CLIAMS:We Claim:
1. A method for high-throughput optofluidic hyperimaging of a biological cell, the method comprising;
- selecting at least one of a cell type;
- initiating a flow of the selected cell type;
- irradiating the flow path with a first light source and at least one second light source;
- simultaneously capturing an image of the cell and a signal specific to the cell type; and
- analyzing the captured image along with the signal detected to obtain a hyperimaging data corresponding to the cell.
2. The method of claim 1, wherein the cell type is a homogenous cell type or a heterogeneous cell type.
3. The method of claim 1, wherein the flow is an uniform flow.
4. The method of claim 1, wherein the wavelength of the first light source is in the range of 300 nm to 900 nm.
5. The method of claim 1, wherein the first light source is for obtaining a bright field and/or flourescence image of the cell.
6. The method of claim 1, wherein the wavelength of the second light source is specific to the cell type and/or chemical composition of the cell.
7. The method of claim 1, wherein the signal is an electrical signal specific to the cell type.
8. The method of claim 1, wherein simultaneous capturing is achieved by splitting the light transmitted through the cell into a first part for capturing an image specific to the cell type and a second part for capturing a signal specific to the cell type.
9. The method of claim 1, wherein the hyperimaging data is at least one selected from a group comprising of size of a cell, shape of a cell, at least one optical characteristic of a cell, an electrical signal corresponding to a cell or a combination thereof.
10. A method for detecting an anomaly in a cell type, the method comprising;
- selecting at least one type of cell;
- initiating a flow of the selected cell;
- irradiating the flow path with a first light source and at least one second light source;
- simultaneously capturing an image of the cell and a signal corresponding to the cell;
- analyzing the captured image along with the signal detected to obtain a hyperimaging data corresponding to the cell;
- comparing the hyperimaging data obtained with a threshold data; and
- establishing a presence of an anomaly from the compared data.
11. The method of claim 10, wherein the anomaly is a physiological anomaly, an anatomical anomaly or a combination thereof.
12. The method of claim 10, wherein the threshold data is obtained from a database of hyperimaging data obtained from a plurality of known cell types.
13. The method of claim 10, wherein the cell type is at least one of a normal cell type or an anomalous cell type.
14. The method of claim 10, wherein the anomaly is established by detecting the number of occurrences of the signal below the determined threshold.
15. An apparatus for high-throughput optofluidic hyperimaging of a biological cell, the system comprising;
an irradiation arrangement;
a microfluidic device;
a collection arrangement;
a primary detection arrangement and at least one secondary detection arrangement coupled to the collection arrangement; and
an analysis unit coupled to each of the detection arrangement.
16. The apparatus of claim 15, wherein the irradiation arrangement comprises of at least one light source and a lens arrangement for collimating the light from the light source.
17. The apparatus of claim 15, wherein the collection arrangement comprises of a lens arrangement; and a beam splitter.
18. The apparatus of claim 15, wherein the primary detection arrangement comprises of a focusing arrangement; a neutral density filter; and a detector.
19. The apparatus of claim 18, wherein the detector is selected from a list comprising of a CCD camera, a CMOS camera, a NMOS camera, a two-dimensional array detector, a liquid-nitrogen cooled CCD, a back-illuminated CCD and an on-chip amplification CCD.
20. The apparatus of claim 15, wherein the second detection arrangement comprises of a lens arrangement; and at least one photodiode.
21. The apparatus of claim 20, wherein photodiode is specific to the wavelength of the light source.
22. The apparatus of claim 15, wherein the analysis unit comprises of
a database for storing the data obtained from the detection arrangement;
a comparison engine for comparing the data stored; and
a rendering engine for displaying the hyperimaging data.

Bangalore NARENDRA BHATTA HL
23rd June 2015 (INTELLOCOPIA IP SERVICES)
AGENT FOR APPLICANT
,TagSPECI:HIGH-THROUGHPUT OPTOFLUIDIC HYPERIMAGING

FIELD OF INVENTION
The invention generally relates to the field of instrumentation and applied physics and particularly to a method and an apparatus for high-throughput optofluidic hyperimaging of a biological cell.

BACKGROUND
Early detection of anomalies relies on high-content screening technologies including but not limited to automated digital microscopy and flow cytometry. Flow cytometry analyses the physical and chemical characteristics of a cell or a particle in a fluid as it passes through an electronic detection apparatus. Flow cytometry is employed to perform several procedures including but not limited to cell counting, cell sorting, biomarker detection and protein engineering. Automated digital microscopy is employed for applications which need high resolution such as the automated imaging of pathology samples, molecular diagnostics or high resolution images of cells.
Imaging flow cytometry combines the high-thoroughput power of flow cytometry with the spatial resolution and quantitative morphology of digital microscopy. Imaging flow cytometry offers advantages over other techniques in terms of live cell imaging and high-thoroughput imaging. Some disadvantages are elaborate, bulky arrangement and high cost of equipment. Further, both flow cytometry and imaging flow cytometry provide single mode characterization data of a population of cells. Flow cytometry measures (optical signal mode) laser based characteristics of cells like scattering, absorption, fluroscence, whereas, Imaging flow cytometry enables (image mode) high-throughput image acquisition. But neither techniques enable acquisition of dual mode data of cells.

BRIEF DESCRIPTION OF DRAWINGS
So that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
FIG.1 shows a schematic representation of an arrangement for high-throughput optofluidic hyperimaging of a biological cell, according to an embodiment of the invention.
FIG.2 shows an arrangement for high-throughput optofluidic hyperimaging of a biological cell, according to an example of the invention.
FIG.3 shows an arrangement for detection of malarial parasite infected RBC, according to an embodiment of the invention.
FIG. 4 shows voltage signal recording of normal cell and infected cell, according to an embodiment of the invention.
FIG. 5 shows voltage signals and the corresponding images of cells, according to an embodiment of the invention.
FIG. 6 shows histogram of percentage absorbance for normal and infected cell, according to an embodiment of the invention.

SUMMARY OF THE INVENTION
One aspect of the invention provides a method for high-throughput optofluidic hyperimaging of a biological cell. The method includes selecting at least one of a cell type, initiating a flow of the selected cell type, irradiating the flow path with a first light source and at least one second light source, simultaneously capturing an image of the cell and a signal corresponding to the cell type, and analyzing the captured image along with the signal detected to obtain a hyperimaging data corresponding to the cell.
Another aspect of the invention provides a method for detecting an anomaly in a cell type. The method includes selecting at least one of a cell type, initiating a flow of the selected cell type, irradiating the flow path with a first light source and at least one second light source, simultaneously capturing an image of the cell and a signal corresponding to the cell type, and analyzing the captured image along with the signal detected to obtain a hyperimaging data corresponding to the cell, comparing the hyperimaging data obtained with a threshold data and establishing a presence of an anomaly from the compared data.
Yet another aspect of the invention provides an apparatus for high-throughput optofluidic hyperimaging of a cell. The apparatus includes an irradiation arrangement, a microfluidic device, a collection arrangement, a primary detection arrangement and at least one secondary detection arrangement coupled to the collection arrangement and an analysis unit coupled to each of the detection arrangement.

DETAILED DESCRIPTION OF THE INVENTION
Various embodiments of the invention provide a method and an apparatus for high-throughput optofluidic hyperimaging of a biological cell. Hyperimaging, herein referred to, is defined as the simultaneous characterization of cells through a plurality of signals. The signals are obtained from a plurality of laser interrogation modes including but not limited to fluorescence, absorption, emission and with bright-field and/or other imaging modalities.
One embodiment of the invention provides a method for high-throughput optofluidic hyperimaging of a biological cell. The method includes selecting at least one of a cell type, initiating a flow of the selected cell type, irradiating the flow path with a first light source and at least one second light source, simultaneously capturing an image of the cell and a signal corresponding to the cell type, and analyzing the captured image along with the signal detected to obtain a hyperimaging data corresponding to the cell. The method described herein above in brief shall be described in detail.
The method includes selecting at least one of a cell type. The cell type is a homogenous cell type or a heterogeneous cell type. The selected cell type is allowed to flow uniformly in a microfluidic channel. The microfluidic channel consists of a straight channel having a width of about 20 µm and depth of about 6 µm. To ensure a single cell flow, a constriction of about 8µm width is provided in the microfluidic channel. The flow path is then irradiated with a first light source and at least one second light source. The wavelength of the first light source is in the range of 300 nm to 900 nm, further the first light source is for obtaining a bright field image of the cell. The wavelength of the second light source is in the ultraviolet-visible-infrared (UV-VIS-IR) range and is specific to the cell type and/or composition of the cell.
Light from the first light source and the second light source is collimated and then focused on the cell. The light transmitted through the cell is captured simultaneously by splitting the light transmitted through the cell into a first part for capturing an image specific to the cell type and a second part for capturing a signal specific to the cell type.
The signal captured is an electrical signal specific to the cell type. The captured image of cell along with the signal corresponding to the cell are analysed to obtain a hyperimaging data corresponding to the cell. The hyperimaging data is at least one selected from a group comprising of size of a cell, shape of a cell, at least one optical characteristic of a cell, an electrical signal (generated as per the optical characteristic of the cell) corresponding to a cell or a combination thereof.
The invention also provides an apparatus for microfluidic hyperimaging of a biological cell. FIG. 1 shows a schematic representation of an arrangement for high-throughput microfluidic hyperimaging of a biological cell, according to an embodiment of the invention. The apparatus includes an irradiation arrangement 1, a microfluidic device 5, a plurality of collection arrangement 17, a primary detection arrangement 19 and at least one secondary detection arrangement 15 coupled to the collection arrangement and an analysis unit 21 coupled to each of the detection arrangement. FIG. 2 shows an arrangement for high-throughput microfluidic hyperimaging of a biological cell, according to an example of the invention. The arrangement includes an irradiation arrangement. The irradiation arrangement includes a first light source S1 and a second light source S2. The wavelength of the first light source S1 is in the range of 300 nm to 900 nm. The examples of the light source includes but are not limited to a coherent light sources like gas lasers, solid-state lasers, diode lasers, fibre coupled lasers and incoherent light sources like light emitting diodes (LEDs), high power LEDs, LED arrays, halogen or tungsten lamps. The wavelength of the second light source S2 is in the ultraviolet-visible-infrared (UV-VIS-IR) range. The examples of the second light source includes but are not limited to a coherent light sources like gas lasers, solid-state lasers, diode lasers, fibre coupled lasers and incoherent light sources like light emitting diodes (LEDs), high power LEDs, LED arrays, halogen or tungsten lamps.
The irradiation arrangement also includes a lens arrangement 3. The lens arrangement 3 includes a plurality of biconvex lenses, namely L1, L2 and L3. The lenses L1, L2 and L3 are arranged coaxially. The cell to be analysed, is allowed to flow in a microfluidic channel 5. The microfluidic channel 5 consists of a straight channel having a width of about 20 µm and depth of about 6 µm. To ensure a single cell flow, a constriction (not shown) of about 8 µm width is provided in the microfluidic channel 5. Alternatively, the single cell flow can also be brought about with the use of 2D/3D microfluidic flow focusing mechanism. The cell in the constriction region is irradiated by the light source S1 and S2. Irradiation is achieved first by collimating a light beam from the light source S1 and S2 on to the lens L1 and L2. The collimated light beam is then focused into a fine spot using lens L3. The focused spot is then used to illuminate the cell. After illumination, light transmitted through the cell is collected by an collection arrangement. The collection arrangement includes an objective lens 7 and a beam splitter 9. The light transmitted through the cell is first expanded by the objective lens 7 and then split into two parts by the beam splitter 9. The splitted light is then simultaneously relayed on a primary detection arrangement and a secondary detection arrangement. The primary detection arrangement includes a tube lens 11, a neutral density filter 13 and a detector 15. The examples of detectors include but are not limited to a CCD camera, a CMOS camera, a NMOS camera, two-dimensional array detector, liquid-nitrogen cooled CCD, a back-illuminated CCD, an on-chip amplification CCD and other such devices for high-thoroughput imaging. One portion of the splitted light is first passed through a tube lens 11 and then through the neutral density filter 13 to the detector 15 to obtain an image of the cell. The secondary detection arrangement includes a condenser lens 17 and a photodiode 19. The photodiode 19 is specific to the wavelength of the light source S2. Other portion of the splitted light is focused on the condenser lens 17 and then on the photo diode 19. The photo diode 19 gives a signal corresponding to the cell. The captured image of cell along with the signal corresponding to the cell is analyzed. The detector 15 and the photodiode 19 are coupled to an analyzer 21. The light intensity measured by photodiode 19 is measured in terms of voltage by the analyzer 21. The analysis unit includes a database for storing the data obtained from the detection arrangement, a comparison engine for comparing the data stored and a rendering engine for displaying the hyperimaging data.
The invention also provides for a method for detection of an anomaly in a cell type. The method includes selecting at least one of a cell type, initiating a flow of the selected cell type, irradiating the flow path with a first light source and at least one second light source, simultaneously capturing an image of the cell and a signal corresponding to the cell type, and analyzing the captured image along with the signal detected to obtain a hyperimaging data corresponding to the cell, comparing the hyperimaging data obtained with a threshold data and establishing a presence of an anomaly from the compared data. The anomaly is a physiological anomaly, an anatomical anomaly or a combination thereof. The threshold data is obtained from a database of hyperimaging data obtained from a plurality of known cell types. The cell type is at least one of a normal cell type or an anomalous cell type.
Example 1: Detection of malarial parasite infected RBC
FIG.3 shows an arrangement for detection of malarial parasite infected RBC, according to an embodiment of the invention. A suspension of cells prepared from a culture of plasmodium falciparum infected RBCs is used for validation. The cells in the suspension is irradiated with a laser diode 1 (? = 405 nm). The light from the laser diode 1 is collimated using two bi-convex lenses (L1; focal length,5 cm and L2; focal length,15 cm) and then focused into a fine spot using another bi-convex lens L3(focal length, 20 cm). The suspension of cells containing Pf-iRBCs is flown through a microfluidic device 5 at a flow rate of 60 µLh-1 using a syringe pump. The microfluidic device 5 consists of a straight channel having a width of about 20 µm and depth of about 6 µm. To ensure a single cell flow, a constriction (not shown) of about 8µm width is provided in the microfluidic channel 5. The focused laser spot is used to illuminate the cell flowing through the constriction.
The light transmitted through the cell is first expanded using a 40X microscope objective 7 (Olympus plan fluorite 0.75NA) and then split into two parts by a beam splitter 9. One part of the transmitted light is relayed onto a High-speed CMOS camera 15 (Mikrotron MC 1362) using a Tube lens 11 in order to enable imaging of the cell. The images are recorded at frame-rate of 13000 fps and exposure time of 10 µs. A neutral density filter 13 is placed between the tube lens 11 and the CMOS camera 15, in order to reduce the intensity of transmitted light to avoid damaging the pixels of the camera. The other part of the transmitted light is focused onto an avalanche photodiode (AD800-11) 19 using an aspheric condenser lens 17, so as to measure the overall intensity of the light after it passes through the cell. The photodiode 19 is interfaced to a micro-controller board 21, to convert and record the light intensity to corresponding voltage levels. The voltage levels are recorded at a sampling rate of 24 KHz.
FIG. 4 shows voltage signal recording of normal cell and infected cell, according to an embodiment of the invention. The figure shows distinct minimas obtained from recorded voltage, corresponding to different cells. The images of cells corresponding to the minimas are labeled: (i) - infected, (n) - normal. At the wavelength of 405 nm, significant optical absorption by the hemoglobin present in the cells is expected. In the images of normal cells (n), a large dark portion corresponding to the hemoglobin (Hb) content of the cell is observed. Whereas, in the case of PfiRBCs(infected “(i)”), the dark region is significantly smaller in size, indicative of decrease in hemoglobin content in the infected cells.
Further, the events which may effect the accuracy in detection and thus the analysis are monitored. For example, when two cells flow very close to each other across the interrogation region, a signal with two overlapping dips is recorded (as shown in figure 5 (a)). Also, when two cells, stuck to each other, simultaneously flow across the interrogation region, a signal with a characteristic very low drop in the voltage is recorded (as shown in figure 5 (b)). The two signals(5a and 5b) are quite different from a signal that is recorded for a normal cell (as shown in figure 5 (c)).
FIG. 6 shows histogram of percentage absorbance for normal and infected cell, according to an embodiment of the invention. In order to validate the method over a large population of cells, two different suspensions of cells containing normal and Pf-iRBCs are separately analyzed. The figure represents absorbance values of about 24023 cells present in normal RBC suspension, and about 18558 cells present in Pf-iRBC suspension. The suspension of normal RBCs only contained uninfected cells, whereas the suspension of Pf-iRBCs contained both infected and healthy cells. The percentage absorbance values for the normal RBC suspension are found to have a lower limit of 5 % (histogram plotted in darker shade), whereas some of the absorbance values for the Pf-iRBC suspension are found to be lower than 5 %, indicating the presence of infected cells.
The invention thus provides for a method for microfluidic hyperimaging wherein the data pertaining to image of cell and signals specific to the cell are obtained and analysed simultaneously.
The invention provides a method for high-throughput optofluidic hyperimaging of a biological cell. The method enables rapid screening of a given biological sample. Further the method also enables detection of anamoly in the sample by analyzing the signal obtained from the simultaneous capture of a image and at least one signal from the cell. A system to achieve this is also provided. The method as described herein along with the appended claims provides a method that does not involve need of a skilled person to visually inspect thousands of cells to establish an anamoly. Further, the system enables detailed inspection of any particular cell of interest obtained, at any given time without the need to re-run the sample through the apparatus.
The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.