WO2018172898A1

WO2018172898A1 - Automated wound scratch imaging and monitoring device and methods employed thereof

Info

Publication number: WO2018172898A1
Application number: PCT/IB2018/051794
Authority: WO
Inventors: Rajesh JAGIRDAR; Srinivas CHINTA
Original assignee: Jagirdar Rajesh; Chinta Srinivas
Priority date: 2017-03-20
Filing date: 2018-03-19
Publication date: 2018-09-27

Abstract

Exemplary embodiments of the present disclosure are directed towards a wound scratch imaging and monitoring device comprising of: a wounding template with a plurality of insertion slots configured to fit into the experimental multi-well plate with a guide positioned at the centre; a wounding device configured to have an array of wounding probes accommodated by an armature, whereby each of the wounding probe is configured to penetrate the monolayer of the plurality of penetrable cells using a probing tip; an array of illuminating device positioned to flash the light rays on the plurality of penetrable cells; an array of imaging device configured to capture the images of the plurality of penetrable cells, whereby the plurality of penetrable cells is the imaging target; an array of ultrasound emission device configured to emit the ultrasound pulse by emission of acoustic waves upon the infliction of wound; and an array of ultrasound receiving device configured to receive the emitted acoustic waves upon the infliction of wound, whereby ultrasound receiving device sends out the pulse and integrates the pulse into the display device.

Description

"AUTOMATED WOUND SCRATCH IMAGING AND MONITORING DEVICE AND

METHODS EMPLOYED THEREOF

TECHNICAL FIELD

[001] The present disclosure generally relates to the field of wound scratch imaging technology. More particularly, the present disclosure relates to wound scratching and multiple imaging with hybrid photo-acoustic method in order to monitor and analyze the migration of cells and interaction between the cells.

BACKGROUND

[002] Movement of cells and their study is an important aspect of wound healing assay.

Imaging of the cellular movements, behaviour and their analysis has caused a significant breakthrough in wound monitoring and cancer research. Conventional wound scratch imaging systems mostly cater to manual wounding and automated microscopy. A wounding assay involves multiple parameter analysis. It becomes essential to have a real time analysis of the cell migration and its implications. The existing technologies normally cut the monolayer into halves. Most of them involve disruption of cells by movement of the experimental plate or by the use of plastic tips of diameters close to 1 mm. It makes the process prolonged when multiple equipment are used for wounding each and every cell monolayer. Lack of uniformity in wounding technique is one of the major concerns which need to be redressed. The shape of the wounding tip also has a significant role to play in the wound analysis. The size of the wounding equipment existing in prior art is bulky and are not static and tend to vibrate. Further, it becomes difficult for the investigator to move the bulk of the equipment from one place to another. Present automated imaging involves microscope lenses and mirrors mounted on a movable gantry to capture visual information or by moving the plate from well to well.

[003] In the light of aforementioned discussion there exists a need for an efficient and compact wound scratch imaging system, which would be configured to encompass the analytical elements as a part of single equipment. BRIEF SUMMARY

[004] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[005] Exemplary embodiments of the present disclosure are directed towards an automated wound scratch imaging and monitoring device and methods employed thereof.

[006] Another exemplary object of the present subject matter is directed towards multiple cell imaging in real time.

[007] Another exemplary object of the present subject matter is directed towards a compact wound scratch imaging and monitoring device.

[008] Another exemplary object of the present subject matter is directed towards perfect alignment of LED lights and array of cameras.

[009] Another exemplary object of the present subject matter is directed towards imaging by acoustic-sonar to assess the thickness of samples by infra and/or ultra sound echo.

[0010] Another exemplary object of the present subject matter is directed towards uniform wounding of the multiple cells at the same time.

[0011] Another exemplary object of the present subject matter is directed towards automated image capturing, image analysis and statistical analysis.

[0012] Another exemplary object of the present subject matter is directed towards affordable technology and a budget friendly and more research output means. [0013] Yet another exemplary object of the present subject matter is directed towards offering a complete solution to scratching, imaging and monitoring of the wound.

[0014] Another exemplary aspect of the present disclosure is directed towards an experimental multi-well plate having a plurality of wells arranged in an array, whereby the plurality of wells houses a plurality of penetrable cells.

[0015] Another exemplary aspect of the present disclosure is directed towards a wounding template with a plurality of insertion slots configured to fit into the experimental multi-well plate with a guide positioned at the centre.

[0016] Another exemplary aspect of the present disclosure is directed towards a wounding device configured to have an array of wounding probes accommodated by an armature, whereby each of the wounding probe is configured to penetrate the monolayer of the plurality of penetrable cells using the probing tip.

[0017] Another exemplary aspect of the present disclosure is directed towards an array of illuminating device positioned to flash the light rays on the plurality of penetrable cells.

[0018] Another exemplary aspect of the present disclosure is directed towards an array of imaging device configured to capture the images of the plurality of penetrable cells, whereby the plurality of penetrable cells is the imaging target.

[0019] Another exemplary aspect of the present disclosure is directed towards an array of ultrasound emission device configured to emit the ultrasound pulse by emission of acoustic waves upon the infliction of wound.

[0020] Another exemplary aspect of the present disclosure is directed towards an array of ultrasound receiving device configured to receive the emitted acoustic waves upon the infliction of wound, whereby ultrasound receiving device sends out the pulse and integrates the pulse into the display device. BRIEF DESCRIPTION OF DRAWINGS

[0021] Other objects and advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments, in conjunction with the accompanying drawings, wherein like reference numerals have been used to designate like elements, and wherein:

[0022] FIG. 1 is a diagram depicting a utility case for automated wound scratch imaging and monitoring, according to an exemplary embodiment of the present disclosure.

[0023] FIG. 2 is a diagram depicting an exploded view of the experimental multi-well plate, according to an exemplary embodiment of the present disclosure.

[0024] FIG. 3A is a diagram depicting a wounding probe array along with simultaneous wounding devices, according to an exemplary embodiment of the present disclosure.

[0025] FIG. 3B is a diagram depicting a leveling gauge design to hold an experimental multi-well plate horizontal along with shock and/or vibration absorbent padding beneath the leveling screws, according to an exemplary embodiment of the present disclosure.

[0026] FIG. 4 is a diagram depicting a longitudinal view of the probe design, according to an exemplary embodiment of the present disclosure.

[0027] FIG. 5 is a diagram depicting a two dimensional view of the wounding template, according to an exemplary embodiment of the present disclosure.

[0028] FIG. 6 is a diagram depicting an arrangement of a wounding template, wounding probe mounted on a level gauge inside a laminar airflow unit, according to an exemplary embodiment of the present disclosure.

[0029] FIG. 7 is a block diagram depicting the cell surface imaging environment, according to an exemplary embodiment of the present disclosure.

[0030] FIG. 8 is a flow chart depicting the method of wound scratching and automated imaging, according to an exemplary embodiment of the present disclosure.

DETAILED DESCRIPTION

[0031] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0032] The use of "including", "comprising" or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms "a" and "an" herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms "first", "second", and "third", and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[0033] Referring to FIG. 1 is a diagram 100, depicting a utility case for automated wound scratch imaging and monitoring, according to an exemplary embodiment of the present disclosure. The utility case may act as a system designed to accommodate the electronic housing (preferably with a Faraday cage protection) along with the experimental set up to have a compact and easy to use set up. The utility case is designed to have an ambience control system integrated with an imaging system. A power button 102 is configured to supply power to the utility case for working of the components housed inside the utility case. An outer frame 104 of the utility case may be transparent, semi-transparent and/or opaque. The material used may be similar to as that in incubators, not limiting to, steel and/or any non-corrosive element. A corrosion resistant element would protect the case from corrosion arising out of external environmental factors and the experimental activities occurring inside the utility case. The utility case is designed to provide maximum space for experimentation and easy configuration. The utility case is configured to perform a task similar to that of an incubator. Oxygen may be supplied through an inlet 106 and Carbon-di-oxide through an inlet 108 into the utility case. The utility case is equipped to have its own dedicated gas supply. The supply of oxygen is maintained similar to as that of fresh air to enable the cell growth. An oxygen starved incubator may not be conducive for cell growth. Further, carbon-di-oxide supply ensures the maintenance of adequate atmosphere for cell growth and maintenance. The sensors, not limiting to, Oxygen sensor 110, carbon-di-oxide sensor 112, water sensor 114 and a temperature sensor 116 may also be housed inside the utility case. The sensors (110-116) are equipped to detect the levels of oxygen, carbon-di-oxide, water and/or moisture and temperature inside the utility case. The percentage of gases supplied may vary as per the requirement of the experiment. These sensors detect and regulate any aberrations in the levels of oxygen, carbon-di-oxide, water and/or moisture and temperature inside the utility case during the course of experimentation i.e. they act as ambience controllers. 4] Cameras 118 are in an array format each corresponding to the cell in the cell wall plate. The array may not be limited to, 48 cameras, 96 cameras and the like. The cameras capture the images in real time and may be available in not limited to, BMP, JPEG and TIFF format. The cameras 118 are integrated with the system and other components in the utility case. The cameras 118 in an array enable them to capture the images of the well plates and share them in real time or store them. An array of LED lights 120 proportional to the array of cameras is positioned to flash the light rays on the array of cells 124 containing the imaging target 126. The number of cameras 118 and LED lights 120 in the array is directly proportional to the number of cells which need to be monitored. The LCD screen 122 is for displaying the images captured by the array of cameras of the imaging target 126. Levelling screw 128 is for adjusting the level of the utility case thus making the platform horizontal. A USB port 130, an Ethernet port 132 and the WIFI antenna 134 forms a part of the software components used for retrieval, exporting and sharing of the images captured by the cameras 118 arranged in an array. It is the ambient condition inside the utility case (which acts as the experimental machine) that ensures the experimentation to continue. The images may be uploaded and/or downloaded and other image metrics, statistical analysis, graphed data and the like via not limited to, Bluetooth, USB memory stick, and the like from the imaging system (to be discussed later) to the utility case. The utility case with the components is configured to operate at specified humidity and preferably at temperatures not limiting to, 32 °C and/or 37 °C and /or 42 °C. A sonar ultra sound emitter 136 is configured to send out ultrasound pulse by emission. This ultrasound pulse is received by the ultrasound receiver 138 which sends out the received pulse and integrates into the onboard computer where, the data obtained can be used to detect the wound by the image generated. The internal temperature is configured to be regulated by an electronic heat source 140 which supplies constant and adjustable temperature. 5] Referring to FIG. 2 is a diagram 200, depicting an exploded view of the experimental multi-well plate, according to an exemplary embodiment of the present disclosure. The multi well plate is configured to have a base 204 on which is housed the wells 206 which are configured to be arranged as an array in rows uniformly divided as per the number of cells in a plate. The wells 206 may be compared to that of small test tubes which act as the house for conducting various experiments. For example the diagram 200, depicts an array of wells arranged in 8 rows and 6 columns thus making it a 48 wells cell wall plate. Each well is accommodated with a specific diameter to have a layer of cells for wounding, healing, monitoring and imaging. The experimental multi- well plate may be preferred to be transparent so that the progress and results may be viewed with clarity during the course of experimentation. The experimental multi-well plate may be made of materials, not limiting to, acrylic, and glass. Acrylic may be preferred as its light in weight and acts as a shatter proof alternative to glass and also satisfies the compatibility clauses while dealing with biological experiments involving cells and tissues and also not limiting to sterilization techniques and the like. A lid 202 of the experimental multi-well plate is configured to exactly fit with the base 204. The lid 202 has grooves etched to cover up the wells 206 when place on top of the experimental multi-well plate. The lid may also be referred to as a cover. The lid 202 is mandatory to avoid any foreign substances entering into the experimental multi-well plate which may result in manipulation of the experimental results. The experimental multi-well plate is made to accommodate lighting falling on it through the array of LED 120 and subsequent imaging by the array of cameras 118. While performing the experiment the wells 206 in the experimental multi-well plate are expected to be full of cells.

[0036] Referring to FIG. 3A is a diagram 300a, depicting a wounding probe array along with a simultaneous wounding device, according to an exemplary embodiment of the present disclosure. Diagram 300a depicts both the top view and the bottom view of the wounding probe array with a simultaneous wounding device. A wounding template 302a is configured to accommodate a knob 304a, a pin 306a and a probe array 308a. The bottom view 310a of the probe array 308a is also depicted. The wounding template 302a is configured to have a guide in the center. The wounding technique may involve a wounding of the cells housed in a single well wounding and/or simultaneous wounding of the cells in all the wells 206 (FIG 2) of the experimental multi-well plate. The mechanism involves placing the template on the experimental multi-well plate (FIG 2) wherein each probe may be configured to enter each well of the experimental multi-well plate simultaneously. By pulling the pin 306a the array of probes 308a go down, not limiting to, by penetrating the cells (monolayer) housed in the wells 206 of the experimental multi-well plate. The penetration is done till the array of probes 308a go down to the touch the bottom of the wells 206. The knob 306a is held by the user at the top and is pulled till the end of penetration i.e till the probe tip travels through the well 206 end to end. The bottom view 310a depicts the template 302a when overturned to face the bottom. An armature accommodates the wounding probe. The cuts in the wounding template 302a in order to help the wounding probe pass by may be available in multiple designs as per the requirement of the user.

[0037] Referring to FIG. 3B is a diagram 300b, depicting a leveling gauge design to hold an experimental multi-well plate horizontal along with shock and/or vibration absorbent padding beneath the leveling screws, according to an exemplary embodiment of the present disclosure. The leveling gauge 308b comprises of plate stabilizing screws 310b and experimental well plate stabilizing restraints 312b that immobilize the plate in order to hold the plate rigidly. The leveling screws 306b are adjusted until the level gauges 308b indicate perfect horizontal orientation by means of two water bubble gauges. The leveling screws are further supported at the base with shock absorbing foam material 304b to minimize vibration transfer from the surface of the laminar flow unit (which would be discussed in the subsequent part of the description).

[0038] Referring to FIG. 4 is a diagram 400, depicting a longitudinal view of the probe design, according to an exemplary embodiment of the present disclosure. The wounding probe is configured to be designed to penetrate the monolayer of the cells housed in the wells 206 (FIG 2) of the experimental multi-well plate. The probe design has a probe handle 402 which is further connected to a probing tip 404. The probe handle 402 helps prevents the user from influencing the experimental results as it prevents the user from contaminating the probing tip 404 which would be brought in contact with the monolayer of the cells in the wells 206. One aspect of the current invention's probe design is that the probing tip 404 is a rectangle. The probing tip could be as small as, not limiting to, microns in size. As discussed in FIG 3A, the wounding may be performed by inserting the probe manually one by one in the grooves provided by the template and/or simultaneously wounding wells in the experimental multi-well plate (FIG. 2). The probing tip 404 is expected to cut through the monolayer of the cells in the wells 206 at defined lengths and ensures that end to end cut is made. The cells are ensured to be disturbed through the movement of the probe through them rather than the movement of the experimental multi-well plate.

[0039] Referring to FIG. 5 is a diagram 500, depicting a two dimensional view of the wounding template with a wounding probe intact, according to an exemplary embodiment of the present disclosure. The wounding template 502 comprises of wounding probe insertion slots 504. The size of the wounding template 502 is such that it fits into the experimental multi-well plate (FIG 2) wherein each probe may be configured to enter each well of the experimental multi-well plate simultaneously and/or each well one by one as per the requirement of the user. The wounding probe (as discussed in FIG 4) passes through, not limiting to, small vertical cuts referred to as wounding probe insertion slots 504 in the wounding template 502. The number of wounding probe insertion slots 504 is in proportion to the number of wells in the experimental multi-well plate. The dimension of each of the wounding probe insertion slots 504 in a single wounding template 502 is to be similar. This is ensured for uniformity in the wounding process. As an embodiment an option to dismantle the wounding probe (as discussed in FIG 4) from the array and put them to use as an individual probe may be desired.

[0040] Referring to FIG. 6 is a diagram 600, depicting an arrangement of a wounding template and the wounding probe, experimental multi-well plate mounted on a leveling gauge inside a laminar airflow unit, according to an exemplary embodiment of the present disclosure. A laminar airflow unit 602 is a sterile closet preferably with HEPA filters from where the air is drawn and is circulated in a laminar way towards the user. The filtered air flow is to ensure the atmosphere inside the laminar airflow unit 602 to be sterile and free of any unwanted microorganisms. External contamination may lead to manipulation of the results and thus corresponding images may get disrupted. As discussed earlier the experimental multi-well plate base 606 is covered with the wounding template 604 with a wounding probe (as discussed in FIG 4). This particular arrangement is done inside the laminar air flow unit 602. The wounding probe (as discussed in FIG 4) cuts the cell monolayers at defined lengths. The scratch is inserted and the probing tip 404 (FIG. 4) is pulled out. Post wounding the wounding template 604 is replaced by the lid of the experimental multi-well plate.

[0041] Referring to FIG. 7 is a block diagram 700, depicting the cell surface imaging environment, according to an exemplary embodiment of the present disclosure. The diagram comprises of a remote display device 702, a display device 704 and an on-board computer 708 are configured to display, not limited to, images, image metrics, statistical analysis, graphed data and the like. The remote display device 702, and the display device 704 may not be limited to a laptop, a computer, a television, a conventional personal computer (PC) and/or other type of conventional user work station and the like having screens not limiting to, LCD screen, a flip LCD screen, LED TV screen and the like. The remote display device 702 is further configured to capture user input and rendering the display updates received from the distant server. The remote display device 702, the display device 704 and the on-board computer 708 are connected through a network 706. An imaging camera array 710 comprises of microscopes which are attached to individual cameras where each camera is configured to capture the images by auto focus and edge detection. The wounded experimental multi-well plate is placed atop the lenses of the microscopes which reads the image and the image is captured by the camera and/or array of cameras from where the images are fed into the on board computer 708 which in turn displays the captured images on to the display device 704. The entire process of capturing the image and corresponding display occurs in real time. An ultra sound emission and/or receiving array 712, whose number is directly proportional to the number of LED lights in the array and the number of cameras in the array, is configured to send out ultrasound pulse by emission. This ultrasound pulse is received by the ultrasound receiver and integrates into the onboard computer where, the data obtained can be used to detect the wound by the image generated after the infliction of the wound. 2] Referring to FIG. 8 is a flow chart 800, depicting the method of wound scratching and automated imaging, according to an exemplary embodiment of the present disclosure. The method starts at step 802 by growing the cells in wells of the experimental multi-well plate. These cells are grown so that they can be wounded at the later part of the experimentation. At step 804 it is visually checked and confirmed that there are no gaps between the grown cells for all the wells at the same time. Further at step 806, wounding of all the monolayers of the cells in the wells is done physically at the same time and/or by aligning a wounding template on the experimental multi-well plate followed by inserting a probe through the gaps in the template and gently dragging the probe on the layer of cells. The wounded monolayers are treated with different combination of drugs at step 808. Further, the experimental multi-well plate is placed inside the imaging machine to take the images and store them. Repeating the capturing of images after regular intervals of experimentation is done at step 812. For example: a reading may be taken at to immediately after wounding. Further, after 8 hours of placing the wounded experimental multi-well plate a further imaging at t₈ may be done and the results may be compared. Removing the experimental multi-well plate post experimentation is done at step 814.

[0043] Although the present disclosure has been described in terms of certain preferred embodiments and illustrations thereof, other embodiments and modifications to preferred embodiments may be possible that are within the principles and spirit of the invention. The above descriptions and figures are therefore to be regarded as illustrative and not restrictive.

[0044] Thus the scope of the present disclosure is defined by the appended claims and includes both combinations and sub combinations of the various features described herein above as well as variations and modifications thereof, which would occur to persons skilled in the art upon reading the foregoing description.

Claims

CLAIMS What is claimed is:

1. A wound scratch imaging and monitoring device comprising of: a wounding template with a plurality of insertion slots configured to fit into the experimental multi-well plate with a guide positioned at the centre; a wounding device configured to have an array of wounding probes accommodated by an armature, whereby each of the wounding probe is configured to penetrate the monolayer of the plurality of penetrable cells using the probing tip; an array of illuminating device positioned to flash the light rays on the plurality of penetrable cells; an array of imaging device configured to capture the images of the plurality of penetrable cells, whereby the plurality of penetrable cells is the imaging target; an array of ultrasound emission device configured to emit the ultrasound pulse by emission of acoustic waves upon the infliction of wound; and an array of ultrasound receiving device configured to receive the emitted acoustic waves upon the inflicted wound, whereby ultrasound receiving device sends out the pulse and integrates the pulse into the display device.

2. The device of claim 1, wherein plurality of inlet ports supply at least one of: oxygen; and carbon-di-oxide.

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3. The device of claim 1, wherein a heat source supplies at least one of: constant temperature; and adjustable temperature.

4. The device of claim 1 , wherein a plurality of sensors are configured to sense the levels of at least one of: Oxygen, carbon-di-oxide, water, and temperature.

5. The device of claim 1, wherein the imaging device is configured to capture the images by auto focus and edge detection.

6. The device of claim 1 , wherein the wounding probe is customized to be used for at least one of: penetrate the monolayer of the plurality of penetrable cells housed in multiple wells; and penetrate the monolayer of the plurality of penetrable cells housed in single well.

7. The device of claim 1, wherein the number of wounding probe insertion slots is in proportion to the number of wells in the experimental multi-well plate.

8. The device of claim 1, wherein the electronic housing comprises of display devices configured to display the images in a computer readable format.

9. The device of claim 1, wherein the probing tip cuts end to end through the monolayer of the plurality of penetrable cells in the wells and at predetermined lengths.

10. The device of claim 1, wherein the probing tip is at least one of rectangular in shape.

11. A method of wound scratch imaging and monitoring comprising of:

growing the cells in wells of the experimental multi-well plate; confirming visually that there are no gaps between the grown cells for all the wells at the same time; wounding of all the monolayers of the cells in the wells is done physically at the same time and/or by aligning a wounding template on the experimental multi-well

2 plate followed by inserting a probe through the gaps in the template and gently dragging the probe on the layer of cells; treating the wounded monolayers with different combination of drugs; placing the experimental multi-well plate inside the imaging machine to take the images and store them; repeating the capturing of images after regular intervals of experimentation; and removing the experimental multi-well plate post experimentation.

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