DESC:A SYSTEM AND METHOD FOR DETECTING PATHOGENS IN A CELL CULTURE
FIELD OF INVENTION
[0001] The embodiment herein generally relates to detection of pathogens in a cell culture. More specifically, the invention provides an improved system and method for detecting pathogens in a cell culture using a portable device.
BACKGROUND AND PRIOR ART
[0002] The Indian patent having application number IN201741010111 discloses devices for rapid, non-invasive, automatic and in-situ detection and classification of pathogens.
[0003] The device comprises of an imaging module, an image processing module, and a display module. The imaging module comprises a plurality of light sources to emit excitation radiation at a predetermined range of wavelengths.
[0004] The imaging module also comprises an optical switch to expose a sample comprising pathogens to the excitation radiation for a predetermined duration at a predetermined periodicity. The imaging module further comprises a detector configured to synchronously capture time-resolved fluorescence emission spectra, time-resolved reflectance, and transmittance spectra, at multiple spectral bands from the sample. The image processing module is coupled to the imaging module.
[0005] The image processing module comprises an image processor to perform spatial and temporal resolution of the time-resolved fluorescence emission spectra, time-resolved reflectance, and time resolved transmittance spectra to obtain a plurality of spectral parameters.
[0006] The image processing module further comprises a library database comprising a set of standard spectral parameters identifiable with reference pathogens. The image processing module is to compare each of the plurality of spectral parameters with the set of standard spectral parameters to detect and classify the pathogen. The device further comprises a display module to display a result of the comparing.
[0007] The device is used for quantification of various pathogens present in a sample. The intensity and time dependent fluorescence information at various spectral bands may be obtained from the sample and compared with the fluorescence intensity data from the library database for intensity quantification.
[0008] The device may also be used for monitoring wound healing and wound closure. The device may also be used to study wound parameters, such as wound size, wound depth, wound temperature distribution, tissue classification, biofilm information, degree of contamination, tissue oxygenation and blood flow.
[0009] However, the above cited patent does not disclose a method and system for identifying contamination in cell cultures. In particular, the cited patent does not disclose a system and method for providing improved and accurate detection of pathogens in a cell culture of islet cells.
[00010] Cell culture is a process of growing cells under controlled conditions, generally outside their natural environment. The cells are isolated from a living tissue and maintained under carefully controlled conditions for various medical purposes. However, there are many chances of contamination of the cell culture during maintenance.
[00011] Contamination of the cell culture affects many of the cell’s biochemical and immunological properties, leading to unreliable results. It is essential to regularly test the cell cultures for contamination with mycoplasmas or bacteria. The existing methodology for detecting contamination in cell cultures is an elaborate procedure, requiring a timeline of at least a few days.
[00012] Therefore, there is a need for a user friendly and faster detection of pathogens in cell cultures. Further, there is a need for an improved system and method for detecting contamination in a cell culture of islet cells and obtaining accurate data.
OBJECTS OF THE INVENTION
[00013] Some of the objects of the present disclosure are described herein below:
[00014] A main object of the present invention is to provide a system and method for faster detection of pathogens in a cell culture.
[00015] Another object of the present invention is to provide a system and method for user friendly detection of pathogens in a cell culture of islet cells.
[00016] Yet another object of the present invention is to provide an improved portable device for accurate detection pathogens in a cell culture.
[00017] The other objects and advantages of the present invention will be apparent from the following description when read in conjunction with the accompanying drawings, which are incorporated for illustration of preferred embodiments of the present invention and are not intended to limit the scope thereof.
SUMMARY OF THE INVENTION
[00018] In view of the foregoing, an embodiment herein provides a system and method for detecting pathogens in a cell culture.
[00019] In accordance with an embodiment, the method includes illuminating a sample of cell culture using a plurality of light sources in UV-A, UV-B, UV-C and visible band, capturing emission from the illuminated sample using a detector, processing the captured emission using an electronic hardware unit, comparing the processed emission with a standard data using the electronic hardware unit; updating a comparison of a sample and comparing the sample with a comparison data for providing improved results using the artificial intelligence module; displaying improved result of the comparison of the sample using the display unit. In an embodiment, the method includes detecting pathogens in a cell culture of islet cells, identifying a difference in auto fluorescence and a threshold to detect contamination of islet cells and/or cell culture and providing updated comparison data by the artificial intelligence module used for comparing with a sample for providing improved and accurate identification of contamination in the sample.
[00020] In accordance with an embodiment, the processing includes applying thresholding and image enhancement on the captured emission obtained in a form of spectral images and processing using machine learning and/or deep learning techniques for detecting and quantifying pathogens based on spectral and spatial properties.
[00021] In accordance with an embodiment, the comparing includes comparing intensities of fluorescence emission and spatial parameters of captured spectral images of the sample with the standard data. In an embodiment, amount of pathogens is quantified based on intensity of emitted fluorescence intensity in various spectral bands of the sample. In an embodiment, excitation sources placed at 450 angle to ensure illumination.
[00022] In accordance with an embodiment, the system consists of a device for detecting contamination in a sample of cell culture including an electronic hardware unit, an optical unit, a display unit, an artificial intelligence module.
[00023] In accordance with an embodiment, the optical unit includes a plurality of light sources producing light in UV-A, UV-B, UV-C, visible band, and a detector. The optical unit is provided for illuminating a sample and detecting emission from the illuminated sample, the electronic hardware unit is connected to the optical unit for processing the emission and comparing the processed emission, an artificial intelligence unit is connected to the electronic hardware unit for updating comparison data of a plurality of samples from the electronic hardware unit and comparing the sample with the comparison data and a display unit is connected to the optical unit for displaying improved result of the comparison of the sample received from the artificial intelligence unit. In an embodiment, the electronic hardware unit includes a controller and a power source.
[00024] In accordance with an embodiment, the system is a portable device, wherein the portable device takes minutes for accurate detection pathogens in a cell culture.
[00025] In accordance with an embodiment, the electronic hardware unit is configured for supplying power to the optical unit using the power source. In an embodiment, the controller in the electronic hardware unit is configured for controlling the optical unit to detect the illuminated sample, wherein the controller synchronizes excitation components and emission components for capturing auto fluorescence. In an embodiment, the controller in the electronic hardware unit is configured for comparing an illuminated sample with a standard non-contaminated sample for identifying contamination of the sample.
[00026] In accordance with an embodiment, the detector includes a hyper spectral camera or a multispectral camera having a detector selected from a group consisting of CMOS detector, CCD detector, photodetector, phototransistor, Avalanche photodiode, Single photon avalanche photodetector, Photomultiplier tube. In an embodiment, the detector is configured for capturing emission from the illuminated sample and placed parallel to the sample.
[00027] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

BRIEF DESCRIPTION OF DRAWINGS
[00028] The detailed description is set forth with reference to the accompanying figures. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. The use of the same reference numbers in different figures indicates similar or identical items.
[00029] Fig.1 illustrates a system a 100 for detecting pathogens in a cell culture according to an embodiment herein;
[00030] Fig.2 illustrates an optical unit for detecting pathogens in a cell culture 200, according to an embodiment herein;
[00031] Fig.3a illustrates an excitation emission spectra of a contaminated cell culture 300a, according to an embodiment herein;
[00032] Fig.3b illustrates an excitation emission spectra of a non-contaminated cell culture 300b, according to an embodiment herein; and
[00033] Fig.4 illustrates a method 400 for detecting pathogens in a cell culture, according to an embodiment herein.
[00034] Fig. 5 illustrates excitation emission spectra of (a) Fetal Bovine Serum, (b) HSF without contamination, (c) HSF contaminated mildly, (d) HSF contaminated heavily; according to an embodiment herein.
[00035] Fig. 6 illustrates Fluorescence emission curve when cells are excited at specific wavelengths, according to an embodiment herein;
[00036] Fig. 7 illustrates Fluorescence emission curve when media is excited at specific wavelengths, according to an embodiment herein;
[00037] Fig. 8 illustrates Fluorescence emission curve when FBS is excited at specific wavelengths, according to an embodiment herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[00038] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
[00039] As mentioned above, there is a need for a user friendly and faster detection of contamination in cell cultures. In particular, there is a need of a system and method for detecting contamination of pathogens in a cell culture within few minutes using a portable device. The embodiments achieve this by providing “A system and method for detecting pathogens in a cell culture”. Referring now to the drawings, and more particularly to FIGS. 1 through 4, where similar reference characters denote corresponding features consistently throughout the figures, there are shown preferred embodiments.
[00040] Fig.1 illustrates a system 100 for detecting pathogens in a cell culture, according to an embodiment.
[00041] The system comprises an electronic hardware unit 101, an optical unit 102, a sample 103, a display unit 104, and an artificial intelligence unit 105.
[00042] The sample 103 is a cell culture created for detecting pathogens. In a preferred embodiment, a cell culture of islet cells from pancreas is created.
[00043] The electronic hardware unit 101 includes a controller, a power source.
[00044] The optical unit 102 is configured for illuminating the sample 103 and detecting emission from the illuminated sample.
[00045] The electronic hardware unit 101 is configured for supplying power to the optical unit 102 using the power source. The controller in the electronic hardware unit is configured for controlling the optical unit to detect the illuminated sample. The controller is also configured for comparing an illuminated sample with a standard non-contaminated sample for identifying contamination of the sample 103.
[00046] In an embodiment, the controller synchronizes excitation components and emission components of the optical unit 102. In an example embodiment, the controller switches ON/OFF LEDs and simultaneously positions emission spectral filters of the optical unit 102 for capturing fluorescence from the sample.
[00047] The artificial intelligence unit 105 is connected to the electronic hardware unit. The artificial intelligence unit 105 is configured for updating comparison data of a plurality of samples from the electronic hardware unit. The updated comparison data is used for comparing with a sample for providing improved and accurate identification of contamination in the sample.
[00048] The display unit 104 is configured for displaying improved result of the comparison of the sample.
[00049] Fig.2 illustrates the optical unit for detecting pathogens in a cell culture 200, according to an embodiment.
[00050] The optical unit 102 includes the detector 203, a plurality of light sources 201, 202.
[00051] In an embodiment, the light sources produce emission in UV-A, UV-B, UV-C and visible band. The light source 201 produces light of UV-B, UV-C wavelength and the light source 202 produces light of UV-A wavelength. The light of UV-A, UV-B, UV-C and visible band (not shown in figure) from the light sources is oriented at an angle X of 45 degrees to the sample 103 for uniform illumination.
[00052] The detector 203 can include a hyper spectral camera or a multispectral camera having a detector including but not limited to one of CMOS detector, CCD detector, photodetector, phototransistor, avalanche photodiode, single photon avalanche photodetector, photomultiplier tube. The detector 203 is configured for capturing emission from the illuminated sample 103 in a form of spectral images. The detector 203 is placed parallel to the sample 103.
[00053] Various example implementations and applications of the devices and methods of the present subject matter have been described herein for illustration purposes. It will be understood that the application of the devices and methods is not restricted to these examples. Further, in different applications, the devices and methods may provide some oral of the advantages disclosed herein and it will be understood that, depending on the implementation, the devices and methods may provide different advantages also.
[00054] Fig. 3a illustrates auto fluorescence spectra 300a of a contaminated sample. The auto fluorescence spectra of the sample 103 obtained from the display 104 having wavelength of excitation in the Y axis and wavelength of emission in the X axis. Fig. 3b illustrates auto fluorescence spectra of a non-contaminated sample. The auto fluorescence spectra of a standard from the display 104 having wavelength of excitation in the Y axis and wavelength of emission in the X axis. Based on a comparison of 300 a and 300 b, a difference in intensity of auto fluorescence of the sample 203 is identified at an area between emission wavelength of 450nm to 500nm and excitation wavelength of 340nm to 400nm. The difference in intensity of the auto fluorescence of the sample 203 is due to the presence of pathogens.
[00055] Fig.4 illustrates a method of detecting contamination, according to an embodiment.
[00056] In an embodiment, the method of detecting contamination in a cell culture includes the steps of:
[00057] At block 401, illuminating a sample 103 of cell culture using a plurality of light sources 201, 202 in UV-A, UV-B, UV-C band.
[00058] At block 402, capturing emission due to auto fluorescence from the illuminated sample using a detector 203. In an embodiment, the detector detects auto fluorescence having pathogen load equal and greater than 10^2 CFU/ml. In an embodiment, a controller synchronizes excitation components and emission components for accurately capturing auto fluorescence.
[00059] At block 403, image processing the captured emission using an electronic hardware unit 101. In an embodiment, image processing is performed across various spectral bands including but not limited UV-A, UV-B. In an embodiment, image processing includes taking input of the captured emission obtained in a form of spectral images from the detector 203 and applying steps of thresholding and image enhancement. In an embodiment, machine learning and/or deep learning techniques are used on the captured images for pathogen detection and quantification based on spectral and spatial parameters.
[00060] In an embodiment, the machine learning technique extract features from the captured spectral images, wherein the features include but not limited to texture features.
[00061] At block 404, comparing the processed emission with a standard data using the electronic hardware unit 101. In an embodiment, the electronic hardware unit 101 compares intensities of fluorescence emission in various spectral bands from the sample, spatial parameters including but not limited to texture features extracted using machine learning algorithms. In an embodiment, deep learning technique automatically generates latent parameters for comparing from the spectral images.
[00062] In an embodiment, the electronic hardware unit 101 quantifies an amount of pathogens present in the sample based on intensity of emitted fluorescence in various spectral bands.
[00063] In an embodiment, the electronic hardware unit 101 classifies the sample as contaminated or un-contaminated based on the amount of pathogens. If contaminated, the quantification of contamination will be indicated in scale of 0-5, wherein the scale refers to increasing amount of pathogens from 0 to 5.
[00064] At block 405, updating a comparison of the sample 203 and comparing the sample 203 with a comparison data for providing improved results using the artificial intelligence module 105.
[00065] At block 406, displaying improved result of the comparison of the sample using the display unit.
[00066] Fig. 5 illustrates excitation emission spectra of (a) Fetal Bovine Serum, (b) HSF without contamination, (c) HSF contaminated mildly, (d) HSF contaminated heavily; according to an embodiment herein.
[00067] According to an embodiment, the figure is to provide better understanding of the auto fluorescence phenomenon depicted by islet cells and Fetal Bovine Serum.
[00068] According to an embodiment, Fetal bovine serum (FBS) is the liquid fraction of clotted blood from fetal calves, depleted of cells, fibrin and clotting factors, but containing a large number of nutritional and macromolecular factors essential for cell growth. Bovine serum albumin is the major component of FBS. Growth factors in FBS are essential for the maintenance and growth of cultured cells. FBS also contains a variety of small molecules like amino acids, sugars, lipids, and hormones.
[00069] According to an embodiment, FBS is used in a wide range of applications. One of the primary uses of FBS is in eukaryotic cell culture, with concentrations up to 20% or even higher, where it provides many essential nutrients and growth factors that facilitate cell survival and proliferation. However, it is important to note that FBS in human cell cultures may introduce research artifacts; human cells cultured with human sera behave differently from those cultured with FBS.
[00070] According to an embodiment, Fetal bovine serum (FBS) is the most widely used growth supplement for cell culture media because of its high content of embryonic growth promoting factors. When used at appropriate concentrations it supplies many defined and undefined components that have been shown to satisfy specific metabolic requirements for the culture of cells.
[00071] According to an embodiment, Prokaryotic and eukaryotic cells exhibit an intrinsic natural fluorescence due to the presence of fluorescent cellular structural components and metabolites. Therefore, cellular autofluorescence (AF) is expected to vary with the metabolic states of cells.
[00072] According to an embodiment, Cells contain molecules, which become ?uorescent when excited by UV/Visible radiation of suitable wavelength. This ?uorescence emission, arising from endogenous ?uorophores, is an intrinsic property of cells and is called auto-?uorescence to be distinguished from ?uorescent signals obtained by adding exogenous markers.
[00073] According to an embodiment, the majority of cell auto-?uorescence originates from mitochondria and lysosomes. Together with aromatic amino acids and lipo-pigments, the most important endogenous ?uorophores are pyridinic (NADPH) and ?avin coenzymes.
[00074] According to an embodiment, in tissues, the extracellular matrix often contributes to the auto-?uorescence emission more than the cellular component, because collagen and elastin have, among the endogenous ?uorophores, a relatively high quantum yield.
[00075] According to an embodiment, changes occurring in the cell and tissue state during physiological and/or pathological processes result in modi?cations of the amount and distribution of endogenous ?uorophores and chemical–physical properties of their micro environment. Therefore, analytical techniques based on auto-?uorescence monitoring can be utilized in order to obtain information about morphological and physiological state of cells and tissues.
[00076] According to an embodiment, the images in Fig. 5 correspond to excitation emission spectra recorded from standard spectrofluorometer to understand the auto-fluorescence response of a) Fetal Bovine Serum, b) HSF without contamination, c) HSF Contaminated mildly and d) HSF heavily contaminated.
[00077] According to an embodiment, the auto-fluorescence observed in red region around 600 nm towards porphyrin production from bacteria suspecting contamination.
[00078] Fig. 6 illustrates Fluorescence emission curve when cells are excited at specific wavelengths, according to an embodiment herein;
[00079] Fig. 7 illustrates Fluorescence emission curve when media is excited at specific wavelengths, according to an embodiment herein;
[00080] Fig. 8 illustrates Fluorescence emission curve when FBS is excited at specific wavelengths, according to an embodiment herein.
[00081] According to an embodiment, it can be interpreted that at 365, 395 and 415 nm excitation wave length an increase in the fluorescence intensity in the contaminated flasks containing cells from 0-96 hrs was observed.
[00082] According to an embodiment, the change in the fluorescence intensity in mild contamination (MC) flask cells was observed to be relatively less.
[00083] According to an embodiment, the Fluorescence intensity starts from the time there is an induction in the contamination in the cells and the fluorescence increases drastically as the contamination peaks
[00084] An advantage of the present invention is that it provides faster detection of pathogens in a cell culture.
[00085] Another advantage of the present invention is that it provides a portable device for detecting pathogens in a cell culture of islet cells.
[00086] Yet another advantage of the present invention is that it provides an improved and efficient system and method for detecting pathogens in a cell culture.
[00087] Still another advantage of the present invention is that it provides a user friendly device for detecting pathogens in a cell culture.
[00088] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
,CLAIMS:We claim:

1. A method 400 for accurately detecting contamination in a sample of cell culture, wherein the method comprising the steps of:
illuminating 401 a sample of cell culture using a plurality of light sources in UV-A, UV-B, UV-C, visible band;
capturing 402 emission from the illuminated sample using a detector;
processing 403 the captured emission using an electronic hardware unit;
comparing 404 the processed emission with a standard data using the electronic hardware unit;
updating 405 a comparison of a sample and comparing the sample with a comparison data for providing improved results using an artificial intelligence module; and
displaying 406 improved result of the comparison of the sample using the display unit; characterized in that
detecting pathogens in a cell culture of islet cells;
identifying a difference in auto fluorescence and a threshold to detect contamination of islet cells and/or cell culture; and
providing updated comparison data 405 by the artificial intelligence module for comparing with a sample for providing improved and accurate identification of contamination in the sample.
2. The method as claimed in claim 1, wherein the processing 402 includes applying thresholding and image enhancement on the captured emission obtained in a form of spectral images and processing using machine learning and/or deep learning techniques for detecting and quantifying pathogens.
3. The method as claimed in claim 1, wherein the comparing 404 includes comparing intensities of fluorescence emission and spatial parameters of captured spectral images of the sample with the standard data.
4. The method as claimed in claim 1, wherein amount of pathogens is quantified based on intensity of emitted fluorescence intensity in various spectral bands of the sample.
5. The method as claimed in claim 1, wherein excitation sources placed at 450 angle to ensure illumination.
6. A system 100 for accurately detecting contamination in a sample of cell culture, wherein the system comprises:
an optical unit 102 provided for illuminating a sample and detecting emission from the illuminated sample;
an electronic hardware unit 101 connected to the optical unit 102 for processing the emission and comparing the processed emission;
an artificial intelligence unit 105 connected to the electronic hardware unit 101 for updating comparison data of a plurality of samples from the electronic hardware unit 101 and comparing the sample with the comparison data; and
a display unit 104 connected to the optical unit for displaying improved result of the comparison of the sample received from the artificial intelligence unit 105.
7. The system as claimed in claim 6, wherein the electronic hardware unit 101 includes a controller and a power source.
8. The system as claimed in claim 6, wherein the optical unit 102 includes a plurality of light sources producing light in UV-A, UV-B, UV-C, visible band and a detector.
9. The system as claimed in claim 6, wherein the system is a portable device.
10. The system as claimed in claim 6, wherein the portable device takes minutes for accurate detection pathogens in a cell culture.
11. The system as claimed in claim 6, wherein the electronic hardware unit 101 configured for supplying power to the optical unit 102 using the power source.
12. The system as claimed in claim 6, wherein the controller in the electronic hardware unit 101 configured for controlling the optical unit 102 to detect the illuminated sample, wherein the controller synchronizes excitation components and emission components for capturing auto fluorescence.
13. The system as claimed in claim 7, wherein the controller in the electronic hardware unit 101 configured for comparing a illuminated sample with a standard non-contaminated sample for identifying contamination of the sample 103.
14. The system as claimed in claim 8, wherein detector 203 includes a hyper spectral camera or a multispectral camera having a detector selected from a group consisting of CMOS detector, CCD detector, photodiode, phototransistor, avalanche photodiode, single photon avalanche detector, photomultiplier tube.
15. The system as claimed in claim 14, wherein the detector 203 configured for capturing emission from the illuminated sample 103 and placed parallel to the sample 103.