Claims:We Claim:

1. A method for rapid probing of an object within a sample, the method comprising;
obtaining an image of the sample;
identifying location of plurality of objects within the obtained image;
determining a centre of the object within the obtained location;
arriving at an intermediate path of the travel of probe between any two successive object positions;
optimizing the total path travelled by the probe from the plurality of intermediate paths obtained; and probing of the objects along the optimized total path.

2. The method of claim 1, wherein the image is an optical image or a scanning electron microscopy image.

3. The method of claim 1, wherein the determination of the centre of the object is achieved by identifying the surface of the object; pixelating the identified surface of the object; identifying a plurality of positions within the pixelated region; and optimizing the final position of the object from the plurality of positions wherein optimization of the final position yields the centre of the object.

4. The method of claim 3, wherein the optimization of the final position is achieved by traversing through the plurality of positions within the pixelated region.

5. The method of claim 1, wherein the optimization of the total path is achieved by calculating the shortest travel path for probing the plurality of objects within the sample.

6. The method of claim 1, wherein the rapid probing of the object is to identify mechanical stiffness.

7. The method of claim 1, wherein the sample is selected from a list comprising of biological cells, large molecules like DNA, nanoparticles and quantum dots.

8. The method of claim 1 wherein, the probing is achieved by probe selected from a list comprising of a scanning probe microscope, an atomic force microscope, a scanning tunneling microscope, a near-field scanning optical microscope, a micro-indenter and a nano-indenter.

Bangalore NARENDRA BHATTA HL
29 April 2016(INTELLOCOPIA IP SERVICES)
AGENT FOR APPLICANT

Description:A METHOD FOR RAPID PROBING OF A SAMPLE

FIELD OF INVENTION

The invention generally relates to the field of cell probing and particularly to a method for rapid probing of a sample.

BACKGROUND

Mechanical properties of cells play important role in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells are often signs of changes in cell physiology or diseases in tissues. Thus, probing of the cells to obtain mechanical stiffness data of the cells provides valuable information relating to various diseases and infections.

Various techniques are available in the art for measuring the mechanical stiffness of cell including but not limited to particle-tracking microrheology, magnetic twisting cytometry, micropipette and micro indentation. Particle tracking microrheology traces the thermal vibrations of submicron fluorescent particles injected into cells, elastic and viscous properties of cells are then calculated from the measured particle displacements. One significant disadvantage of the technique is that injecting fluorescent particles into cells may lead to changes in cellular function, cytoskeleton structure, and hence the cell mechanics.

The micropipette aspiration method applies negative pressure in a micropipette to suck a small piece of cell membrane into the pipette. Cell stiffness is calculated from the applied negative pressure and cell membrane deformation.

One significant disadvantage of the method is that the heterogeneous distribution of stiffness across the cell is not detected.

Magnetic twisting cytometry applies magnetic field to generate torque on super paramagnetic beads attached to the cell membrane. Cell stiffness is calculated from the relationship between the applied torque and the twisting deformation of the cell membrane. One significant disadvantage of the technique is difficulty in controlling the location of magnetic beads.

Micro indentation technique requires an indenter with well-defined geometry to punch into the cell. The indenting force and the resulting indentation in the cell are used to measure mechanical stiffness of the cell. Among the many devices for micro indentation, the Atomic Force Microscope (AFM) is most widely applied to characterize mechanical properties of cells. AFM has several advantages in measuring mechanical stiffness of the cells including provide stiffness data on multiple points within a cell, provide lateral stiffness and viscosity data of the cell, and since the cells are on fixed locations, the AFM data can be correlated with fluorescent data. One significant disadvantage of the AFM is slow speed of probing and hence the use of AFM is restricted to as a research tool.

There are several methods available in the art which provide methods to improve the speed of the AFM. But all the available methods focus on the improvement in the mechanics, optical detection, controls and cantilever size to increase the scanning speed of the AFM.

Alternatively, the scanning speed of the AFM can be improved by optimizing the number of points for collection of data from a cell. Patent, CN103123362 B, assigned to Shenyang Inst Automation, discloses a method for rapid positioning of an AFM tip. The method includes optical imaging of the cells to create a circle using edge detection and the centre position of the circle is used to rapidly move the AFM tip. A method further describes scanning along specific lines in the cell circle to obtain mechanical stiffness data of the cell. One significant disadvantage of the method is that it still requires scanning multiple lines which will make it slow. Further, it approximates cell images into circles which in many cases may be a poor approximation.

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.1shows an optical image of the cells within a sample, according to an embodiment of the invention.

FIG.2a shows edges of the cell, according to an embodiment of the invention.

FIG.2b shows a two dimensional mesh created in the
cell image and the centroids of the triangles within the cell, according to an embodiment of the invention.

FIG.2c shows a travel path from one centroid to another centroid, according to an embodiment of the invention.

Fig. 3 showsthe edges and centroids of the cells and a travel path from one cell to another cell, according to an embodiment of the invention

SUMMARY OF THE INVENTION

One aspect of the invention provides a method for rapid probing of a plurality of objects within a sample. The method includes imaging of the sample to obtain location of plurality of objects within the sample, computing the position of the objects within the obtained location, computing an intermediate path of the travel of probe between any two successive object positions, optimizing the total path travelled by the probe from the plurality of intermediate path obtained and probing of the objects along the optimized total path.

DETAILED DESCRIPTION OF THE INVENTION

The invention as described herein provides a method for rapid probing of a sample. The method is based on optimization of the path travelled by the AFM probe to scan and collect the mechanical stiffness data from specific locations on the plurality of objects within a sample. Optimization of path is done for scanning each individual objects and for scanning plurality of objects within the sample. The method optimizes the time of travel and data collection so as to allow investigation of large number (100s or even 1000s) of objects within the sample. The method for rapid probing of a sample includes optical imaging of the sample to obtain location of plurality of objects within the sample, computing the location of the objects within the obtained location, computing an intermediate path of the travel of probe between any two successive object positions, optimizing the total path travelled by the probe from the plurality of intermediate path obtained and probing of the objects along the optimized total path.

The method described in brief herein above shall be described in detail with various examples. The method for rapid probing of a sample includes imaging of the sample to obtain location of plurality of objects within the sample. Examples of imaging technique include but are not limited to optical imaging and scanning electron microscopy. The examples of sample include but are not limited to biological cells, large molecules like DNA, nano particles and quantum dots. In one example of the invention, the biological cells are selected as sample for probing. For probing of the sample, the probes include but are not limited to a scanning probe microscope, an atomic force microscope, ascanning tunneling microscope, a near-field scanning optical microscope, a micro-indenter and a nano-indenter. FIG.1shows an optical image of the cells, according to an embodiment of the invention. The cells 1 in the sample are optically imaged by an optical microscope of the tip 2 of the AFM cantilever 3. The field of the optical image ranges from about 100 microns x 100 microns to about 1mm x 1mm. The AFM tip 2 could be visible in the optical image or be hidden under the cantilever 3. When the AFM tip is kept invisible, the co-ordinates of the tip 2 with respect to the cantilever 3 are known and can be estimated. The cells 1 are either live or are fixed. In one example of the invention, the cells are fixed by formaldehyde. The sample is prepared by spreading a suspension containing the cells on a flat substrate 4. The number of cells spread on the substrate is kept such that the cells typically do not touch with each other. Optionally, to avoid disturbance from outer environment or to maintain controlled conditions, the substrate 4, the AFM cantilever 3, the tip 2 and the cells 1 are kept inside a liquid chamber.
Subsequent to optical imaging, the position of the objects within the obtained location is computed. The computation of the position is achieved by first identifying the surface of the object. Identification of the surface of the object is done by edge detection technique (FIG.2a). After identifying the surface of the object, the identified surface is pixelated to obtain various pixel regions. In one embodiment of the invention, pixelation of identified surface is achieved by creating a two dimensional mesh of triangles. FIG.2b shows a two dimensional mesh created in the cell image and the centroids of the triangles within the cell, according to an embodiment of the invention. The mesh density is controlled by the desired maximum size of the edges of the triangles which in turn controls approximately the number of specific points from which AFM data is to be collected. Each cell is typically meshed into about 10 to about 100 triangles. After pixelating the identified surface, the pixel regions are normalized to obtain one position within each pixel region. In one example of the invention, normalization is achieved by obtaining one position as centroid 6 from the pixel region (FIG.2b). The normalized position is then optimized to obtain the final positions of the object. The centroids 6 of the triangle are used to plan an optimum path 7 for the AFM tip 2 (FIG.2c). The AFM tip 2 is then moved to the centroids 6 along the optimum path. During this movement, the AFM tip 2 is maintained slightly above the surface of the sample so that the tip 2 can be moved fast without the risk of touching the surface of the cell 1. At every centroid 6 of the triangle, the AFM tip 2 is stopped and moved in z direction (towards the surface). Additionally, the tip is stopped when a parameter approaches a threshold value. Subsequent to attaining the threshold value, the tip is retracted away from the surface. As the tip 2 comes in contact with the surface, the signals are recorded. The signal is either a deflection of the cantilever, frequency, amplitude, phase, excitation or a combination thereof. Subsequent to the optimization of path within the object, the path for the travel of probe between any two successive objects is computed. Various paths obtained after computation are then optimized by selecting shortest path for probing the plurality of objects within the sample.
From the optical image of the plurality of objects, the edges of the objects, centroids of the objects and the position of the AFM tip 2 are detected using edge-detection algorithms. Fig. 3 shows the edges and centroids of the cells and a travel path from one cell to another cell, according to an embodiment of the invention. The edges 5 of the cells and the centroids 8 of the cells and the position of the AFM tip 2 are detected using edge-detection algorithms as available in the art. The co-ordinates of the centroids 8 are then used to plan an optimum path 9 for the AFM tip 2. The optimization of the path is achieved by an optimisation algorithm. Examples of optimization algorithm include but are not limited to a genetic algorithm, an ant colony optimization algorithm and a greedy algorithm.
In one example of the invention, the path 9 is optimized by using a genetic algorithm. Initially, an even number (about 10) of random paths is generated. A path is represented by the sequence of centroids (e.g. ….ABCD….). The path length is calculated for each path and a fitness probability function is defined for the ith path as =1/path_length_i/[1/path_length_1 + 1/path_length_2 + …. 1/path_length_n]. New set of n paths are created from the previous set in which a previously defined path is repeated in proportion to its fitness probability. Then n/2 pairs of paths, parent paths, are selected for ‘crossover’. A random part of the sequence from the first of the pair is selected [….ABCD….] and its corresponding part is found in the second of the pair [..C.D..A..B..]. Then a first path, ‘child’ path, is created as [..A.B..C..D..]. A second path is then created as [….CDAB….].

Further, three types of ‘mutations’ are applied to the new paths:

(a) random exchange of two sites […A..B…] to […B..A…],

(b) random exchange of two pieces of paths
[…ABCD…EFGH…] to […EFGH…ABCD…] and

(c) random flip of a piece of path […ABC…] to […CBA…].

The entire ‘evolution’ process is repeated N number of times. N could be about 1000, and is determined by computation time and the accuracy of minimum path desired.

In another example of the invention, the path 9 is optimized using an ant colony optimization algorithm. Initially, about 20 virtual ants are positioned randomly on the centroids 8. Then a path is developed for each ant to cover all the centroids 8. When an ant is at a given centroid, the next centroid is selected based on a probability derived from the nearness of the centroid and the pre-existing desirability (pheromone trace). In one example, all the ants find a path to cover all the centroids 8. A cost function (total distance) is calculated for each path. The pheromone trace is updated for each possible section (A-B) based on the cost function. An evaporation coefficient for the pheromone trace ensures that the probability for paths that are high cost are gradually diminished. The steps are repeated for about 20 numbers of iterations. The shortest path from the last iteration is selected.
Subsequent to the optimization, the plurality of objects is probed along the optimized path. The AFM tip 2 is moved to the centroids 8 according to the optimum path 9. During this movement, the AFM tip 2 is maintained slightly above the surface of the sample so that the tip 2 can be moved fast without the risk of tip touching the surface.

The invention provides for a method for rapid probing of a sample. Optimization of path for probing of the individual objects/cells at specific points and for probing all the objects within the sample reduces the time taken to probe cells. Hence, more number of cells can be probed in a given time thus, increasing the speed and performance of the probe. The method as described herein can be employed for measuring mechanical stiffness of cells, specifically to determine the metastatic potential of cancer cells. The method can also be applied in diagnostics examples of which include but are not limited to identification of metastatic potential of cells from a biopsy and sepsis.

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. It is to be noted, however, that the aforesaid description illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to equally effective embodiments.