Indian Patents
Title of Invention

A SPECTROPHOTOMETRIC SYSTEM FOR MEASUREMENT OF VERTICAL VELOCITY OF CLINICAL AND BIOLOGICAL SAMPLES
Abstract A spectrophotometric system for measurement of vertical velocity of clinical and biological samples, the system comprising: (a) a light source being provide light into a light path leading to the sample whose vertical velocity is to be measured; (b) a filter means operatively associated with said light source; ( c) an optical system connected to said filter means to transmit filtered light along the light path to the said sample; (d) a cuvette housing system such as herein described, being connected to the said optical system at one end and comprising a housing within which a cuvette movable in a plurality of predetermined height positions being provided mounted on a cuvette holder which in turn being mounted on an angular disc cam and connected to a cuvette drive means through a coupling sleeve; (e) a light detection and analysis means connected to the cuvette housing system at an opposing end and connected to a display means.
Full Text COMPUTER BASED SPECTROPHOTOMETRIC SYSTEM TO DETERMINE VERTICAL VELOCITY OF CLINICAL AND BIOLOGICAL SAMPLES
Field of the invention
The present invention relates to the development of a unique computer-based, spectrophotometer with vertical cuvette movement and associated analytical software with new mathematical modeling, for objective measurement of the vertical velocity of clinical and biological samples More particularly, the present invention also relates to a method for the determination of vertical velocity of spermatozoa using the novel spectrophotometnc system of the invention Background of the invention
Several clinical and biological applications require determination of velocity of samples For example, velocity of spermatozoa is considered a primary determinant factor to predict the fertilizing ability of the semen sample Further, vertical velocity, in comparison with horizontal velocity, is expected to be a better identifying parameter for grouping of various semen samples as per the respective qualities Because of this reason, measurement of vertical velocity is of significant importance The instrumentation system of the invention (Sperm Motility Analyzer - SPERMA) has tremendous global market potential and commercial uses in various research institutions and research organizations, universities and medical colleges, hospitals and human fertility clinics, drug design and pharmaceutical laboratones etc Capability of objective determination of vertical velocity of spermatozoa of this instrument is an important parameter for comparing with horizontal velocity obtained from any other instrument of sperm analyzer, such as CAS A etc Thus, it has the potential to generate remarkable impact in the field of Reproductive Biology In India as well, this instrumental system has got great market potentiality and commercial uses for the human fertility clinics, various research Institutions/Organizations, various animal and cattle breeding centers in rural and urban areas etc, particularly because of its inherent accuracy and preciseness Second advantage of this automated system is it's low cost Since a simple visible range spectrophotometer with higher resolution is sufficient to implement this new technique, the cost of such a computer aided sperm motility analyzer is much lower than that of presently available instruments Thirdly, it is adoptable to any spectrophotometer with little modification of the cuvette holder of the spectrophotometer By adopting this technique, it is expected that a spectrophotometer with variable and multiple height exposure of the sample would be useful in many other fields of research and development, besides
determination of sperm motility Thus it is strongly felt that various other usages (with little modification/ variation of this technique) will be opened/ revealed, once this instrumental system is marketed and gets exposure Thus, in short, it has got great global market potentiality because of its accuracy, low cost, adoptability, flexibility and capability of determination of vertical velocity along with other sperm motility parameters
To the best of our knowledge, a spectrophotometer with vertical movement of the cuvette (within the light path) has not yet been developed till date Thus in reality this idea itself is altogether a new and unique concept The associated mathematical models and software for calculating and analyzing sperm motility are new and unique in nature Objective determination of vertical velocity of spermatozoa, following direct method of velocity calculation, is a remarkable achievement of this instrumental system National and International patent data bases were searched, using, US Patent website, http //patft uspto gov, European Patent website, http //v3 espacenet com and CSIR website, http //www patestate com and no such spectrophotometnc sperm analyzing system was found Similar result has evolved when various other search engines (such as, www google com, www sciencedirect com, etc) were referred The instrumental system of the present invention (SPERMA) includes mainly of two fields of scientific applications, computer controlled instrumentation using a spectrophotometer and measurement of sperm motility parameters So, Extensive literature survey has been conducted in both of these two fields In the turbidimetnc method, semen is layered on the bottom of the cuvette filled with buffer, and motility is formulated as the rate of change in optical density as the sperm swim upward into the light path The visual sperm motility rating did not correlate with the turbidimetnc rating On the basis of these studies it is believed that the turbidimetnc analysis of sperm motility is a much more accurate measure of sperm motility than is visual microscopic analysis This increase in absorbance is dependent on the amount of motile cells and their average velocity (1, 2, 3, 4) Its major advantages are that 1) it provides a quantitative measure of sperm motility, 2) it does not rely on arbitrary rating by an individual, and 3) the sperm must perform work in order to be measured (l e, leave the semen and swim upward into the light path) (5) The method is based on the fact that sperm cells so endowed will be the first to swim upward into clear medium from a concentrated cell suspension at the bottom of an optical cuvette This results in a time-dependent increase in turbidity in the medium, which is recorded spectrophotometrically as an increase in absorbance The first slope of the curve represents an index of the velocity of the population of cells showing the fastest motility When measured in this system forward motility activity
(expressed as units) increased linearly with cell concentration Ficoll had no effect on the values recorded (6) The vigorously motile cells may be the best candidates for fertilization, and samples with a high fraction of such cells should have high fertilizing capacity It is suggested that this simple turbidimetnc test be used in evaluation of human semen as a possible indicator of fertilizing capacity (7) The spectrophotometric values did not necessarily correlate with light microscope assessments of forward motility, because the former method provides an assessment of numbers of motile cells and their rate of progression (6) There are objective methods such as cinemicrography, time-lapse photography, and videomicrography, are available There was a statistically significant positive relationship between sperm motility and morphology rated on a one-by-one basis, but the relationship was too small to influence the visual rating of human sperm motility (8, 9) The digital imaging system can adequately and rapidly quantitate a large number of cells with heterogeneous motility patterns. This technique may prove to be useful in defining motihty characteristics associated with capacitation, the acrosome reaction, and fertility of human sperm (10) A new, non-destructive, objective technique for measuring collective motility of highly concentrated ram and bull semen is described The principle is based on changes in the reflected light scattered by motile spermatozoa (11) The MEP (Multiple Exposure Photography) method enables concurrent accurate estimation of the spermatozoal concentration, the rate of motile spermatozoa and the velocity of individual spermatozoa (12) The Sperm Quality Analyzer provides an estimate of the overall quality of sperm by reflecting sperm concentration, viability, and motility This index reflects both the concentration of motile sperm cells and the intensity of their motility and cannot be determined by conventional semen analysis The sperm motility index shows a good correlation with sperm density, motility, morphology, which are crucial parameters for male fertility (13, 14, 15, 16) CAS A or Computer Assisted Semen Analyzer is based on microscopic video photography and corresponding analysis of the photography by a specific sophisticated software The CASA gives various types of analytical data but analyzes the sperm cells placed horizontally only (17, 18, 19, 20, 21, 22) Thus it is concluded that the determination of elaborate motility characteristics as obtained by CASA systems is of limited value to optimizing the evaluation of male fertility status (22) Movement characteristics of spermatozoa in culture medium, especially the average path velocity are of prognostic value in prediction of human oocyte fertilization rates (23) Low significant negative correlation was found between number of spermatozoa/ml and spermatozoal velocity (24) A constant motility profile was found in the sperm of fertile men after 8 h, whereas a different profile
was found in 94% of the infertile male groups (25) Sperm velocity is a more sensitive indicator of sperm function when compared to standard semen analysis results (26) Computer controlled instrumentation integrates computation, instrumentation and control or manipulation of the environments (experimental process) Uses of robotic techniques, automation and remote controlled manipulation have attained remarkable impact in various fields of applied science on the present day scenario Ferrell and Sheridan (38) described the concept of supervisory control and its implication for remote manipulations Bejczy (39) presented an exhaustive survey on the application of various types of sensors for the remote control of manipulators Sahchs et al (40) presented the development scheme of "Man-Machine-Interface" for actuating a mobile robot through rigorous software augmentation Brown et al (41) designed and developed a four degree-of-freedom planar joystick to control a three degree-of-freedom revolute type remote manipulator Luh (42) reviewed the principles of control circuit design for multi-joint multi-degree-of-freedom manipulators with position and/or velocity feedback schemes using various compensation techniques Pfeffer et al (43) implemented a robotic control system by taking sensory feedback of the joint-torques, enumerating the details regarding design of the joint torque sensor, analysis and information of the control system and finally computer simulation of the output responses Remote manipulation of robot can be manifested delicately for gross and fine movements with the help of various kinds of multi-fingered hands (44-46) A robot manipulator is characterised by a set of nonlinear, time-varying, and highly coupled dynamics equations (47) The real time control of a manipulator is difficult for its nonhnearity and complexity Most of the time the dynamic parameters are not precisely known and in most cases these parameters are time-varying The works reported in (48) and show that Fuzzy logic is suitable for the robot control system Artificial neural networks offer some advantages such as learning, adaptation, fault tolerance, parallelism and generalization A great interest in artificial neural networks has taken place during the past decade Neural networks have made strong advances not only in the area like continuous speech recognition and pattern recognition, but also in the field of automatic control systems These aspects are described in (49-50) Hardware and Computer related Topics are described in (51-55) Application of robots are found in automation of painting works (56-57) also In the medical science, automation and robotic techniques are extensively used, particularly in various surgical operations Cardiac surgery, endoscopic coronary artery bypass grafting, determination of blood flow, advanced endoscopic procedures, Urologic Laparoscopy etc are some of the specific examples where robotic automations are very commonly used (58-63)
The principles of measurement of sperm motility of some of the presently available instruments are briefly discussed here The most primary method of sperm motility detection and measurement is the microscopic method where it is entirely dependent on the visual observation and thus a chance of error always remains The turbidimetnc method uses a spectrophotometer for the estimation of sperm motility, where the sperm cells passing across the light path are analyzed in terms of change in absorbance or optical density (6, 7) The present invention is based on this method There is also an optical method where the changes in the reflected light scattered by motile spermatozoa are taken into consideration for the motility evaluation (11) There is an instrument available called as AutoMARQER which estimates the sperm count by measuring the colour intensity produced after the semen sample has been stained with Thiazme blue Motility and average velocity estimates are calculated after shining a laser beam through the sample (27) The Semen Quality Analyzer or SQA uses the fiber optic sensor and phototransistor for recording the small changes in light caused by the passage of sperm cells (28, 29) The Accucell Photometer of IMV Technologies works on a halogen bulb photometer principle and gives real time simultaneous display of the concentration and absorption after a user definable delay (30, 31) Time-Lapse or Time-Exposure photography is also used in the sperm motility assessment (8, 32) The Micro-cinematographic or Video-micrographic analysis is based on microscopic video photography where the morphology of the sperm cells are also detectable (8, 9) The Multiple Exposure Photography (MEP) system is an advanced system where light pulses were generated with the aid of a multipulser using a xenon stroboscope for the sperm motility analysis (12) The most costly system to date for the sperm motility assessment and velocity estimation is the CASA or Computer Aided Semen Analyzer where the microscopic video photographic method and stroboscopic methods are merged and with the help of computer software the entire analysis is done for the determination of velocity and other parameters (17-22, 33 -37) Except the spectrophotometnc / turbidimetric method (6, 7), in all the presently available methods, horizontal movement of the sperms are only considered Again, presently known turbidimetnc method considers the vertical movement of the spermatozoa against gravity for objective determination of cell motility, since it follows an indirect method of velocity determination A spectrophotometer with a fixed position of the cuvette produces single set of data, which restricts in the accuracy of the sperm quality analysis Moreover, this method deals with the vertical velocity of the most rapidly moving cells only Presently available sperm analyzers, such as CASA, are very costly All these drawbacks of the presently available sperm analyzers are considered and sufficient care has been taken to overcome
these shortcomings in the present invention In the present invention, vertical movement of the sperms is determined, considering entire population of cells in the cuvette This is possible by employing this unique technique of exposing the cuvette at multiple heights within the light path In the present invention, vertical velocity of the sperms is measured following a direct and unique technique Further, there are few motility parameters, which are new in reality Moreover the cost of the Instrumental system of the present invention (SPERMA) will be very less compared to any other sperm analyzer including CASA Present invention has evolved from this necessity Objects of the invention
The main object of this present invention is to develop a low cost, computer based, spectrophotometnc system to determine vertical velocity of the spermatozoa accurately for clinical and biological applications
Another object of the present invention is to develop a modified spectrophotometer which is capable of moving the cuvette vertically within the light path at multiple and variable heights
Yet another object of the present invention is to develop associated mathematical models and analytical software for calculating and analyzing sperm motility of the entire population of cells in the spermatozoa sample very precisely and accurately Summary of the invention
The present invention provides a low cost spectrophotometnc instrumental system which is capable of a) objective measurement of vertical velocity following a direct method b) moving the cuvette vertically within the light path of a spectrophotometer, thus exposing the sample at variable and different heights, c) calculating and analyzing sperm motility of the entire population of cells in the spermatozoa sample very precisely and accurately
Accordingly the present invention provides a spectrophotometnc system for measurement of vertical velocity of clinical and biological samples, the system comprising
(a) a light source to provide light into a light path leading to the sample whose vertical velocity is to be measured,
(b) a filter means operatively associated with said light source,
(c) an optical system connected to said filter means to transmit filtered light along the light path to the sample,
(d) a cuvette housing system connected to the optical system at one end and comprising a housing within which a cuvette movable in a plurality of predetermined height positions is provided mounted on a cuvette holder which
in turn is mounted on an angular disc cam and connected to a cuvette drive means through a coupling sleeve, (e) a light detection and analysis means connected to the cuvette housing system at an opposing end and connected to a display means
In one embodiment of the invention, the spectrophotometer is selected from a UV-VIS spectrophotometer, ordinary visible range spectrophotometer, and any other conventional spectrophotometer
In another embodiment of the invention, the cuvette is movable in four different predetermined height positions, such that the light path from the light source traverses through each of said different height positions
In another embodiment of the invention, the cuvette drive means is a stepper motor
In another embodiment of the invention, the display means is selected from an analog display means or a digital display means or a personal computer
In another embodiment of the invention, the spectrophotometnc system is provided with automated control means to regulate the speed of movement of the cuvette and to detect and analyse results of vertical velocity of the sample loaded therein
In another embodiment of the invention, the sample is a semen sample and the vertical velocity of spermatozoa is measured
The present invention also provides a cuvette housing system for a spectrophotometnc measurement system for use in measurement of vertical velocity of clinical or biological samples, the cuvette housing system comprising a housing within which a cuvette movable in a plurality of predetermined height positions is provided mounted on a cuvette holder which in turn is mounted on an angular disc cam and connected to a cuvette drive means through a coupling sleeve
In one embodiment of the invention, the cuvette dnve means is a stepper motor
In another embodiment of the invention, the cuvette drive means comprises a gear and pinion means
In another embodiment of the invention, the cuvette holder is provided rigidly connected to a rotatable circular base provided inside the housing, said rotatable base being connected to a stepper motor through a coupling sleeve to dnve the rotatable base and result in an up-down movement of the cuvette held rigidly in the cuvette holder
In another embodiment of the invention, the sample is a semen sample and the vertical velocity of spermatozoa is measured
In another embodiment of the invention, the circular base of the cuvette holder is provided with a gradation of height by means of a circular and angular disc cam Of varying and predetermined heights
In another embodiment of the invention, a single cuvette holder, attached with a proportionate constructed and freely movable metallic load, rolls over a vertically hanging slide, the entire assembly being hinged together at the top of the interior of he cuvette housing such that the base of the cuvette-holder can slide over the circular disc cam with varying heights
The present invention also relates to a method for measurement of vertical velocity of a clinical or biological sample using a spectrophotometnc system with vertical movement in a plurality of different height positions for a cuvette located therein, said method comprising actuating a light source along a predetermined light path through a filter means and an optical system, and therethrough to a spectrophotometer provided with a multiple height position enabled cuvette provided with the sample, such that the cuvette is exposed to the light path in said plurality of different height positions, due to the vertical shding-movement of the cuvette-load attachment over a circular base of varying height, as soon as a stepper motor is actuated
In one embodiment of the invention, the results are collected and analysed by a detection means provided downstream of the light path and then collated and displayed through a display means
In another embodiment of the invention, the sequence of data obtained is stored in in a personal computer.
In another embodiment of the invention, the spectrophotometer is selected from a UV-VIS spectrophotometer, ordinary visible range spectrophotometer, and any other conventional spectrophotometer
In another embodiment of the invention, the cuvette is movable in four different predetermined height positions, such that the light path from the light source traverses through each of said different height positions
In another embodiment of the invention, the cuvette drive means is a stepper motor
In another embodiment of the invention, the display means is selected from an analog display means or a digital display means or a personal computer
In another embodiment of the invention, the spectrophotometnc system is provided with automated control means to regulate the speed of movement of the cuvette and to detect and analyse results of vertical velocity of the sample loaded therein
In another embodiment of the invention, the sample is a semen sample and the vertical velocity of spermatozoa is measured
In another embodiment of the invention, the spectrophotometer is an ordinary visible range spectrophotometer and the method comprises generation of programmed pulses from a computer transmitted to a cuvette control unit in order to drive the stepper motor through a driver circuit, in turn mechanically driving a coupled cuvette holder and resulting in an up and down movement of the cuvette within the light path thereby exposing the sample to light in varying predetermined heights, the analog output from the spectrophotometer being then fed to a DSP/ ADC card, mounted on a PCI slot of a personal computer, for conversion into digital signal Brief description of the accompanying drawings
Fig 1 and Fig 1A show the schematic block diagram of a general spectrophotometer with fixed vertical position of the cuvette where only a single set of Absorbance Vs Time graph obtained This method was adopted by Sokoloski et al to determine the motility parameters for the most vigorously motile cells (7, 6)
Fig 2 and Fig 2A show schematic block diagram of a modified spectrophotometer with variable vertical position of the cuvette where multiple sets of Absorbance vs Time data are plotted In the present invention, four variable heights are considered, but it could be of any steps of heights within the span of the base of the cuvette and the light path The present invention is capable of calculating sperm motility for the entire population of the cells contained in the cuvette and as such this instrumental system will be able to determine sperm motility more accurately and precisely, in comparison with presently available sperm motility analyzing instruments
Fig 3 is a schematic diagram of a modified cuvette holder with a modified cuvette loading arrangement for a sophisticated spectrophotometer
Fig 3 A is a schematic diagram of a integrated instrumental system consisting of a UV-VIS spectrophotometer, modified cuvette holder and a computing means with interfacing
Fig 4 is one example of user interfaces for experimental data acquisition software
Fig 4 A is a flow diagram of the data acquisition software according to the invention
Fig 5 is an integrated block diagram of a computer based instrumentation system for measurement and analysis of sperm motility using an ordinary visible range spectrophotometer
Fig 6 is a schematic representation of an assembly of a modified cuvette holder with stepper motor for an ordinary spectrophotometer
Fig 6A is a schematic diagram of an integrated instrumental system consisting of an ordinary visible range spectrophotometer, modified cuvette holder which is stepper motor driven and a computing means with interfaces therebetween
Fig 7 is a circuit diagram of a cuvette actuator unit containing the stepper motor driving circuit
Fig 8 is a block diagram of a cuvette controller unit containing DAS/ADC card
Fig 9 are depictions of various user interfaces for experimental data acquisition software according to one example (Example II hereinbelow)
Fig 9A is a flow diagram of data acquisition software according to Example II
Fig 10 is a depiction of a user interface for data analysis software for both Examples I and II
Fig 10A is a flow diagram of a data analysis software for both Examples I and II
Fig 11 is a graphical representation of absorbance versus time over four different heights
Fig 12 is a graph correlation between absorbance (optical density) with number of sperm cells per ml
Fig 13 shows cell concentrations/ ml Vs time graph at four different heights of exposures along with the average plot
Fig 13 A shows cell concentration/ ml Vs time graph for single height exposure and the average curve of four heights of exposures (from Fig 13)
Fig 14 shows Increment in Cell Concentration/ ml Vs Time Graph for Four Different Heights of Exposures, along with the Average Plot
Fig 14A shows Cell Concentration/ ml Vs Time graph for Single Height Exposure and the Average Curve of the Four different Heights of Exposures (From Fig 14)
Fig 15 shows pictonally, the instant of times at which same number of cells appear at the four different heights
Fig 16 is a Graphical Presentation of vertical velocity (from Base to four different heights) Vs Time, along with the Plot for the Average
Fig 16A shows Vertical Velocity (from base to four different heights) Vs Time graph for Single Height Exposure and the Average Curve of Velocities of the Four Heights of Exposures (From Fig 16)
Fig 17 is a graphical presentation of Vertical Velocity (in between adjacent heights) Vs Time, along with the Average Plot
Fig 17A shows the Average Vertical Velocity (in between two adjacent heights) Vs Time graph (obtained From the Fig 17) For single Height Exposure, this Vertical Velocity is not available
Fig 18 shows the Plots of Vertical Velocity for the Incremented Group of Cells Vs Time for four different heights of exposures along with the Plot for the Average
Fig 18A shows the Average Vertical Velocity (for the Incremented Group of Cells) Vs Time graph and the Average Curve of Vertical Velocities of the Four Heights of Exposures Vs Time (From Fig 18)
Fig 19 shows the Plots of PFM Vs Time for four different heights of Exposures along with the Plot for the Average
Fig 19A shows PFM Vs Time graph for Single Height Exposure and the Average PFMVs Time Curve obtained from the Four Heights of Exposures (From Fig 19)
Fig 20 shows Bar Diagrams of PFM (At Saturation) Vs Time for four Different Heights of Exposures, along with the Plot for the Average
Fig 20A shows Bar Diagrams of PFM (At Saturation) Vs Time for Single Height Exposure (HO) and the Average PFM Vs Time Bar obtained from the Four Heights of Exposures (From Fig 20)
Fig 21 shows Plots of FMU Vs Time for four different heights of Exposures along with the Plot for the Average
Fig 21A shows Plots of FMU Vs Time for Single Height Exposure (HO) and the Average FMU Vs Time Graph obtained from the Four Heights of Exposures (From Fig 21)
Fig 22 shows Bar Diagrams of FMU (At 60 Sec) Vs Time for four different heights of Exposures along with the Plot for the Average
Fig 22A shows Bar Diagrams of FMU (At 60 Sec) Vs Time for Single Height Exposure (HO) and the Average FMU Vs Time Bar obtained from the Four Heights of Exposures (From Fig 22)
Fig 23 shows the plots of FMU (in terms of No of Cells) Vs Time, along with the Average Graph
Fig 23 A shows the Plots of FMU (in terms of No of Cells) Vs Time for Single Height Exposure (HO) and the Average FMU (in terms of No of Cells) Vs Time, obtained from the Four Heights of Exposures (From Fig 23)
Fig 24 shows Bar Plots of Horizontal Velocities in the CAS A and Vertical Velocities (from Base to Heights) in the method of the mvention, using Same Samples simultaneously Gradation (1,2,3 and 4) of Four Samples are also Shown for Comparative Studies
Fig 25 shows Bar Plots of Horizontal Velocities in the CAS A and Vertical Velocities (in Between Heights) in the method and system of the invention, using Same Samples simultaneously Gradation (1,2,3 and 4) of Four Samples are also Shown for Comparative Studies
Fig 26 shows graphically the effect of sperm motility Inhibitor on sperm velocities using the CAS A and the system of the invention
Fig 27 shows graphically the effect of sperm motility Activator on sperm velocities using the CAS A and the system of the invention Detailed description of the invention
The main features of this instrumental system are described here In an embodiment of the present invention, the conception of vertical movement of the cuvette may be adopted in any spectrophotometer (ordinary or sophisticated) with little addition, alteration or modification of the cuvette-holder assembly The idea of vertical movement of the cuvette in a spectrophotometer is altogether a new and unique conception, which exposes the sample in multiple heights, thus acquiring multiple sets of data In contrast, presently available in any sophisticated or ordinary spectrophotometer, the cuvette position is fixed at a particular height only and as such exposes the cuvette-sample at a single height only, there by acquiring only a single set of data Present invention (SPERMA) is thus capable of measuring sperm motility (including vertical velocity) more accurately and precisely, in comparison with any other method that uses fixed height spectrophotometer (6, 7) Since a simple visible range spectrophotometer with higher resolution is sufficient to implement this new technique, the cost of such a computer aided sperm motility analyzer (using techniques of present invention) is much lower than that of presently available sperm analyzers Multiple height spectrophotometnc method of the present invention generates information about the entire spermatozoa sample. In contrast to the spectrophotometnc method (using single height spectrophotometer) adopted by Sokoloski (7) informs only about the characteristics of the most vigorously motile cells within the entire sample Vertical velocity of the spermatozoa sample against gravity can be measured using this instrumental system It is to be mentioned here that Sokoloski described subjective determination of vertical velocity of sperm, following an indirect method, measuring the slope of the Absorbance vs Time curve Where as in the present invention, a direct method velocity measurement (distance traveled / time
elapsed) has been followed for objective determination of sperm velocity, which is a new and unique concept Mathematical models for measuring various motility parameters applied in the present invention are new and unique in nature Software for calculating and analyzing sperm motility, are also new and unique in nature, which determines sperm quality very precisely A pictorial view of the upward moving cells with the passage of time can be ascertained, using the acquired database, 1 e number of cells present at different instants of time and at different heights This is a new and unique parameter At a particular instant of time, how the cell concentration varies at different heights, can be obtained Some of the recorded and derived parameters of this instrumental system are unique, which specifies the characteristics of the moving sperm cells more precisely This instrumental system, because of its low cost, will be very useful to various human infertility clinics, animal breeding centers in rural and urban areas, and also to various research laboratories In order to facilitate Conservation of Endangered Species, this instrumental system can be used for semen quality evaluation This instrumental system considers the vertical movement of the spermatozoa, whereas, a CASA measures the horizontal movements Thus, a new avenue in the research-field of reproductive biology could be seen in which horizontal and vertical characteristics of the spermatozoa could be compared for analysis Practically, this low cost, instrumental system can be considered as an effective alternative of the Computer Aided Semen Analyzer (CASA), which is very expensive It is expected that various other fields of usage (with little modification/ variation of this technique) will be opened or revealed, once this instrumental system is marketed and gets exposure
In order to materialize the above mentioned objectives of developing a modified spectrophotometer which is capable exposing the cuvette/ sample at variable heights (within the light path), two different types of spectrophotometers were considered deliberately, 1) a Sophisticated Micro-processor/ computer based, UV-VIS spectrophotometer with high resolution, and n) an Ordinary visible range spectrophotometer The purpose of taking two types of spectrophotometer is to exhibit the flexibility and adoptability of the technique The cuvette holders of both the spectrophotometers have been designed and developed and assembled into them Description of these two modified cuvette assemblies are given in details in Method -1 and Method -II During conduction of tests, on line data acquisition is carried out and data are analyzed subsequently The experimental procedure of the present invention is given in this paragraph Goat cauda epididymis brought from nearby slaughterhouse and minced into small pieces on a petri dish along with little amount of modified ringer phosphate buffer solution The sperm is then extracted by sieving through
cheesecloth Cell number is counted microscopically and the concentration is made to a certain level with buffer for better result Variable concentration may also be used but higher the concentration, better the result 10% Ficoll-400 was mixed with a particular quantity of the sample so that only the motile cells swims up and not the dead cells The total assay volume becomes 500 ul this makes the final solution for application in the cuvette Epididymal Plasma (0 6 to 1 2 mg / ml) may be used as an anti-sticking agent to avoid the sticking of sperm cells with the wall of the cuvette However the cell concentration and assay volume of 500 ul is maintained The cuvette is then filled with 1 5 ml (1500 of) of the modified ringer phosphate buffer solution and placed in the cuvette holder of the spectrophotometer This is important because the light beam in normal condition must pass through the uppermost part of the solution The computer interfacing software is initiated at this stage when the spectrophotometer takes the reference data and PAUSES for adding sperm sample After this with the help of a 500 ul Hamilton Syringe 50 ul of the prepared sample from the test tube is layered slowly and delicately at the bottom of the cuvette The cuvette thus contains a sample of 1550 ul volume The shutter of the spectrophotometer is closed as quickly as possible and the associated computer interfacing software of the instrument is initiated To start with, the initial conditions of the spectrophotometer are required to be adjusted The wavelength of the spectrophotometer is set at 545 nm (produces highest peak for sperm sample) The total scan time is given within a range of about 5 to 20 minutes (normally 10 minutes scan time is set) to obtain a saturation curve for Absorbance Vs Time The time interval between the start of each set of scan is set at 1 minute (60 seconds) Thus, to complete one cycle of scanning for four different heights remains within 6 seconds The experimental data, Absorbance vs Time, is acquired at four different heights of the cuvette dunng each cycle of time scanning At the end of the test, the content of the cuvette is mixed thoroughly and Absorbance data are collected once again This represents Total Absorbance (AT), which is necessary for calculating cell concentration as well as percentage forward motility (PFM) Detailed description of the invention Mathematical Model For Sperm Analysis
Let, HO, HI, H2, and H3 are the heights at which the cuvette is exposed to the light path (Fig.2) and H_AVG is the average of all these four heights The Fig. 2A show the graphical representation of ABS vs. Time data for all the heights of exposure of the sample Further, average values of all the four ABS data for four different heights are also calculated at each interval of time and plotted, as shown in Fig. 11 In the present invention, the
hardware is designed in such a manner that HO = 8 mm, HI = 6mm, H2 = 4 mm, h3 = 2 mm and thus the H_AVG = 5 mm But, these heights are hardware dependent and alterable
The motility parameters measurable with this instrumental system are given as follows -
A Total Cell Concentration of the Sample
B Cell Concentration at Different Heights of the Cuvette at a Given Time
C Variation or Increment in Cell Concentration with Time for a Given Height of the
Cuvette D Movement of Same Number / Group of Cells through Different Heights of the
Cuvette at Different Interval of Time E Vertical Velocity (From Base to Different Heights of the Cuvette and their Average),
V-BTH F Vertical Velocity (Point to Point or In-between Different Heights of the Cuvette and
their Average), V-IBH G Vertical Velocity of the Incremented Number of cells of Parameter-3, at Different
Heights and their Average H Percentage Forward Motility (PFM) at Different Interval of time or the gradual
change of PFM with Time at Different Heights and their Average I Forward Motility Unit (FMU) at Different Interval of time or the gradual Change of
FMU with Time at Different Heights and their Average J Number of Cells Passing Through the Light Path with respect to the Forward Motility Unit at Different Height and Time and their Average But, among these ten motility parameters, measurement of vertical velocities (V-BTH, V-IBH) of spermatozoa is the primary interest of the present invention A. Total Cell Concentration :
It is experimentally established, 1 O D (Optical Density or Absorbance) = K Cells / ml Where, K is a constant for a particular species and it varies from species to species Fig 12 shows the correlation between Absorbance (Optical density) and Number of Sperm Cells for goat So, the total cell concentration of the sample could easily be calculated as the Average Total Absorbance value and the relation between Absorbance and Number of Cells per ml is known Mathematically this can be expressed as follows
N = K X C X AT_AVG Cells/ ml Where, N = Number of cells/ ml
K = A constant that depends on the correlation between the O D and number of sperm cells
C = A constant that depends on the volume of buffer solution and sample taken AT_AVG = Average value of Total absorbance obtained from all the heights
B. Cell Concentration at Different Heights of the Cuvette at a Given Time
Fig. 13 shows that graphical presentation of Cell Concentration/ ml vs time In order to determine number of cells/ ml that appeared at different heights of the cuvette for a given time, a grid line on the X-axis is considered The intersecting points of this grid line with the plots for height HO, HI, H2, H3, and HAVG represents the corresponding Cell Concentration / ml
C. Variation or Increment in Cell Concentration with Time for a Given Height of the
Cuvette
The cell concentration/ ml, for a particular height and at a particular time is calculated as the relation between absorbance and number of cells per ml is known (1 O D = K Cells / ml) This parameter is calculated for all the heights and time and thus the Cell Concentration / ml vs Time data could be obtained for any height and time From this data the Increment in Cell Concentration/ ml is calculated by subtraction method and thus the gradual increase in the number of cells per ml at a particular height and at a particular time could be estimated Mathematically, the increment in Cell Concentration/ ml is calculated as follows -
[(ABS_H_T(n+6o)) - (ABS_H_Tn] X (K) Where, ABS_H_Tn)= Absorbance at a particular height and time and n = 0, 60, 120, 180, etc
K = A constant that depends on the correlation between the O D and number of sperm cells Increment in Cell Concentration/ ml vs Time graph has been plotted in Fig. 14.
D. Movement of Same Number / Group of Cells through Different Heights of the
Cuvette at Different Interval of Time
In order to determine the time at which the same number of cells (since absorbance value is convertible to number of cells / ml) appears at different heights of the cuvette, a grid line on the Y-axis is considered The intersecting points of this grid line with the plots for height HO, HI, H2, H3, and H_AVG represents the corresponding time of passing of the cells through those heights of the cuvette Unitary method is applied for calculating the Time It is graphically shown in Fig. 15.
E. Vertical Velocity (From Base to Different Heights of the Cuvette and their Average).
V-BTH
Here, in general, Vertical Velocity of spermatozoa is calculated using direct method
(parameters mentioned in E, F and G) as given in the following equation -
(Equation Removed)
Following the same procedure described above for parameter D and Fig 15, time required to for the same group of sperm cells to reach H3, H2, HI, HO and HAVG from the base are calculated Since the Heights and the corresponding Times are known, the velocity from base to the particular height (V-BTH) of the cuvette can easily be calculated using the basic formula, given above This is shown graphically in Fig. 16
F. Vertical Velocity (Point to Point or In-between Different Heights of the Cuvette and
their Average), V-D3H
Following the procedure mentioned above for the parameter D and Fig 15, the time required for the same group of sperm cells to reach H3, H2, HI, HO and HAVG from the base are calculated and then by the method of subtraction the time required for the sperm cells to travel from one height to the adjacent height is calculated As the differences in heights are known (2 mm, in this instrumental system) and the time required is calculated, thus, the vertical velocity in between different heights (from Base to H3, from H3 to H2, from H2 to HI, from HI to HO and average of these velocities) is calculated following the basic formula given before This is shown graphically in Fig. 17
G. Vertical Velocity of the Incremented Number of cells of Parameter-3, at Different
Heights and their Average
Velocities of the particular group of incremented cells/ ml have been calculated by dividing the particular height (8 mm, 6 mm, 4 mm, 2 mm and 5 mm) by the corresponding time In real sense, the plot represents the vertical velocities of the group of cells/ ml that are incremented with the variation of height and time as calculated for the Parameter-C This is shown graphically in Fig. 18
Vertical velocity of sperm is also measurable by the method of Sokoloski et al (7) Where the formula used is VRM = (dA / dT) / Al Where, Al and VRM are the turbidity mduced by the rapidly moving cells and velocity of the rapidly moving cells respectively Sokoloski et al used the above formula for a single set of absorbance data acquired from a single height of the cuvette only This formula can also be used with the instrumental system of the present invention, where four sets of absorbance vs time data are acquired for four different heights And as such, it is expected that the present instrumental system would
calculate vertical velocity more accurately (using Sokoloski et. al. 's formula also) than that of
a single set of data used by Sokoloski et. al Sokoloski et aVs formula can be used in any
of the four sets of data as well as for the average absorbance vs time data However, the
method applied by Sokoloski et al calculates the vertical velocity of the most rapidly motile
cells, where as the method used in the present invention considers the entire population of
cells Moreover, the system of the present invention is much more subjective and direct in
comparison to the objective and indirect method of Sokoloski et al
H. Percentage Forward Motility (PFM) at Different Interval of time or the gradual
change of PFM with Time at Different Heights and their Average
Percentage forward motility (PFM) represents the percentage of forward motile cells
available in a given sperm cells sample under consideration It is calculated by dividing the
absorbance at saturation/ equilibrium level, AEQ, (where no more cells are coming up) by the
total absorbance, AT (after mixing the entire content of the cuvette) Here the fraction of the
total forward motile cells within the whole sample could be estimated This could even be
done for all the absorbance data obtained with time, so that the gradual increase in the
fraction of forward motile cells with time could be seen This is shown graphically in Fig. 19
and 20. And also the saturation point where no more cells are coming up could also be
obtained from the plot The formula used for calculating PFM is given as follows -
(Equation Removed)
I. Forward Motility Unit (FMU) at Different Interval of time or the gradual Change of FMU with Time at Different Heights and their Average
Forward motility unit (FMU) is the rate of change of absorbance or the rate of change
of number of cells that are passing through the cylindrical light beam It is calculated by
obtaining the slope of the curve at any particular time A 0 01 increase in absorbance (O D) is
considered as 1 FMU This calculation can be done for the rate of change of absorbance of
every minute and thus the gradual change of FMU with time could be calculated This is
shown graphically in Fig. 21 and 22 FMU is calculated using the following formula -
(Equation Removed)
J. Number of Cells Passing Through the Light Path with respect to the Forward Motility Unit at Different Height and Time and their Average
The light beam of the spectrophotometer through the cuvette is considered as a cylindrical structure and the volume of buffer along with sperm in it is calculated using the formula πr2l, where r = the radius of the cylinder and 1 = length of the beam Therefore, the FMU can be expressed in terms of number of cells, as the relation between the absorbance and the numbers of cells are known This is shown graphically in Fig. 23.
The software for analyzing sperm motility is written in Visual Basic Studio 6 0, Enterprise Edition (VB) at the front end and MS-Excel/ MS-Access, MS-Office 2000, is used for database management at the back end Report generation is accomplished by using Seagate Crystal 8 0, Developer Edition User Interface for Data Analysis Software (for both Example I and II) is shown in Fig. 10 and Flow Diagram of the Data Analysis Software (for both Example I and II) is given in Fig. 10A.
Statistical analysis was carried out on the acquired data where two types of protein factors were used for verification of the capability of the newly invented instrumental system One of the factors was a sperm motility inhibitor and the other was a sperm motility activator The effect of these above mentioned factors were tested on goat cauda-epididymis sperm against normal control The effects (unpublished) on the vertical velocity and other motility parameters were calculated by the newly invented data analysis software Results were compared with that of a CASA Statistical analysis was carried out on the acquired data and they were found to be encouragingly significant Please refer to Fig. 26 and 27
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the invention
Example -1
Design and Development of the Modified Cuvette Holder Assembly for a Sophisticated UV-VIS Spectrophotometer
The technique of exposing the cuvette to the light path in four different heights is accomplished by vertical sliding-movement of the cuvette-load attachment over the circular base of varying heights All the six-cuvette holders from the circular base of the cuvette holder were removed and a circular and angular disc cam made of ebonite with varying heights was fabricated and mounted on the outer surface of the circular base of the cuvette holder The variation in height is made in such a manner that the cuvette-holder can be exposed to the light path in four different heights of the cuvette A single cuvette holder, attached with a proportionately designed and freely movable metallic load, rolls over a vertically hanging slide The entire attachment is hanged together at the top of the inner-body of the motor-cuvette enclosure, in such a manner that the base of the cuvette-holder can slide
over the circular disc cam with varying heights As soon as the stepper motor is actuated, the circular base (mounted on the motor) rotates along with the variable height circular disc cam attached to it through a coupling sleeve The freely movable cuvette holder then slides upward due to free movement of the cuvette-load attachment, since the base of the hanging cuvette holder is positioned on the circular disc cam During its downward movements, the cuvette slides down over the circular disc cam due to the own weight of the load attachment (under the gravitational force) This makes the cuvette-holder slide over the circular disc cam upward and downward, thus exposing the cuvette-holder / cuvette in four different heights Schematic Diagram of the Modified Cuvette-holder along with Modified Cuvette-load Arrangements for Sophisticated Spectrophotometer is shown in Fig. 3 And the Schematic Diagram of the Integrated Instrumental System Consisting of Sophisticated UV-VIS Spectrophotometer, Modified Cuvette Holder and the Computer with interfacing is exhibited in Fig. 3A
Data Acquisition Software: Using a RS-232 serial port, sequence of data is stored in a Personal Computer The spectrophotometer has been used in Time Scan Mode, for a single sample of spermatozoa After scanning Reference the system pause for adding sample sperm Once the sperm cells are added and the system takes about 6 seconds time to scan and acquire data (Absorbance vs Time) during the exposure of the sample at four different heights, HO, HI, H2, and H3 This completes one cycle of scanning It then waits for about 54 seconds, thus it takes a total of 60 seconds to complete a full period At the 60th second, the process is repeated till the total Scan Period, say, 600 seconds (10 minutes, which is pre-adjustable) is completed User Interfaces for experimental Data Acquisition Software is shown in Fig. 4 and the flow chart of the data acquisition software is given in Fig. 4A.
Calibration: This is a sophisticated spectrophotometer with high resolution It has an accuracy and repeatability of the order of ± 0 005 Abs at 1 0 Abs Example - II
Design and Development of the Modified Cuvette Holder Assembly for an Ordinary Visible Range Spectrophotometer
Robotics technique of the simplest form has been adopted in the automated instrumental system of the present invention Fig. 5 shows the Schematic block diagram of the entire Instrumental system, comprising of the following six sub-systems
I Spectrophotometer IV Cuvette Controller Unit
II Modified Cuvette Holder with Stepper V A/ D Converter and Data Acquisition
Motor (DAS) Card
III Cuvette Actuator Unit VI Personal Computer
Programmed pulses are generated from the computer, which arrives to the Cuvette Controller Unit (CCU) and drives the stepper motor (installed in sub-system II modified cuvette holder with stepper motor) through the stepper motor driver circuit, CAU This in turn drives the mechanically coupled cuvette holder, which in effect make the cuvette move in upward and downward directions within the light path, thus exposing the sample in variable heights Analog output from the spectrophotometer is then fed to the DSP/ ADC card, mounted on a PCI slot of a personal computer, for conversion into digital signal Thus, spectrophotometer output (Absorbance Vs Time data) is stored in the personal computer Modified Cuvette Holder with Stepper Motor: Subsystem II
This is primarily an electromechanical manipulator driven by a stepper motor This unit consists of four parts The first part is a metallic platform upon which two metallic stands are erected Two metallic slides are then attached vertically with the metallic stands in such a manner that the cuvette-holder could be just accommodated inside them for accomplishing vertical movement of the cuvette This part of the subsystem is positioned inside the spectrophotometer and between the light source and the detector The second part is the mechanical coupling with a vertical lead screw (made of brass), which passes through the nut of the cuvette holder slide The vertical up-down motion of the cuvette holder is accomplished by the bi-directional motion of the stepper motor The brass-made screw passes through a threaded-hole located on the platform, such that the screw is freely movable through the hole On the other end of the screw is rigidly coupled with the shaft of the stepper motor The third part is the stepper motor, which is activated by the driving signal generated by the personal computer/ CCU The fourth part consists of the gear assembly, coupled with the motor shaft This is for conversion of angular movement to vertical movement The purpose of this part is to measure the height of the cuvette exposed to the light path of the spectrophotometer The gear is coupled with a precision multi-turn-potentiometer powered by a precision power supply for measuring height of the cuvette within the light path The output of the potentiometer is fed to the computer for on line data acquisition Assembly of Modified Cuvette-holder with Stepper Motor for an Ordinary Spectrophotometer is shown in Fig. 6 And Fig. 6A exhibits the Schematic Diagram of the Integrated Instrumental System Consisting of an Ordinary Visible Range Spectrophotometer, Modified Cuvette Holder (Stepper Motor Driven) and the Computer with interfacing Cuvette Actuator Unit: Subsystem in
Fig. 7 shows the Cuvette Actuator Unit, Containing Stepper Motor Driving Circuit Stepper Motor Dover Card contains stepper motor driving circuits, which drives the stepper
motor attached to the subsystem II The inputs to this circuit are the control pulses generated by the associated software through stepper motor controller unit, CCU
Cuvette Controller Unit And A/ D Converter and Data Acquisition (DAS) Card: Subsystem IV and V
Fig. 8 shows the Block Diagram of the Cuvette Controller Unit, Containing DAS/ ADC Card The function of the CCU is to provide control and driving signals for activating the stepper motor housed in subsystem II The function of Data Acquisition (DAS) Card is to acquire on line data, analog or digital In order to acquire on line analog data from the spectrophotometer, a standard data acquisition card has been procured and installed in a personal computer (PCI bus) It has got 16 analog 1/ P, 16 digital I/P and 16 digital O/P On line analog signal/ data from the spectrophotometer is fed to the DAS card for analog to digital conversion and acquire data in the personal computer in accordance with the associated software Parameters, which are measured and stored in the computer on line, are ABSORBANCE (ABS), TIME, VERTICAL HEIGHT, and TEMPERATURE In order to calibrate the spectrophotometer, data, like, 0 % ABS, 100 % ABS and REFERENCE are also acquired on line Personal Computer: Subsystem V
This personal computer (PC) contains the Data Acquisition (DAS) Card The PC generates control signals, acquires digitized data and stores in the hard disk, contains data acquisition and data analyzing software and generates output The software is written in Visual Basic and the data is stored in text mode, so that the same could be used in MS-Excel The programme is written in such a way that the height of the cuvette could be set to HO, HI, H2 and H3 respectively Thus, the sample is exposed to the light beam at different heights This is one complete cycle of operation and the time for completion of one cycle is about 6 sees The software makes the motor rotates for say, 100 turns, to make a vertical movement of one step of the cuvette Starting at time TO, the software acquires data during one cycle of operation and then waits and restarts at the 60 sec, when it repeats the cycle of operation and acquires data In this way the operation is repeated for 600 sees or 10 minutes (which is pre-adjustable according to requirement) User Interfaces for experimental Data Acquisition Software is shown in Fig. 9 and the flow chart of the data acquisition software is given in Fig. 9A.
Calibration: This is an ordinary visible range spectrophotometer which has got accuracy and repeatability of the order of ± 0 005 ABS, ± 0 5 % T, ±03 %
Performance Analysis of SPERMA
Using the same samples, experiments were conducted simultaneously in a single height commonly available spectrophotometer, multi height modified spectrophotometer of the present invention (SPERMA) and a sophisticated CASA Based upon the experiments conducted, two comparative charts/ tables have been prepared which describes the advantages of the present invention Table 1 and Table 2 are given below Table 1 : Advantages of Multi-height Spectrophotometer (SPERMA) over Single-height Spectrophotometer and the CASA

(Table Removed)

Table 2 : Comparative Advantages of Multi-height Modified Spectrophotometer over The Standard Spectrophotometer with Single Height Exposure

(Table Removed)
INVENTION (SPERMA) Vs CASA: Comparative Performances
Presently available all the sperm motility analyzers consider sperm movements on a horizontal plane for measuring sperm motility parameters Since the instrument of the present invention (SPERMA) measures the Vertical Velocity along with other Vertical Motility Parameters, it is thus not possible to exhibit any direct comparison of the efficiency of the SPERMA with any other instrument Still, to assess the performance-level of the SPERMA, simultaneous tests were conducted on a CASA and the SPERMA, using the same samples However, such comparisons were made in terms of velocities only The two Vertical Velocity parameters of the SPERMA, known as Average Vertical Velocity from Base to Heights (Avg-V-BTH) and Average Vertical Velocity In Between Heights (Avg-V-IBH), were compared with the Horizontal Velocity parameters, known as Average Path Velocity (VAP) and Straight Line Velocity (VSL), of CASA and gradations of the samples were made accordingly Fig. 24 and 25 compare the gradation of the samples, which are almost similar in both the instruments. As because the vertical movement of sperm cells against the gravity requires more energy than horizontal movement, the value of the vertical velocity was observed to be lesser than the corresponding horizontal velocity Tests were also conducted by applying a Sperm Motility Inhibitor and a Sperm Motility Activator using the same three tissue samples in each case against the control and statistical analysis were done It is evident from the results that the effects of the Inhibitor and Activator are almost equally effective in both the instruments (Fig 26 and 27). So the SPERMA, the instrument of the present invention could easily and effectively be used for the sperm analysis purpose in the infertility clinics, animal breeding centers, and research organizations Moreover the vertical movement parameters are expected to be rendering much better quality of analysis, as the vertically movable cells are more energized/ motile and the best candidates for fertilization
Multi Height Exposure (SPERMA) vs. Single Height Exposure :_Comparative Performances
Table - 1 and 2 show the advantages of the multiple height exposure over the single height exposure of the sample/ cuvette Some parameters calculated by SPERMA such as, Velocity In Between Heights, Cell Concentration at Different Heights of the Cuvette at a Given Time, Movement of Same Number/ Group of Cells through Different Heights of the Cuvette at Different Interval of Time, etc are not possible when only a single height data is taken from the single height exposure Fig. 13A, 14A, 16A, 17A, 18A, 19A, 20A, 21A, 22A, and 23A show the comparative graphical presentation of the parameters, calculated from the single height and the average of multi height exposures Furthermore, a good statistical estimation
requires multiple sets of data and also random sampling Thus, in the present invention, multiple sets of data are taken into consideration, which are acquired from the different heights of the cuvette This follows the law of random sampling and as such better statistical analysis is possible Since, in the present invention, the entire populations of cells are considered, the SPERMA serves with a more relevant result in comparison with single height exposure
The main advantages of the present invention are summarized here The automated system is a spectrophotometer with variable and multiple height exposure of the cuvette in the hght path for the determination of sperm motility This system acquires multiple sets of data for different heights of exposures and as such it eliminates the inaccuracies and limitations of the present spectrophotometnc method (6,7) with fixed and single height cuvette exposure Thus, an inherent accuracy and preciseness exit in the present invention Since a simple visible range spectrophotometer with higher resolution is sufficient to implement this new technique, the cost of such a computer aided sperm motility analyzer would be much lower than that of presently available any instruments Thirdly, it is adoptable to any spectrophotometer with little modification of the cuvette holder of the spectrophotometer This instrumental system, the SPERMA, is capable of generating a few new and unique motility parameters Direct way of measuring vertical velocity of spermatozoa is a remarkable advantage of this instrumental system Since, velocity is considered to have great importance on the fertilizing ability of spermatozoa, great importance has been given for the measurement of vertical velocity Various other needs of this automated system may be opened in many other scientific apphcations It has got great market potentiality because of its accuracy, low cost, adoptability, flexibility, and capability of determination of new sperm motility parameters, including vertical velocity REFERENCES
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We claim;
1. A spectrophotometric system for measurement of vertical velocity of clinical and
biological samples, the system comprising:
(a) a light source being provide light into a light path leading to the sample whose vertical velocity is to be measured;
(b) a filter means operatively associated with said light source;
( c) an optical system connected to said filter means to transmit filtered light along the light path to the said sample;
(d) a cuvette housing system such as herein described, being connected to the said optical
system at one end and comprising a housing within which a cuvette movable in a plurality of
predetermined height positions being provided mounted on a cuvette holder which in turn
being mounted on an angular disc cam and connected to a cuvette drive means through a
coupling sleeve;
(e) a light detection and analysis means connected to the cuvette housing system at an
opposing end and connected to a display means.
2. A system as claimed in claim 1 wherein the spectrophotometer is selected from a UV-VIS spectrophotometer, ordinary visible range spectrophotometer, and any other conventional spectrophotometer.
3. A system as claimed in claim 1 wherein the cuvette is movable in four different predetermined height positions, such that the light path from the light source traverses through each of said different height positions.

4. A system as claimed in claim 1 wherein the cuvette drive means is a stepper motor.
5. A system as claimed in claim 1 wherein the display means is selected from an analog display means or a digital display means or a personal computer.
6. A system as claimed in claim 1 wherein the spectrophotometric system is provided with automated control means to regulate the speed of movement of the cuvette and to detect and analyse results of vertical velocity of the sample loaded therein.

7. A system as claimed in claim 1 wherein the biological sample is a semen sample and the vertical velocity of spermatozoa is measured.
8. A system as claimed in claim 1 wherein cuvette housing system comprising a housing within which a cuvette movable in a plurality of predetermined height positions is provided mounted on a cuvette holder which in turn is mounted on an angular disc cam and connected to a cuvette drive means through a coupling sleeve.
9. A system as claimed in claim 8 wherein the cuvette drive means comprises a gear and pinion means.
10. A system as claimed in claim 8 wherein the cuvette holder is provided rigidly connected to a rotatable circular base provided inside the housing, said rotatable base being connected to a stepper motor through a coupling sleeve to drive the rotatable base and result in an up-down movement of the cuvette held rigidly in the cuvette holder.
11. A system as claimed in claim 8 wherein the circular base of the cuvette holder is provided with a gradation of height by means of a circular and angular disc cam of varying and predetermined heights.
12. A method for measurement of vertical velocity of a clinical or biological sample using a spectrophotometric system with vertical movement in a plurality of different height positions for a cuvette located therein, said method comprising actuating a light source along a predetermined light path through a filter means and an optical system, and there through to a spectrophotometer provided with a multiple height position enabled cuvette provided with the sample, such that the cuvette is exposed to the light path in said plurality of different height positions, due to the vertical sliding-movement of the cuvette-load attachment over a circular base of varying height, as soon as a stepper motor is actuated.

13. A method as claimed in claim 15 wherein detection means provided downstream of the light path and then collated and displayed through a display means.
14. A method as claimed in claim 15 wherein the sequence of data obtained is stored in in a personal computer.
15. A method as claimed in claim 15 wherein the cuvette is movable in four different predetermined height positions, such that the light path from the light source traverses through each of said different height positions.
16. A method as claimed in claim 15 wherein the cuvette drive means is a stepper motor.
17. A method as claimed in claim 15 wherein the display means is selected from an analog display means or a digital display means or a personal computer.
18. A method as claimed in claim 15 wherein the sample is a semen sample and the vertical velocity of spermatozoa is measured.
19. A method as claimed in claim 15 wherein method comprises generation of programmed
pulses from a computer transmitted to a cuvette control unit in order to drive the stepper
motor through a driver circuit, in turn mechanically driving a coupled cuvette holder and
resulting in an up and down movement of the cuvette within the light path thereby exposing
the sample to light in varying predetermined heights, the analog output from the
spectrophotometer being then fed to a DSP/ ADC card, mounted on a PCI slot of a personal
computer, for conversion into digital signal.
20. A spectrophotometric system for measurement of vertical velocity of clinical and biological samples substantially as herein described particularly with reference to the examples and the drawings accompanying this specification.

Documents:

1605-DEL-2004-Abstract-(27-07-2011).pdf

1605-del-2004-abstract.pdf

1605-DEL-2004-Claims-(27-07-2011).pdf

1605-DEL-2004-Claims-191214.pdf

1605-del-2004-claims.pdf

1605-DEL-2004-Correspondence Others-(22-09-2011).pdf

1605-DEL-2004-Correspondence Others-(27-07-2011).pdf

1605-DEL-2004-Correspondence-191214.pdf

1605-del-2004-correspondence-others.pdf

1605-DEL-2004-Description (Complete)-(27-07-2011).pdf

1605-del-2004-description (complete).pdf

1605-del-2004-description (provisinal).pdf

1605-del-2004-drawings.pdf

1605-DEL-2004-Form-1-(22-09-2011).pdf

1605-del-2004-form-1.pdf

1605-del-2004-form-18.pdf

1605-DEL-2004-Form-2-(27-07-2011).pdf

1605-del-2004-form-2.pdf

1605-DEL-2004-Form-3-(27-07-2011).pdf

1605-del-2004-form-3.pdf

1605-del-2004-form-5.pdf

1605-DEL-2004-OTHERS-191214.pdf


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Patent Number 264556
Indian Patent Application Number 1605/DEL/2004
PG Journal Number 02/2015
Publication Date 09-Jan-2015
Grant Date 06-Jan-2015
Date of Filing 26-Aug-2004
Name of Patentee COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH
Applicant Address ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 DEBASHIS PAUL INDIAN INSTITUTE OF CHEMICAL BIOLOGY, 4 RAJA S C MULLICK ROAD, JADAVPUR KOLKATA-700 032, INDIA.
2 GOPAL CHANDRA MAJUMDAR INDIAN INSTITUTE OF CHEMICAL BIOLOGY, 4 RAJA S C MULLICK ROAD, JADAVPUR KOLKATA-700 032, INDIA.
3 SUDIPTA SAHA INDIAN INSTITUTE OF CHEMICAL BIOLOGY, 4 RAJA S C MULLICK ROAD, JADAVPUR KOLKATA-700 032, INDIA.
4 ASHUTOSH MUKHERJEE INDIAN INSTITUTE OF CHEMICAL BIOLOGY, 4 RAJA S C MULLICK ROAD, JADAVPUR KOLKATA-700 032, INDIA.
5 SOMNATH BANERJEE INDIAN ASSOCIATION FOR THE CULTIVATION OF SCIENCE, E, RAJA S C MULLICK ROAD, JADAVPUR KOLKATA-700 032, INDIA.
PCT International Classification Number G01N 21/25
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA