow cone angles. Furthermore, the narrow cone angle light scatter signals that start late are not useful

for gating weak fluorescence signals. Finally, the narrow cone angle light scatter

signals that return to baseline early cannot be used to denote the position of weak

fluorescence along the axis of the worm. The signals that return to baseline early can

also be confused with the passage of two or more separate organisms when actually

only one passed through the analysis beam.

The present invention does not employ the usual single cell, light scatter

detection methods, and instead uses light scatter collection over very wide cone angles

when analyzing and sorting multicellular organisms. One aspect of the invention is to

collect scattered light over a wide cone angle such as 20 degrees or more. This

provides a light scatter signal that becomes positive accurately at the time the organism

enters the beam, remains unambiguously above baseline while the organism is in the

beam, and returns to baseline accurately at the time the organism exits the beam. This

aspect of the invention enables another aspect of the invention, which is to use

accurate, unambiguous, light scatter signals collected over wide cone angles to mark

the linear position of weak and noisy fluorescence signals along the axis of the

organism. The width of the cone angle needed depends upon the type of organism.

The present invention uses the unambiguous light scatter signal from a wide

acceptance angle, light scatter (WACLS) detector as a gate and a timing method for

the analysis of fluorescence along the axis of the organism. The location of

fluorescence along the axis of the organism is an important parameter for analysis and sorting. For example, with C. elegans, it is important in many cloning applications to

separate males from hermaphrodites. This can be accomplished with a fluorescently

labeled lectin (wheat germ agglutinin) that binds to the vulva of the hermaphrodite and

the copulatory bursa of the male. These two structures are not easily distinguishable in

brightness, but the vulva is located near the midpoint of the organism and the

copulatory bursa is located in the tail. Thus, axial location of fluorescence becomes the

parameter for differentially analyzing and sorting males and hermaphrodites. This is

illustrated schematically in Figure 1 where two oscilloscope traces are shown for single

organisms. One trace (Fig. IB) has a fluorescent peak near the midpoint, and the other

(Fig. 1C) has a fluorescent peak at the tail.

Since there is no fluorescent signal to mark the beginning of the organism in

the oscilloscope traces of Figure 1 , a means must be established to mark the beginning

and end of the passage of the organism through the light beam. This is done by the use

of the wide acceptance cone, light scatter (WACLS) signal. The start of this signal

triggers a clock in the electronic processor that, in turn causes fluorescent data to be

sampled at regular intervals in time while the wide acceptance cone, light scatter signal

remains above a preset threshold level. Sampling stops when the WACLS signal drops

below threshold, denoting the end of the organism.

The following is a parametric representation of a multicellular organism that

can be employed through the use of a WACLS signal to gate the sampling of

fluorescence along the organism's axis. Consider a WACLS detector that produces signal SI and a timing mechanism that samples signals from all other detectors every T

microseconds. Assume that there are other light scatter or light absorption detectors

situated at various angular positions with respect to the analysis beam. Let the signals

from these detectors be denoted by S2, S3, ... Sn. Further assume that there are

fluorescence detectors sensitive to various emission wavelengths producing signals FI,

F2, F3, ... Fn. The matrix below has columns of data for each detector and rows of

data for each sampling interval.

SI S2 S3 Sn FI F2 F3 Fn

Tl 0 0 0 0 0 0 0 0

T2 al 0 cl 0 e2 0 0 0

T3 a2 b2 c2 d3 0 f3 g3 0

T4 a3 0 c3 d4 0 f4 0 0

T5 a4 b4 0 0 0 f5 0 0

Tn-1 an-1 0 0 0 0 0 gn-1 0

Tn 0 0 0 0 0 0 0 0

The matrix example above shows a WACLS signal SI with non-zero entries

from time intervals T2 to Tn-1. This is the independent timing signal for all other

detector channels. The other light scatter detectors S2 to Sn are not necessarily

WACLS detectors, and therefore have zero values during the time T2 to Tn-1. The

fluorescence feature with emission wavelength FI is small and localized within interval

T2. This represents a feature that can be used to mark the "tail" of the organism (see

Fig. 1). The fluorescence feature with emission wavelength F2 is not as small (along

the axial direction) and occurs at a different location than the FI feature. The relative

location of the feature is established by reference to the timing initiated by the WACLS

detector signal SI. If the velocity of the organism is known and the "tail" marker is

used, then the absolute location of this feature can be determined as well. The

fluorescence feature with emission wavelength F3 shows up in two small locations

indicated in the WACLS timing sequence as T3 and Tn-1.

Each scanned organism can be represented by a parametric matrix of this kind.

While not containing as much information as a microscope image of the organism, the

data acquisition times for such matrices are of the order of five microseconds to 250

microseconds, depending on the length of the organism. This high speed is achieved

because simple, fast photomultipliers collect the scattered light and no image is

formed. In cytometers images are usually stored by CCD cameras, which are

inherently less sensitive than photomultipliers, and therefore require more time to

collect enough photons to form an image. Imaging times for fluorescence analysis of

organisms such as C. elegans are of the order of 50 milliseconds, which is from 200 to

10,000 times slower than the time required to collect and store the parametric data

described above. The sampling time and the speed of the organism determine the

spatial resolution of the parametric method. For example, when the organism typically

travels at about 500 cm/sec through the analysis beam, then for a five microsecond

sampling time the spatial resolution is approximately 25 μm. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a diagrammatic representation of optics, flow cell, command

electronics, and fluid switch.

Figure 2 shows a diagrammatic representation of the optical beams of the

instrument of Figure 1

Figures 3A and 3B show diagrams relating fluorescence signals (gated by one

of the methods of the invention) related to hermaphroditic (Fig. 3 A) and male (Fig.

3B) C. elegans as measured by the instrument of the present invention.

Figure 4A shows an actual oscilloscope traces from a NACLS (lower trace)

forward light scatter detector placed at a 45 degree forward light scatter angle and

fraction of a degree below the to the optical axis and a fluorescence detector (upper

trace) at right angles to the optic axis.

Figure 4B shows an actual oscilloscope traces from a NACLS (lower trace)

forward light scatter detector placed at 45 degrees from the to the optical axis and a

fluorescence detector (upper trace) at right angles to the optic axis.

Figure 5 shows actual oscilloscope traces from an extinction detector (lower

trace)placed on the optical axis and a fluorescence detector at right angles to the optical

axis (upper trace). Figure 6A shows actual oscilloscope traces from a WACLS forward light

scatter detector (lower trace) and a fluorescence detector at right angles to the optical

axis (upper trace); the C. elegans samples scanned showed several discreet points of

fluorescence.

Figure 6B shows actual oscilloscope traces from a WACLS forward light

scatter detector (lower trace) and a fluorescence detector at right angles to the optical

axis (upper trace); the C. elegans specimens scanned showed a small additional

fluorescence at one end..

DETAILED DESCRIPTION

OF THE PREFERRED EMBODIMENTS

The following description is provided to enable any person skilled in the art to

make and use the invention and sets forth the best modes contemplated by the inventor

of carrying out his invention. Various modifications, however, will remain readily

apparent to those skilled in the art, since the general principles of the present invention

have been defined herein specifically to provide optical gating devices and methods for

use with an optical analyzer/sorter designed for elongated multicellular organisms.

The flow scanning experimental system

An instrument such as that shown schematically in Fig. 1 was constructed with

an interchangeable pair of lasers (argon ion and helium-neon) as the light source. Detection was carried out variously with silicon photodetectors and photomultipliers.

The flow cell was rectangular with a square cross-section capillary measuring 250 μm

on a side for use with C. elegans. The flow cell capillary was 1000 μm on a side to

accommodate first through third instar, D. melanogaster larvae. Sheath flow is used to

orient these elongate organisms as they emerge from the sample nozzle and enter the

flow cell capillary.

This capillary flow cell is located at the line focus of the laser beam. Fig. 2

diagrammatically shows the geometric relationship of the flow and the various optical

beams. The fluorescent light is collected by simple aspheric lenses or microscope

objectives and passed through emission filters to photomultipliers. By virtue of the

focused laser beam and the collection lenses, the flowing organism is optically scanned

as it passes through the focus.

Simultaneous NACLS and fluorescence from C. elegans

A light scatter sensor was placed at various angular positions with respect to

the optical axis in the forward scatter direction. The collection cone angle was

approximately six degrees (NACLS). A photomultiplier with a 20X-collection lens and

a barrier filter optimized for fluorescence from GFP was used on the fluorescence

detector. The C. elegans that were used for this illustration expressed GFP at two

locations in the "head" and nowhere else. The oscilloscope traces for light scatter and

fluorescence are shown in Figs 4A and 4B. The traces show the passage of the organism through the line focus laser beam.

The lower trace 1 is the light scatter signal and the top trace 2 is the fluorescence

signal. The x-axis is time. Figure 4A is typical of a class of light scatter trace observed

with a NACLS detector. The detector was placed at a 45 degree forward light scatter

angle directly below the laser beam axis (below the horizontal plane in Fig. 2) as it

emerged from the flow cell. No scattered light from the flow cell structures themselves

was incident on the detector. The NACLS signal appears to rise at the proper time.

The onset of the NACLS trace and the weak autofluorescence trace from the anterior

structures of the nematode coincide. The NACLS signal appears to return to baseline

after the fluorescent head passes. Unfortunately, the trace returns to baseline

approximately during the middle of the passage of the nematode as well. This would

give the false impression that two organisms had passed rather than one. This NACLS

signal demonstrates the need for a new, unambiguous trigger and timing signal.

Figure 4B illustrates another problem associated with improper placement of a

light scatter detector for triggering. In this example, the same detector was placed in

the horizontal plane of Fig. 2, but at an angle of 45 degrees to the forward direction.

In this case, stray, scattered light from the capillary was incident on the detector. A

baseline restoration circuit was used to zero out this light level. The NACLS trace

shows a false return to baseline that is caused by the acceptance cone angle being too

small, and in addition a place where the signal becomes negative. The negative going

region is caused when stray light from the flow cell is blocked by the nematode to an extent that there is more light blockage than there is light scatter. This signal could not

be used as a trigger or timing signal for two reasons. The first is that the detector

acceptance cone was too small and the second was that stray light on the detector

became blocked by the passage of the nematode.

Problems associated with optical extinction signals as trigger and timing signals

Fig. 5 illustrates another problem associated with improper placement of a light

scatter detector for triggering. In this case a sensor was place directly on axis and in

the laser beam. The object was to measure light blockage (extinction)by the organisms.

Light extinction is a possible alternative to the preferred WACLS trigger of the present

invention. The test C. elegans had a single weak region of fluorescence at a neuronal

location in the head located slightly posterior to the tip of the "nose" . A 40X objective

was used to collect more light since this organism was very weakly fluorescent. The

extinction sensor collected light over a two degree cone. In this case extinction trace

returns to baseline during the passage of the nematode, and even becomes slightly

negative. Therefore, this signal could not be used as a trigger or timing signal.

Simultaneous WACLS and fluorescence from C. elegans

A photodetector was placed on the optic axis with a collection cone angle of

approximately 30 degrees (WACLS). A mask was placed over the center front of the

detector to block any directly transmitted light or stray scattered light from the flow

cell capillary. This way, the detector collected light scatter from the organisms over a several times wider cone angle than in the previous examples. The photomultiplier

with a 40X collection lens and a barrier filter for green fluorescence protein was used

to detect fluorescence since the fluorescence signal was very weak.

Figure 6A shows a WACLS signal on the lower trace and the associated

fluorescence signal on the upper trace. Note that the WACLS signal begins and ends at

the proper time and does not return to baseline during the passage of the nematode.

This was a consistent and systematic observation so long as the acceptance angle was

sufficiently wide and light from the illuminating beam or the scatter detector did not

collect stray light. The particular C. elegans used for this example expressed

fluorescence along its entire length with 5 to 6 points along the axis where the

expression was locally stronger. Some evidence for these local peaks can be seen in the

fluorescence trace. The WACLS signal begins and ends at the proper time and does

not return to baseline during the passage of the nematode through the laser beam.

There were no exceptions to this observation when over 500 nematodes were analyzed.

In the examples of useless trigger signals described above almost half of the signals

returned to baseline improperly.

Fig. 6B also shows the traces for a C. elegans with very weak fluorescent

protein expression. There is a low level of auto fluorescence throughout the length of

the organism and two local regions of weak expression near the tail. The WACLS

signal begins and ends at the proper time and does not return to baseline during the

passage of the nematode through the laser beam. The fluorescence signal is far too noisy to serve as a self trigger and timing signal, however the onset and end of the

WACLS signal is strong and unambiguous, and could be used to time and guide an

analysis of the fluorescence trace to the location of the two weak peaks.

In addition to the equivalents of the claimed elements, obvious substitutions

now or later known to one with ordinary skill in the art are defined to be within the

scope of the defined elements. The claims are thus to be understood to include what is

specifically illustrated and described above, what is conceptually equivalent, what can

be obviously substituted and also what essentially incorporates the essential idea of the

invention. Those skilled in the art will appreciate that various adaptations and modifi-

cations of the just-described preferred embodiment can be configured without

departing from the scope and spirit of the invention. The illustrated embodiment has

been set forth only for the purposes of example and that should not be taken as limiting

the invention. Therefore, it is to be understood that, within the scope of the appended

claims, the invention may be practiced other than as specifically described herein.

Claims

CLAIMSWhat Is Claimed Is:

1. An instrument for analyzing and selectively dispensing elongate multicellular organisms comprising: a source containing multicellular organisms in a fluid suspension; means for causing the fluid suspension to move in a direction of flow; means for aligning the elongate multicellular organisms relative to the direction of flow; a light source for producing an optical beam through which the elongate multicellular organisms pass after becoming aligned; a first optical detector for detecting light over a solid angle of at least 20 degrees for receiving light scattered by the elongate multicellular organisms for detecting passage of said organisms through said optical beam; and a fluid switch downstream of a point where said organisms pass through said optical beam, said switch responsive to the first optical detector to allow detected objects to pass to a sample container.

2. The instrument of Claim 1 further comprising additional optical detectors for detecting sequential optical characteristics arrayed along a length of the multicellular organism wherein outputs of said detectors are gated by an output of the first optical detector to produce gated outputs.

3. The instrument of Claim 2 further comprising a data representation of the sequential optical characteristics comprised of the outputs of the additional optical detectors.

4. The instrument of Claim 3 further comprising a controller connected to the fluid switch and operative to cause said switch to select multicellular organisms showing data representations meeting predetermined criteria.

5. A method of selectively dispensing elongate multicellular organisms comprising the steps of: centering and orienting the sample objects in a flowing fluid stream; passing the fluid stream through a sensing zone; optically detecting the presence of a multicellular organism passing through the sensing zone by means of a light scatter sensor that has an acceptance angle of at least

20 degrees; creating a data representation of sequential optical characteristics of the multicellular organism comprising output signals from additional optical sensors; diverting at least some portion of the fluid stream with a switched fluid stream based on the data representation so as to collect ones of the multicellular organisms remaining in portions of the sample stream that were not diverted.

6. The method of Claim 5 further comprising the step of exposing the multicellular organisms collected in the step of diverting to a test chemical or test environment.

7. The method of Claim 5 further comprising the step of exposing the multicellular organisms to a test chemical or a test environment prior to the detecting step to determine whether the data representation is altered by the test chemical or the test environment.

8. A data structure representative of an oriented elongate multicellular organism containing indicia of sequential optical characteristics disposed along a length of said organism, said data structure comprised of stored sequential outputs derived from optical sensors arranged to receive optical energy emanating from the elongate multicellular organism as said organism passes through an optical beam wherein a signal from a light scatter sensor that has an acceptance angle of at least 20 degrees is used to create or utilize the data structure.