OPTICAL TECHNIQUE FOR ANALYZING INSECTS, SHRIMP AND FISH

Abstract

A novel technique for automated analysis of organisms like insects, shrimp and fish. The technique comprises detection of structures and/or organs in the organisms in a flow-system. Alternatively or in addition, the technique may comprise sorting of the organisms, for example sex separation of the organisms in the flow system. The technique may comprise steps of: (a) illuminating the organisms in a detection region, (b) imaging the organisms in the detection region, with an extended depth of field that spans at least one-fourth of their thickness and from at least two different directions; (c) analyzing the images for the presence of one or more features, structures and/or organs of interest.

Claims

1. A computer implemented method for automated detection of features, structures and/or organs in organisms being shrimp, fish or pre-adult insects, the method comprising: a) providing a flow-system comprising a fluidic channel with an inlet, a detection region and at least one outlet, an electro-optical module for monitoring the detection region and a processor; b) ensuring passage of the organisms via a fluidic channel towards the detection region, c) imaging an individual organism in the detection region by one or more sensors of the electro-optical module, thereby acquiring optical data on the organism, d) transmitting the acquired optical data to the processor, e) analyzing the obtained optical data by the processor, for detecting presence of one or more of said features, structures and/or organs of interest.

2. The method of claim 1, performing the imaging of the individual organism in the detection region with an extended depth of field that spans at least one-fourth of the organism's thickness, from at least two different observation directions.

3. The method of claim 1, further performing sorting of the organisms by the following steps: performing classification of the individual organism, using the acquired optical data, wherein said classification being based on one or more morphologic features and/or one or more color-related features and/or one or more sex-related features, and sorting said organism based on its classification.

4. A system for automated detection of one or more features, structures and/or organs in organisms being shrimp, fish or pre-adult insects, the system comprising: a) a fluidic channel comprising an inlet, a detection region and at least one outlet, said channel is configured to allow flowing there-though of the organisms suspended in liquid media; b) an electro-optical module for monitoring the detection region, c) a processor in communication with the electro-optical module, wherein said electro-optical module comprises at least one sensor and is configured to acquire optical data by imaging an individual organism, to transmit said optical data to the processor, and the processor is configured to receive, process and analyze the optical data acquired by the electro-optical module so as to detect said features, structures and/or organs of interest in the organism.

5. The system of claim 4, wherein the electro-optical module is configured for capturing images of an individual organism in the detection region from at least two different observation directions and with an extended depth of field that spans at least one-fourth of the organism's thickness.

6. The system of claim 4, configured for sorting of said organisms, further comprising a controller in-communication with said processor, wherein the processor being further configured to classify the organism based on the analysis results, on one or more morphologic features and/or one or more color-related features and/or one or more sex-related features, and to instruct the controller to sort said organism based on the classification.

7. The method of claim 1 wherein said pre-adult insects are in the shape of a larva or a pupa or a nymph; said shrimp are in the shape of a larva or a post-larva or a juvenile; and said fish are in the shape of a larva or a fry or a fingerling or an adult.

8. The method of claim 1, wherein the organisms at the detection region are illuminated by pulsed or strobed illumination.

9. The method as claimed in claim 8, wherein the pulse duration is less than 50 microseconds.

10. The method as claimed in claim 1, performed with broad spectrum illumination at the detection region.

11. The method as claimed in claim 1, wherein the organisms flow through the fluidic channel one at a time.

12. The method as claimed in claim 2, wherein the angle between said two observation directions is of about 90°.

13. The method as claimed in claim 1 wherein the imaging of the individual organism is taken from different locations along the fluidic channel at the detection region.

14. The method as claimed in claim 1, wherein the optical data not containing the organisms of interest is used for adjusting and pre-processing the optical data containing said organisms.

15. The method of claim 1, wherein the organisms are classified at least into two classes, wherein said two classes being selected from at least the following pairs: male and non-male, female and non-female, male and female, alive and dead, healthy and sick, strong and weak, quickly developing or retarded.

16. The method of claim 1 further comprising steps of reducing or elevating the temperature of the liquid media so as to control wriggling movements of the organisms flowing within the fluidic channel.

17. The method of claim 1, wherein said steps of detecting and/or sorting comprise using a trained neural network.

18. The method as claimed in claim 1, wherein the imaging provides smearing of the obtained images by less than 0.25% of the full frame.

19. The method as claimed in claim 1, wherein said features for analyzing or sorting the organisms include one or more features from at least one of the following non-exhaustive groups: a first group of morphologic features comprising: overall size of the organism, morphology of the organism, shape, segment size ratio, a second group of color-related features comprising: absorption, transmission, IR (Infrared) absorption, IR transmission, color, fluorescence, a third group of sex-related features comprising: gonad disc morphology, secondary sex organ morphology, gonad size; gonad morphology, gonad autofluorescence, size of testes, size of male accessory glands, morphology of testes, morphology of male accessory glands, autofluorescence of testes, autofluorescence of male accessory glands, size of the developing male and female primary or secondary reproductive structures, primitive sex organs, morphology of the developing male and female primary or secondary reproductive structures, autofluorescence of the developing male and female primary or secondary reproductive structures and any combination thereof.

20. The system as claimed in claim 4, wherein the electro-optical module comprises at least one of; one or more light sources configured to illuminate the detection region, one or more acoustic sources configured to generate sound waves that pass through the detection region, one or more image sensors, at least one optical element and an internal control unit in-communication with the processor.

21. The system as claimed in claim 4, comprising at least one blocking structure associated with a suitable sensor, such that only light from the detection region can reach it.

22. The system as claimed in claim 6, wherein the fluidic channel further comprises a separation region, wherein said separation region is located between the detection region and the at least one outlet; and wherein said separation region comprises at least one guiding tool controlled by the controller and configured to guide said organism to an outlet associated with the organism's classification.

23. The system as claimed in claim 4, wherein the fluidic channel further comprises a destruction region; wherein said destruction region comprises at least one destroying tool in-communication with the controller; and wherein said destroying region has “on” and “off” configurations operated by the controller.

24. The system as claimed in claim 4, further comprising an additional sensor in-communication with the controller, wherein the controller is further configured to control the amount of organisms at the entrance to the fluidic channel based on the data acquired by said sensor, to thereby allow passage of single organism at a time to the fluidic channel.

25. The system as claimed in claim 4, configured to provide illumination pulses with duration substantially close to that determined in the following equation:
dT=(N*p*PS)/(v*M)  (1) where dT is the pulse duration, N is the size of the organism of interest in the flow direction, p is the % of allowed smearing for the object of interest, PS is the pixel size of the imaging sensor, v is the flow velocity of the organism of interest in the channel, M is the optical magnification of the imaging system.

26. The system as claimed in claim 4, wherein the fluidic channel is a transparent capillary having a square or rectangular cross-section.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0194] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting examples only, with reference to the accompanying drawings, in which:

[0195] FIG. 1 illustrates one example of the proposed flow-system for determining structures or organs of interest and/or for classifying organisms such as pre-adult insects, shrimp or fish.

[0196] FIGS. 2A-2C schematically illustrate non-limiting examples of a flow system with integrated detection/classification region and sorting region (FIGS. 2A-B) and optionally destruction region (FIG. 2C), according to some embodiments of the present invention;

[0197] FIG. 3 is an illustrative flow chart of one version of the proposed technology, describing an exemplary method and an exemplary flow system for classifying a specific type of the organisms of interest and sorting them accordingly;

[0198] FIG. 4 illustrates a non-limiting example of an imaging unit of an electro-optical module according to the present invention;

[0199] FIGS. 5A-5B schematically illustrate two exemplary embodiments of the imaging units arranged along the flow system;

[0200] FIG. 6 schematically illustrates an exemplary embodiment of the optical arrangement in the flow system, where a single camera can image more than one direction with the use of mirrors:

[0201] FIG. 7 schematically illustrates an exemplary embodiment of the optical arrangement in the flow system, where a camera can image more than one direction by rotating it about the detection region.

[0202] FIG. 8 schematically illustrates the proposed way of imaging, where the depth of field is at least ¼ of the thickness of the organism of interest.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0203] Embodiments of the proposed technique will be illustrated and commented by referring to a non-limiting example of detecting sex organs in pre-adult organisms, and on non-limiting example of further sex separation of the organisms.

[0204] The invention is applicable to other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0205] The invention provides, in some of its aspects, a novel fluidic channel comprising an inlet and outlet(s); a detection/classification region or unit or zone or module. Optionally, it also provides a separation region or unit or zone or module and/or a destruction region or unit or zone or module. The channel is configured to receive pre-adult insects or shrimp or fish in any stage (whether in the larva or fry or fingerling or adult developmental stages). suspended in a liquid. In more specific examples, the pre-adult insects may be in the shape of a larva or a pupa or a nymph; the shrimp may be in the shape of a larva or a post-larva or a juvenile; and the fish may be in the shape of a larva or a fry or a fingerling or an adult.

[0206] The organisms, pass through the detection/classification region where they are imaged, analyzed and optionally classified according to their sex. According to this classification, in some embodiments, the organisms are separated into different outlets.

[0207] FIG. 1 shows a schematic block diagram of one embodiment of the present invention, intended for analyzing organisms like pre-adult insects, shrimp and fish in a flow system 10. The figure shows a fluidic channel 24 through which individual organisms 26 flow from left to right. Their flow may be passive or active. The channel 24, or part of it, may be manufactured of glass. (The organisms may be introduced in the fluidic channel from a container 22 which is shown in dashed lines). When the organisms pass through the detection region 28 of the channel, they are monitored. The monitoring may be provided by an electro-optical module which is schematically marked with the same number 28. For monitoring, the organisms in the channel 24 are usually illuminated. The electro-optical module then performs imaging of an individual organism using one or more optical sensors, and transmits the obtained optical data to a processor 32. The processor analyzes the received optical data (the images) and detects, for example, presence or absence of structures and/or organs of interest on the imaged organism. Additional steps may be taken according to results of the detection process (for example, steps of sex classification and separation) while the imaged organism continues flowing though the channel. Such steps will be illustrated and described in the following figures.

[0208] FIGS. 2A-C show schematic illustrations of exemplary flow-systems for sorting organisms like pre-adult insects, shrimp and fish according to the present invention. Upon the imaging of individual organisms within the detection region 28 (which may also be called a classification region), the processing unit/processor 32 performs analysis and classification thereof while the organisms continue flowing until reaching a separation region 30. In the separation region 30, the individual organisms are guided to different channels (for example, by a number of controlled flows) according to the classification performed by the processor 32. The processor 32 may be in control communication with a controller and/or suitable devices (not shown) of the flow system. Classification attributes exist for any organism of interest. For example, such attributes may include, but are not limited to the overall size of the organism, morphology of the organism, shape, segment size ratio, absorption, transmission, IR (Infrared) absorption, IR transmission, color, fluorescence, gonad disc morphology, secondary sex organ morphology, gonad size, gonad morphology, gonad autofluorescence, size of testes, size of male accessory glands, morphology of testes, morphology of male accessory glands, autofluorescence of testes, autofluorescence of male accessory glands, size of the developing male and female primary or secondary reproductive structures, primitive sex organs, morphology of the developing male and female primary or secondary reproductive structures, autofluorescence of the developing male and female primary or secondary reproductive structures and any combination thereof.

[0209] FIG. 2A schematically illustrates a flow system 20, where the individual organisms are guided to either a ‘male-only’ or ‘female-only’ channel 34 or an ‘anything else’ channel 36. FIG. 2B shows a schematic illustration, where the individual organisms may be guided to either a ‘male-only’ channel 34, a ‘female-only’ channel 38 or to an ‘anything else’ channel 36. FIG. 2C shows a schematic illustration, where after the classification region only a single class of organisms continue flowing, undamaged, through the main channel. The rest are destroyed in a destruction region 40.

[0210] FIG. 3 illustrates a flow chart schematically showing one version of the process according to the present invention. A method for male-female sorting in a flow system may begin with the provision of an unsorted organism (for example, fish) as shown in step 50.

[0211] Within the system, the individual organism flows preferably in a single-file towards the detection region (which may also serve as a classification region), as shown in step 52.

[0212] There, each individual fish is imaged for detection and/or classification purposes as shown in step 54 of the flow chart.

[0213] In step 56 each individual is classified. Classes used may include males and the rest (other, unspecified, unclassified or anything else), or males, females and the rest (other, unspecified, unclassified or anything else) or females and the rest (other, unspecified, unclassified or anything else). Next, the individual organisms reach a separation region, where they are guided, according to their classification, to a predefined flow outlet (step 58). Alternatively, if only a single class is needed, the unnecessary individuals may be destroyed in the channel (step 60). The outcome of the above described process is sex-sorted organisms (step 62).

[0214] Further in the present invention, a compact, cost effective, rapid and easy to use technique for detection of structures and/or organs in pre-adult insects, shrimp and fish, is described. Preferably, the organisms of interest are transparent or partially transparent (like larvae or the like), though they may be non-transparent (like some adult fish).

[0215] A first exemplary embodiment of automated detection of structures in the mentioned organisms (say, pre-adult) in a flow channel by imaging is shown in FIG. 4.

[0216] FIG. 4 is a schematic cross-section of the flow system at its detection region. The detection region includes a portion of the fluidic channel 102 (24) surrounded by components of an electro-optical module schematically marked 28.1. The organisms, for example in the form of larvae, flow one at a time through a transparent square capillary (102, 24), where they are illuminated, from two different directions, by light sources (104, 106) [all shown from top-view]. Images of the organisms are captured by image capturing units (108, 110), such as CCD or CMOS sensors, after being focused by a single lens or an array of lenses (112, 114). When two image capturing units are used, the angle between their observation directions is preferably 90° as this is the angle with the greatest information gain. In another particularly preferred embodiment of the present invention, observation takes place from three or more directions. The acquired images are transmitted to a processing unit (116, 32) where they may be preprocessed before being analyzed by an image recognition algorithm. The processing unit 116 may also be in communication with the flow-system control module (30, see FIG. 2A). As mentioned, it can alter, based on the algorithm's results, the flow path or the condition of the organisms downstream of the detection region (e.g., separate the larvae into different containers according to their sex, destroy some classified organisms, sterilize some classified organisms by irradiation, etc.)—The color of the illumination is chosen according to the level of penetration in the organism analyzed and the level of highlighting of the structures and/or organs of interest. In some instances, it is advantageous to use white or broad spectrum illumination, in conjunction with a multichannel image capturing unit (e.g., RGB sensor) which provides more information than a grey-scale sensor. In such cases, the recognition of the structures and/or organs of interest may be improved according to their broad spectrum absorbance profile. In other instances, using Infra-red (LR) or near-IR illumination, may be beneficial when visible light is strongly attenuated as it passes through the organism (for example when the organisms are non-transparent or only partially transparent). In such instance, the IR or near-IR illumination could be used as the sole illumination source(s) or alongside visible illumination as well.

[0217] In this embodiment, a blocking structure (118), for example an aperture associated with the image capturing units ensures that only light (depicted by dashed line) from the flow-channel can reach them. Furthermore, the image capturing units and light sources are placed one opposite the other, with the capillary in between.

[0218] Since the image capturing units 108, 110 (cameras, sensors) are imaging the moving (flowing) larvae, steps should be taken to make sure that the resulting images are sharp and not smeared. One approach would be to use a camera with a fast, global shutter (<50 uSec) and a light source constantly turned on. The problem with this approach is that as the camera sensor is illuminated for only a short time during each frame acquisition, either the light source needs to be powerful or the camera sensor extremely light-sensitive. Both these options are expensive and generate additional problems. Light-sensitive sensors typically have lower resolution (less pixels) while powerful light sources generate a lot of heat. The Inventors' solution is to use pulsed (strobed) illumination. The outcome is the same as in the previous case—the camera sensor is illuminated for only a short time during each frame acquisition—yet much simpler and cheaper to implement. First, one could use a common LED light source to generate light bursts (<50 uSec). Second, so long as the light source duty cycle is small (for example, it may be less than 1:100), there is very little heat generation. Finally, there is no special requirement on the camera in the system (e.g., light sensitivity, speed of shutter (could be in the millisecond range) and type of shutter (a simple rolling shutter would work fine)).

[0219] An RGB sensor/camera can be used.

[0220] The illumination pulses may be synchronized to take place in the periods when the image sensor performs the imaging (and not when the image sensor saves the pictures to memory).

[0221] Smearing of the obtained images may be minimized down to 1-2 pixels by selecting a suitable duration of the illumination pulses, for example using an equation (1). In percent, the smearing of the obtained images may be less than 0.25% of the full frame. The illumination pulse duration may be less than 50 microseconds.

[0222] FIG. 5 shows two different embodiments for the automated detection of structures in the organisms of interest in a flow-channel [both in isometric-view]. The detection is performed using measurements of the electro-optical module indicated 28.2. In FIG. 5A, the different image capturing units (two are shown) image the organism at the same location along the capillary (120). In FIG. 5B, the different image capturing units (two are shown), belonging to the electro-optical unit 28/3, image the organism at different locations along the capillary (122, 124).

[0223] The images of one and the same object taken from different locations along the flow channel may be not all simultaneous.

[0224] If the images are taken while one and the same light source is used, and the light source provides pulsed illumination, the images will be naturally synchronized by this light source.

[0225] In FIG. 5B, the two image capturing units image one and the same organism at locations 122 and 124. The imaging system typically works in video mode, such that all passing objects are recorded. If two objects pass one after the other, the images associated with each one will be analyzed separately. It is also possible the two objects are so close to one another that it is not feasible to separate them downstream and so they may both be discarded. Preferably, the organisms are conveyed one by one with a space there-between. The safe space can be measured in units of time. In the proposed embodiment, it is ˜0.5 sec or more between successive larvae. Unsorted larvae may be fed into the flow system through an entrance container, like in FIG. 1. The larvae in the container may be gently mixed, to overcome their self-movement and prevent clustering, through bubbling of air. The air may also be used to pressurize the container and drive the larvae through its outlet and into the flow system. Once a larva exits the entrance container into the flow system, it is detected by a sensor (e.g., photodiode). This triggers the system to switch a valve, such that (1) no more larvae (or water) can flow out of the entrance container, and (2) water flows into the system from a second, different container which contains only water. (No containers and valves are shown in this drawing). The water flow from the second container drives the larva that entered the system through the flow system and passes the detection region. Once the system is ready to accept a new larva, the valve is switched again so that (1) water and larvae can flow again out of the entrance container, and (2) no water can flow from the second container.

[0226] In different embodiments, imaging of the pre-adult insects, shrimp and fish in the flow-channel can be done from N>1 different directions, while using M image capturing units, with M<N.

[0227] FIG. 6 depicts one such embodiment 28.4 of the electro-optical module, where a single image capturing unit (108) images the pre-adult insects, or shrimp or fish in any stage from two different directions [shown from top-view]. Whereas, the first direction faces the sensor of the image capturing unit, the second direction is at an angle to it and the light is guided to the sensor by a setup of mirrors (126, 128) and a beam splitter or dichroic mirror (130). The optical path from the organism to the sensor will not necessarily be the same along the two directions. This may be corrected with an optical element (e.g., retarder) placed along the shorter path (132) to ensure that the images associated with the different directions are all in focus. Note that in such a setup, the images from the different directions will be formed simultaneously on the image capturing unit sensor. Also note that in order to avoid significant image data overlap from the two directions, each image will be formed on a different region of the sensor (e.g., by adjusting the angle of the beam splitter or dichroic mirror (130). Alternatively, the different illumination sources associated with different imaging directions captured on the same image capturing sensor, may be toggled on and off such that for any given image, only one illumination source will be switched on.

[0228] If different cameras are imaging the same region of interest and associated with different light sources, then the corresponding light pulses should be synchronized.

[0229] FIG. 7 depicts yet another embodiment where the number of imaging directions is bigger than the number of image capturing units [shown from side-view]. Theoretically, the number of imaging directions may be more than four.

[0230] In the embodiment of FIG. 7, the flow-channel is stationary, whereas the electro-optical unit 28.5 with the illumination and imaging elements, is rotatable about the flow channel.

[0231] In the example shown, a single image capturing unit together with its focusing optics, blocking element and illumination source are all attached to a rotating stage (134). The rotation itself may either be continuous or back-and-forth. The former option is less strenuous on the motor, but requires the use of a slip-ring or the like if any wires are attached to the rotating system (e.g., electronic, data) to prevent their twisting. This problem is also circumvented if the latter option of back-and-forth rotation is used.

[0232] FIG. 8 illustrates how the imaging units may operate according to one option of the proposed imaging technology. The figure shows a portion of the electro-optical module at the detection region. A transparent organism (say, larva 136) flowing via a fluidic channel 102 (24) is illuminated by while light from light source 104. The imaging unit or sensor 108 (say, an RGB camera) images the larva using the depth-of-field D. Dash lines 140 indicate the borders of D, within which sex organs of the larva are expected to be found. The organism 136 is imaged from different directions. Here, for simplicity, only one direction is shown using camera 108, with the extended depth of field D (138). In this example, D spans one-fourth of the organism thickness and is marked as the region between the dashed lines (140).

[0233] According to one embodiment the present invention provides a system for sex separation of organisms such as insects fish and shrimp, comprising. (a) a fluidic channel having an inner space and an outer wall, the fluidic channel comprising an inlet and at least one outlet, wherein the channel is configured to allow flow of the organisms suspended in liquid media; (b) a processor; and, (c) a controller in-communication with the processor; wherein the fluidic channel comprises a classification region, and the classification region comprises an electro-optical module in-communication with the processor, the electro-optical module comprises at least one sensor configured to acquire optical data of an individual organism and to transmit the optical data to the processor, further wherein the processor is configured to process the acquired optical data, to classify the organism based on the acquired optical data, and to instruct the controller to sort the organism based on the classification of the organism.

[0234] According to a further embodiment of the system of the present invention, the inner space of the fluidic channel is adjustable to the dimensions of a single organism.

[0235] According to a further embodiment of the system of the present invention, the fluidic channel is designed to allow passage of a single organism at a time.

[0236] According to a further embodiment the system is further configured to control movement of the organism when passing through the fluidic channel.

[0237] According to a further embodiment of the system of the present invention, the flow of the organism suspended in the liquid media through the fluidic channel is a passive flow.

[0238] According to a further embodiment of the system of the present invention, the flow of the fish suspended in the liquid media through the fluidic channel is an active flow.

[0239] According to a further embodiment of the system of the present invention, the flow is driven by a pump, vacuum, pressurizing means such as pressurized gas, gravity forces or any combination thereof.

[0240] According to a further embodiment the system comprises more than one outlet, wherein each outlet is independently operated by the controller.

[0241] According to a further embodiment of the system of the present invention, each of the one or more outlets is associated with a classification category selected from the group consisting of male, female and other.

[0242] According to a further embodiment of the system of the present invention, the at least one outlet having an “open” and a “closed” configuration.

[0243] According to a further embodiment of the system of the present invention, the outlet is operably coupled to a container.

[0244] According to a further embodiment of the system of the present invention, the fluidic channel further comprises a separation region, wherein the separation region is located between the detection region and the at least one outlet; and wherein the separation region comprises at least one guiding tool configured to guide the organism to an outlet associated with the classification.

[0245] According to a further embodiment of the system of the present invention, the guiding tool is selected from the group consisting of one or more valves, a transducer configured to generate acoustic radiation forces, electrodes configured to apply forces to the liquid media, electrodes configured to apply dielectrophoretic forces to the organism, controlled, liquid flow from one more additional directions and any combination thereof.

[0246] According to a further embodiment of the system of the present invention, the fluidic channel further comprises a destruction region; wherein the destruction region comprises at least one destroying tool in-communication with the controller; and wherein the destroying region has an “on” and “off” configurations operated by the controller.

[0247] According to a further embodiment of the system of the present invention, the at least one destroying tool is selected from the group consisting of a laser beam, a high-power electric field, ultrasonic blasts, a grinding tool, a squashing tool and any combination thereof.

[0248] According to a further embodiment of the system of the present invention, the controller is configured to execute functions selected from the group consisting of: receiving instructions from the processor, controlling flow rate, switching on the guiding tool, switching off the guiding tool, switching on the destroying tool, switching off the destroying tool, switching configuration of the outlet from “open” to “close”, and, switching configuration of the outlet from “closed” to “open”.

[0249] According to a further embodiment of the system of the present invention, the sensor is selected from the group consisting of an optical detector, a camera, a photodiode, a photomultiplier, an image acquisition sensor, an optical acquisition sensor, an electro-optical sensor, light detector, a photon sensor, a reflectometer, a photodetector, a spectral image sensor, and any combination thereof.

[0250] According to a further embodiment of the system of the present invention, the sensor is an imaging sensor selected from the group of consisting of RGB frequency spectrum, broad spectrum, hyperspectral, visible light frequency range, near infrared (NIR) frequency range, infrared (IR) frequency range, monochrome, specific light wavelengths (e.g., LED and/or laser and/or halogen and/or xenon and/or fluorescent), UV frequency range and any combination thereof.

[0251] According to a further embodiment of the system of the present invention, the electro-optical module of the classification region comprises at least one of: one or more light sources configured to illuminate the classification region, one or more image sensors, one or more acoustic sources configured to generate sound waves that pass through the classification region, at least one optical element and an internal control unit in-communication with the processor. According to a further embodiment of the system of the present invention, the electro-optical module comprises more than one light source, wherein each light source is configured to emit light with predetermined spectrum and/or intensity.

[0252] According to a further embodiment of the system of the present invention, the electro-optical module is configured to obtain multiple images of the organism from different angles.

[0253] According to a further embodiment of the system of the present invention, the image acquisition sensor is a high-frame rate.

[0254] According to a further embodiment of the system of the present invention, the light source illumination is stroboscopic or pulsed.

[0255] According to a further embodiment of the system of the present invention, the optical element is selected from the group consisting of a lens, a mirror, a polarizer, an excitation filter, an emission filter, a dichroic mirror, an optical coating such as antireflective coating, an optical grating and any combination thereof.

[0256] According to a further embodiment of the system of the present invention, the optical element comprises at least one stereoscopic microscope, at least one fluorescence microscope, or a combination thereof.

[0257] According to a further embodiment of the system of the present invention, the sensor is attached to the at least one microscope.

[0258] According to a further embodiment, the system of the present invention further comprises a sensor in-communication with the controller, wherein the controller is further configured to control the number of individual organisms at the entrance to the fluidic channel based on the data acquired by the sensor, to thereby allow passage of single organism at a time to the fluidic channel.

[0259] According to a further embodiment of the system of the present invention, the processor is configured to perform functions selected from receiving the optical data of an individual organism acquired by the sensor of the electro-optical module, processing the optical data, extracting a set of parameters indicative of the sex of the organism from the optical data, classifying the organism based on the set of parameters extracted from the optical data, providing instructions to the controller based on the classification of the organism.

[0260] According to a further embodiment of the system of the present invention, the processor is configured to classify the individual organism into at least two classes selected from the group consisting of male and non-male.

[0261] According to a further embodiment of the system of the present invention, the processor is configured to classify the individual fish into at least two classes selected from the group consisting of female and non-female.

[0262] According to a further embodiment of the system of the present invention, the processor is configured to classify the individual fish into three classes selected from the group consisting of male, female, and other.

[0263] According to a further embodiment of the system of the present invention, the features of the organism including the features (parameters) indicative of the sex of the organism are selected from at least one of a first, a second and a third group respectively comprising the following: [0264] the first group: overall size of the organism, morphology of the organism, shape, segment size ratio, [0265] the second group of color-related features comprising: absorption, transmission, IR (Infrared) absorption, IR transmission, color, fluorescence, [0266] the third group: gonad disc morphology, secondary sex organ morphology, gonad size, gonad morphology, gonad autofluorescence, size of testes, size of male accessory glands, morphology of testes, morphology of male accessory glands, autofluorescence of testes, autofluorescence of male accessory glands, size of the developing male and female primary or secondary reproductive structures, primitive sex organs, morphology of the developing male and female primary or secondary reproductive structures, autofluorescence of the developing male and female primary or secondary reproductive structures and any combination thereof.

[0267] According to a further embodiment of the system of the present invention, the detection/classification region further comprises a glass capillary through which the organism flows.

[0268] According to a further embodiment, the system of the present invention further comprises a thermostat optionally operated by the controller, wherein the thermostat is configured to control the temperature in the fluidic channel.

[0269] According to a further embodiment of the system of the present invention, the liquid media is water or isotonic aqueous solution.

[0270] According to a further embodiment of the system of the present invention, the sorting rate is about 0.1 to 100 organisms per second.

[0271] According to a further embodiment of the system of the present invention, the inner space of the fluidic channel has the size in the range of 0.1 mm to 10 mm.

[0272] According to a further embodiment of the system of the present invention, the sorting accuracy is in the range of 50% to 100%, particularly 80% to 100%, more particularly at least 90%.

[0273] According to a further embodiment of the system of the present invention, the sorting specificity is in the range of 50% to 100%, particularly 80% to 100%, more particularly at least 90%.

[0274] According to a further embodiment of the system of the present invention, the sorting selectivity is in the range of 50% to 100%, particularly 80% to 100%, more particularly at least 90%.

[0275] According to a further embodiment of the system of the present invention, the organism is a pre-adult insect or shrimp or fish larva or fish fry or fish fingerling or adult fish.

[0276] According to a further embodiment, the system of the present invention further comprises a container in fluid connection with the fluidic channel, the container contains unsorted organisms suspended in a liquid media, the container has an outlet configured to allow passage of a single organism at a time to the fluidic channel, the container further comprises pressurizing means, such as pressurized gas, vacuum, gravity forces and/or one or more pumps to drive a flow of the unsorted organisms towards the outlet of the container to the fluidic channel.

[0277] It is further within the scope of the present invention to provide a container in fluid connection with fluidic channel for sex separation of organisms, the container contains unsorted organism suspended in a liquid media, the container has an outlet configured to allow passage of a single organism at a time to the fluidic channel, the container further comprises pressurizing means, such as pressurized gas, vacuum, gravity forces and/or one or more pumps to drive a flow of unsorted organisms towards the outlet of the container to the fluidic channel.

[0278] It is further within the scope of the present invention to provide a computer implemented method of sex separation of organisms such as pre-adult insects, fish or shrimp, the method comprising: (a) providing the system for sex separation of organism as defined in any of the above; (b) streaming the organism suspended in the liquid media through the inlet into the fluidic channel towards the classification region; (c) acquiring optical data of the individual organism at the classification region by one or more sensors of the electro-optical module; (d) transmitting the optical data of the individual organism to the processor; (e) processing the optical data and classifying the individual organism based on a set of parameters indicative of the sex of the organism extracted from the optical data: (f) providing instructions to the controller based on the classification of the individual organism; and, (g) sorting the organism according to the instructions received by the controller.

[0279] It is further within the scope of the present invention to provide the method as defined above, wherein the step of sorting of the organisms according to the instructions received by the controller comprises at least one of: controlling flow rate, controlling sorting rate, switching on the guiding tool, switching off the guiding tool, switching on the destroying tool, switching off the destroying tool, switching the configuration of the outlet from “open” to “closed”, and switching the configuration of the outlet from “closed” to “open”.

[0280] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the organisms are classified into two classes, wherein the two classes are male and non-male.

[0281] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the organisms are classified into two classes, wherein the two classes are female and non-female.

[0282] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the organisms are classified into three classes, wherein the three classes are male, female and other.

[0283] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the individuals classified as non-male are directed to the destruction region to be destroyed by the at least one destroying tool.

[0284] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the individuals classified as non-female are directed to the destruction region to be destroyed by the at least one destroying tool.

[0285] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the organism is a pre-adult insect or shrimp or fish in any stage (whether larva, fry, fingerling or an adult).

[0286] It is further within the scope of the present invention to provide the method as defined in any of the above, further comprises steps of adjusting the dimensions of the inner space of the fluidic channel to allow passage of a single organism at a time.

[0287] It is further within the scope of the present invention to provide the method as defined in any of the above, further comprises steps of adjusting the dimensions of the innerspace of the fluidic channel such that a single organism extends parallel to the channel walls.

[0288] It is further within the scope of the present invention to provide the method as defined in any of the above, further comprises steps of pressurizing or directing by gravity forces unsorted organisms towards the outlet of the fluidic channel.

[0289] It is further within the scope of the present invention to provide the method as defined in any of the above, further comprises steps of reducing or elevating the temperature of the liquid media so as to control wriggling movements of the organism flowing within the fluidic channel.

[0290] It is further within the scope of the present invention to provide the method as defined in any of the above, further comprises steps of reducing the temperature of the liquid media so as to reduce wriggling movements of the organism flowing within the fluidic channel.

[0291] It is further within the scope of the present invention to provide the method as defined in any of the above, comprising steps of acquiring optical data by a sensor selected from the group consisting of an optical detector, a camera, a photodiode, a photomultiplier, an image acquisition sensor, an electro-optical sensor, an optical acquisition sensor, a light detector, a photon sensor, a reflectometer, a photodetector, a spectral image sensor, and any combination thereof.

[0292] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the electro-optical module of the classification region comprises at least one of: one or more light sources configured to illuminate the classification region, one or more acoustic sources configured to generate sound waves that pass through the classification region, one or more image sensors, at least one optical element and an internal control unit in-communication with the processor.

[0293] Additionally, it is within the scope of the present invention to provide the method as defined in any of the above, wherein the classifying comprises using a trained neural network.

[0294] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the step of processing comprises steps of analyzing the optical data using computer implemented algorithm trained to generate output based on the optical data.

[0295] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the computer implemented algorithm is trained to generate output based on predetermined feature vectors or attributes extracted from the optical data.

[0296] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the method comprises steps of implementing with the algorithm a training process according to a training dataset comprising a plurality of training images of a plurality of organisms captured by the at least one imaging sensor, wherein each respective training image of the plurality of training images is associated with the sex determination of the organism depicted in the respective training image.

[0297] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the training process comprises steps of (a) capturing images of organism using an imaging sensor; (b) classifying images into classification categories by applying a tag associated with parameters or attributes indicative of the sex of the organism extracted from the optical data; and (c) applying a computer vision algorithm to determine a set of feature vectors associated with each classification category.

[0298] It is further within the scope of the present invention to provide the method as defined in any of the above, further comprising steps of applying a machine learning process with the computer implemented trained algorithm to determine the sex of the imaged organism.

[0299] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the algorithm is implemented with a machine learning process using a neural network with the processed data.

[0300] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the machine learning process comprises computing by the at least one neural network, a tag of at least one classification category for the at least one organism, wherein the tag of at least one classification category is computed at least according to weights of the at least one neural network, wherein the at least one neural network is trained according to a training dataset comprising a plurality of training images of a plurality of organisms captured by the at least one imaging sensor, wherein each respective training image of the plurality of training images is associated with the tag of at least one classification category of at least one organism depicted in the respective training image; and generating according to the tag of at least one classification category, instructions for execution by the controller.

[0301] It is further within the scope of the present invention to provide the method as defined in any of the above, wherein the sensor is configured for image capturing and processing, with or without using Artificial Intelligence (AI) and/or machine learning and/or neural networks.

[0302] It is further within the scope of the present invention to provide a computer implemented algorithm comprising code to perform the steps of the method as defined in any of the above.

[0303] According to a further embodiment, the computer implemented algorithm may be a machine learning algorithm. In still a further embodiment, the machine learning algorithm may use verified training data.

[0304] In other words, there is provided a software product comprising computer-implementable instructions and data stored on a non-transitory computer readable storage medium and designed to cause performing steps of the method as described in any of the described embodiments.

[0305] There is also provided a non-transitory computer readable storage medium, accommodating the software product stored thereon.

[0306] In order to understand the invention and to see how it may be implemented in practice, some exemplary embodiments will now be described, by way of non-limiting examples only.

Example 1: A System for Sex-Sorting of Fish

[0307] An exemplified sex-sorting system essentially is based on the following: fish larvae or fries or fingerlings or adults are kept in their natural environment, namely, water. Unsorted fish flow, under pressure or gravity, towards the outlets of the system. The inner dimensions of the flow channel were chosen such that only a single fish can pass at a time. Furthermore, these dimensions constrict the fish to extend parallel to the channel walls. The temperature of the liquid may also be cooled so as to reduce wriggling movements. As the fish pass through a classification region, they are imaged.

[0308] Additional Optional Features:

[0309] Aqueous medium used to flow the fish (typically water) may be cooled in order to reduce their movements during the sorting process.

[0310] The unsorted fish may be kept dispersed in the liquid before entering the sorting system through agitation. This may help fish enter the system one by one and at a constant rate.

[0311] A sensor at the entrance to the system may be used to detect if two or more fish have entered it at close proximity. If this occurs, the larvae are diverted outside to prevent possible mistakes.

[0312] Pressurized gas may be used to mix the fish and drive them through the system.

[0313] Imaging with a color camera enables to improve differentiation by using the different absorption characteristics of the different organs.

[0314] Imaging from two or more distinct angles (observation directions) in order to improve detection of objects of interest (e.g., gonads should be visible from one of the angles). This may be done using two or more cameras or by using a single camera and directing the light from different regions on different parts of the imaging sensor.

[0315] High frame rate (>30 frames per second) and stroboscopic illumination may be used in order to image the flowing larvae without having to stop them before capturing the image.

[0316] In the imaging region/unit, the fish may flow through a glass capillary to improve image quality. A square or rectangular capillary may be used, for a more uniform background and to remove spherical aberrations.

[0317] Both male and female primary and secondary primitive (not fully developed) sex organs can be used.

[0318] With respect to the imaging platform, image quality was compared between an inverted Leica DM IRE2 microscope (×10, NA 0.3 objective), an Olympus SZH10 stereo microscope and a Raspberry Pi with V1 camera module and macro lens. No significant differences were found between the three systems, and the gonads, when visible, were clearly discernible in all three.

[0319] The following operations were observed:

[0320] The system receives a container with water and unsorted larvae,

[0321] The larvae pass one-by-one into a flow system,

[0322] The flowing larvae are imaged,

[0323] After imaging, the system switches the larvae to appropriate outlets (male and female containers) according to a predesigned computer algorithm,

[0324] The larvae are not harmed by the process.

[0325] It can be concluded by the experimental results that:

[0326] No damage was done to the larvae that were sex sorted by the system of the present invention and all of the larvae developed properly to adulthood.

Example 2: Sorting Criteria

[0327] In some embodiments of the technique, the viability of an organism is determined by its light absorption pattern, i.e., its general opacity and the level of uniformity in light absorption. In most cases, but not exclusively, live organisms are less opaque (less absorptive) and less uniform in their absorption profile than dead ones.

[0328] In other embodiments, the growth potential of an organism is determined by a combination of its overall size and shape (e.g., width to length ratio) and/or number of specific structures like head and mouth parts.

[0329] In further embodiments, the health of an organism is determined by its color under a broad spectrum illumination. The observed color and color ratios of the organism are partly the result of light refraction characteristics of its exterior and/or morphological signals such as the presence or absence of structures caused by disease or the organism's response to a pathogen, or a combination thereof.

[0330] In embodiments where sex is being sorted, the determining factor may be the existence or shape of parts of the reproductive system. For example, the existence of secondary male glands in the ninth (anal) segment of mosquito larvae correlates with male outcome.

[0331] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0332] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as are commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods are described herein.

[0333] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the patent specification, including definitions, will prevail. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0334] It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described hereinabove.

[0335] Features described for a method apply mutatis mutandis to the system corresponding to the method, and vice versa. Additional features described for the method of determining features, structures and/or organs in the organisms of interest are also applicable to the method of sorting such organisms, and vice versa. Additional features described for the system for determining features, structures and/or organs in the organisms are also applicable to the system for sorting the organisms, and vice versa. Only part of possible combinations have been claimed in the present set of claims at this stage.

[0336] The scope of the present invention is generally defined by the appended claims and includes some of combinations and sub-combinations of the various features described hereinabove, as well as some variations and modifications thereof, which would occur to persons skilled in the art upon reading the foregoing description. More combinations and sub-combinations may be constructed based on the description and may be brought to the claims at a later stage.