Patent Publication Number: US-2017368365-A1

Title: Device and method for assaying the application of energy to samples in vitro

Description:
RELATED APPLICATIONS 
     This application claims priority under 35 U.S.C. §119 to U.S. Provisional Patent Application No. 62/355,543 filed on Jun. 28, 2016, the entire contents of which is incorporated herein by reference. 
    
    
     FIELD OF INVENTION 
     This invention relates to assaying the application of energy to samples. More specifically, this invention relates to a device for applying various amounts of energy, such as electromagnetic energy, to different wells of a culture plate. 
     BACKGROUND 
     Applying electromagnetic energy to living cells and tissues has been shown to have biological effects on the cells and tissues. Various types of energy have been used for treatment, such as non-thermal plasma, radio frequency, light, electrical fields, and magnetic fields. Pulsing, or modulating, the energy at different frequencies can change the effect of the treatment. However, clinical studies for determining optimum frequencies for specific treatments have been difficult to justify because the physical mechanism behind the biological interaction is not well understood. Clinical studies are also very expensive and the protocol for selecting an optimum frequency is poorly defined. 
     Conventionally, in vitro studies have been used to determine an optimal dosage of a medicament that causes a desired outcome. In one approach, cells or tissue samples are deposited in a multi-well culture plate and each well is dosed with a different amount of the medicament. The dosage is preferably applied with a programmed robotic device to increase speed and accuracy of the testing. The cells or tissue samples of each well may then be individually analyzed to determine the effects of the applied dosage. 
     While many methods and devices exist for studying medicament dosages applied to cells or tissue samples, little exist for studying energy applied to cells or tissue samples in an inexpensive and accurate manner. Thus, it would be desirable to have a device for performing in vitro studies with various types of energy. 
     SUMMARY 
     Some embodiments of the invention provide an assay device for a culture plate including a plurality of culture wells. The assay device includes a housing, an array, and a control module. The housing includes a socket sized to receive the culture plate. The array includes a plurality of emitters and is positioned within the housing adjacent to the socket so that the plurality of emitters are each aligned with at least one of the culture wells when the culture plate is received in the socket. The control module is operably coupled to the array and is configured to individually drive the plurality of emitters. The control module is configured to drive at least two of the plurality of emitters at different frequencies simultaneously. 
     Some embodiments of the invention provide a method of assaying the application of energy to a plurality of samples in vitro. The method includes applying the plurality of samples to individual wells of a culture plate and aligning at least one energy emitter with each individual well of the culture plate. The method also includes applying energy at a separate frequency toward each individual well via each of the at least one energy emitters for a first time period and incubating the wells for a second time period. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of an assay device according to one embodiment of the disclosure. 
         FIG. 2  is a schematic diagram of the assay device of  FIG. 1 . 
         FIG. 3  is top view of an RF array for use with one embodiment of the assay device. 
         FIG. 4  is a cross-sectional view of a portion of the RF array of  FIG. 3 . 
         FIG. 5  is top view of an electric field array for use with one embodiment of the assay device. 
         FIG. 6  is a cross-sectional view of a portion of the electric field array of  FIG. 5 . 
         FIG. 7  is a cross-sectional view of a portion of a magnetic field array for use with one embodiment of the assay device. 
         FIG. 8  is a cross-sectional view of a portion of an LED array for use with one embodiment of the assay device. 
         FIG. 9  is top view of a plasma emitter array for use with one embodiment of the assay device. 
         FIG. 10  is a cross-sectional view of a portion of the plasma emitter array of  FIG. 9 . 
         FIG. 11  is a flow chart of a method of assaying the application of energy to a plurality of samples according to one embodiment of the disclosure. 
     
    
    
     DETAILED DESCRIPTION 
     Before any embodiments of the disclosure are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms “mounted,” “connected,” “supported,” and “coupled” and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, “connected” and “coupled” are not restricted to physical or mechanical connections or couplings. 
     The following discussion is presented to enable a person skilled in the art to make and use embodiments of the disclosure. Various modifications to the illustrated embodiments will be readily apparent to those skilled in the art, and the generic principles herein can be applied to other embodiments and applications without departing from embodiments of the invention. Thus, embodiments of the invention are not intended to be limited to embodiments shown, but are to be accorded the widest scope consistent with the principles and features disclosed herein. The following detailed description is to be read with reference to the figures, in which like elements in different figures have like reference numerals. The figures, which are not necessarily to scale, depict selected embodiments and are not intended to limit the scope of embodiments of the invention. Skilled artisans will recognize the examples provided herein have many useful alternatives and fall within the scope of embodiments of the invention. 
     Generally, embodiments of the present disclosure provide an assay device that enables treating multiple wells of a culture plate simultaneously with different pulse frequencies of energy. The device may receive the culture plate in a socket so that the plurality of wells are aligned in close proximity to an array of energy emitters, each of which emits a different pulse frequency simultaneously. This enables many frequencies to be tested simultaneously, speeding up the search for a frequency that provides a desired outcome on samples in the culture wells. 
       FIG. 1  illustrates an assay device  10  according to some embodiments. As shown in  FIG. 1 , the assay device  10  can include a housing  12  with a socket  14 , a user interface  16  (such as one or more push buttons  18  and a display  20 ), and one or more input/output interfaces  22 . Internally, the device  10  may include an array  24 , an array driver  26 , a control module  28 , one or more thermal control devices  30 , a heat sink  32 , a fan  34 , and a power supply  36 , as shown schematically in  FIG. 2 . Additionally, in one embodiment, as shown in  FIG. 2 , the input/output interfaces  22  can include a memory card slot  38  and an RS-485 interface  40 . As further described below, the assay device  10  is configured to emit energy at different frequencies toward individual wells  42  of a culture plate  44  simultaneously. 
     As shown in  FIG. 1 , the socket  14  may be sized to receive a culture plate  44  that includes a plurality of wells  42 . For example, the socket  14  may be a square or rectangular cutout in the housing  12  approximately equal to a size of the culture plate  44 . It should be noted that, while  FIG. 1  illustrates the culture plate  44  with ninety-six wells  42 , it is within the scope of this disclosure to include an assay device  10  sized and adapted to receive culture plates  44  including more or less wells  42 , such as six wells, twelve wells, twenty-four wells, forty-eight wells, etc. Optionally, the socket  14  may further include a structural support (not shown) around its perimeter to support the culture plate  44 . In other embodiments, the culture plate  44  may be received within the socket  14  and rest upon a top outer perimeter of the array  24 . 
     Accordingly, the array  24  can be positioned within the housing  12  adjacent to (e.g., below) the socket  14 . As described in more detail below with respect to  FIGS. 3-10 , the array  24  can include a board  46  with a plurality of energy emitters  48  driven by the array driver  26  to emit energy, such as radio frequency (RF), electric field, magnetic field, optical emission, and non-thermal plasma, toward the culture plate  44 . The plurality of emitters  48  may be arranged on the board  46  in an array pattern so that each emitter  48  aligns with at least one culture well  42 . As a result, one or more emitters  48  may be dedicated to emit energy at a specific frequency to a single well  42  of the culture plate  44 . Generally, in some embodiments, the number of emitters  48  may equal the number of wells  42  for which the device  10  is adapted (such as six, twelve, twenty-four, forty-eight, ninety-six, etc). However, in other embodiments, the array  24  may include more or less emitters  48  than wells  42 . For example, the device  10  may be configured so that patterns or groupings of multiple emitters  48  may be dedicated to a single well  42 . 
     According to some embodiments, the control module  28  can be operably coupled to the array  24  (e.g., through the array driver  26 ) and can be configured to individually drive each emitter  48  at a specific frequency for a set time period. For example, the control module  28  can control the array driver  26  to drive each emitter  48  at a different frequency, or to drive groupings of more than one emitter  48  at different frequencies. The control module  28  may include an application-specific integrated circuit (ASIC), a programmable logic controller (PLC), or, preferably, a field-programmable gate array (FPGA), such as the Xilinx Artix 7. A signal generator for each emitter  48  (e.g., a square wave signal generator) can be implemented in VHDL code as a phase accumulator, or numerically controlled oscillator (NCO). In a preferred embodiment, control module output to the array driver  26  consists of the most significant bit of the phase accumulator. Using only a single bit of the output may add deterministic phase jitter, but it allows the device to define individual emitter units (e.g., up to ninety-six units) with reasonable FPGA resources. Furthermore, using a 200 MHz clock and both edges of the clock, the peak timing uncertainty can be about ±2.5 nanoseconds (ns). 
     The control module  28  can also be operably coupled (e.g., electrically connected) to the memory card slot  38 , the user interface  16 , the RS-485 interface  40 , the fan  34 , the power supply  36  and/or other connections (not shown). More specifically, the memory card slot  38  can be sized to receive a memory device (such as a data card) allowing, for example, the control module  28  to upload control instructions and test data from the memory card and/or download test data to the memory card. Alternatively, in some embodiments, the memory card slot  38  can be sized to receive a USB flash drive. Also, in some embodiments, the control module  28  can include internal memory accessible via one of the I/O interfaces. 
     In some embodiments, the control module  28  can connect to an external computer via the RS-485 interface  40 . The RS-485 interface  40  may also be used to connect multiple devices  10  (e.g., up to 128 devices) in a daisy-chain configuration, for example, using a MODBUS communication protocol. The interface can be designed so that the first device  10  in the cable chain the lowest address, and the subsequent devices  10  have sequentially higher addresses. 
     In some embodiments, the device  10  can include more than one RS-485 interface  40 . Additionally, it should be noted that, while the memory card slot  38  and the RS-485 interface  40  are shown and described herein, it within the scope of this disclosure to include additional input/output interfaces and/or other communications circuits. For example, the device  10  can include a power supply interface (not shown), permitting the device  10  to be connected to and powered by an external power supply. 
     Accordingly, the control module  28  can receive instructions via the memory card slot  38  (i.e., via an inserted memory device), the RS-485 interface  40  (i.e., via a connected external computer), or additional input/output interfaces. Furthermore, the control module  28  can receive user inputs via the user interface  16 . As shown in  FIG. 1 , the user interface  16  can include one or more push buttons  18 . As such, the control module  28  can receive user input via a user pressing one of the push buttons  18 . For example, the push buttons  18  can be pressed to control power of the device (i.e., a power button), to toggle between displays or programs (e.g., via “up” and/or “down” buttons), to start a treatment program (e.g., a “start” button), to end a treatment program (e.g., a “stop” button), or for other types of device control. Additionally, in some embodiments, the device  10  can include user inputs other than push buttons, such as rotary dials, switches, or the like. 
     Furthermore, the user interface  16  can include the display  20 . The display  20  may be, for example, a light emitting diode (LED) display, an organic light emitting diode (OLED) display, a digital display, or another suitable display. In one embodiment, the display  20  is a 2×20-character OLED display. The control module  28  may control the display  20  to display to a user, for example, program status, on/off status, device status, present temperatures, a timer, test data, programs, or the like. In one example, the device  20  can display test data or programs in a list format, and a user can toggle the lists and/or select a desired program using the push buttons  18 . Alternatively, the control module  28  may automatically execute a program upon receiving a memory card within the memory card slot  38  and being powered on. 
     Internally, the control module  28  can control operation of the fan  34  as part of a thermal control system of the device  10 . More specifically, the thermal control system can include the fan  34 , the heat sink  32 , the thermal control devices  30  (such as thermoelectric coolers, TECs), and one or more temperature sensors (not shown). For example, the array  24 , the array driver  26 , and/or the control module  28  can be thermally coupled to the heat sink  32 , the TECs  30 , and the fan  34 . The heat sink  32  may act as a passive cooling device, while the fan  34  may be operated for active cooling, and the TECs  30  may be operated for heating or cooling. 
     The control module  28  can control operation of the fan  34  and/or the TECs  30  based on a specific treatment program (e.g., the control module  28  may operate the device  10  at a desired temperature or within a desired temperature range based on received temperature control data for a specific program). For example, during and/or after energy application to the culture wells  42 , the control module  28  can control the thermal control system (in particular, the fan  34  and the TECs  30 ) to incubate the culture wells  42  for a preset time period. 
     More specifically, the temperature sensors can be positioned on the array  24 , the housing  12 , and/or the heat sink  32  and can be in communication with the control module  28 . The control module  28  can monitor the temperatures of these components (i.e., by retrieving a current temperature from one or more of the temperature sensors) and determine whether passive or active cooling is necessary or whether heating is necessary. That is, the control module  28  can operate the fan  34  based on input from one or more of the temperature sensors. In one embodiment, the fan  34  can draw in air from one or more bottom cutouts of the housing  12 , past the heat sink  32  and up through the socket  14  (i.e., past the culture plate  44  received in the socket  14 ). Additionally, in some embodiments, the control module  28  may display an alarm (e.g., via the display  20 ) if a temperature of one of the components exceeds a high or low temperature threshold for a set time period despite heating or cooling. 
     Furthermore, it should be noted that other thermal control devices are contemplated within the scope of this disclosure. Additionally, the heat sink  32  and all internal electronics of the device  10  can be thermally insulated from the housing  14 . 
     The following paragraphs describe specific arrays  24  for devices  10  configured to treat culture wells  42  with RF energy, electric fields, magnetic fields, light, and non-thermal plasma, respectively. For example, the above-described components may be common to each device  10 , with the exception of the arrays  24  and array drivers  26 , which may be specific to the type of energy emission of the device  10 . Thus, some embodiments provide different build options for an RF-specific device, an electric field-specific device, a magnetic field-specific device, a light-specific device, and a non-thermal plasma-specific device. Alternatively, in some embodiments, a device  10  can include all the above common components and can be configured to receive different arrays  24  and array drivers  26  in an interchangeable manner. Thus, such a device  10  can be used for all types of energy emissions. 
       FIGS. 3 and 4  illustrate an RF array  24  according to one embodiment of the disclosure. The RF array  24  comprises an RF emitter board  46  driven by an RF driver board  26 . The RF emitter board  46  can be a printed circuit board with rows and columns of individual RF emitters  48 . In one embodiment, the RF array  24  can be a 2.4 gigahertz (GHz) array. As such, an RF emitter  48  can be a resonant structure consisting of a printed spiral inductor and series capacitor, which presents a good impedance match at 2.44 GHz. While not an efficient antenna (since most of the power is dissipated in the spiral structure) the emitter  48  is meant to couple short transmissions of RF energy upward and into a culture well  42 , similar to that used in near-field communication devices. The matching is relatively narrowband, and the control module  28  can adjust the output RF frequency for each emitter  48  to determine optimum frequencies for certain treatments. 
     The RF driver board  26  can be a separate printed circuit board that drives the RF array  24 , as shown in  FIG. 4 . A circuit for measuring the forward and reverse power on a single emitter  48  can also be included, on the assumption that all the forward and reverse power on other emitters  48  will be very similar. The RF array driver  26  can include Wilkinson power splitters, RF switches, and load resistors. The control module  28  can route digital signals to the RF driver  26  to create a distinct modulation frequency underneath each culture well  42 . The RF array driver  26  can also include a temperature compensated crystal oscillator and frequency synthesizer, as well as amplifiers and attenuators for controlling the RF level. The power into the modulation section can be −10 dBm to +17 dBm, but the maximum radiated power from the array can be about 1 mW. Additionally, as shown in  FIG. 4 , the RF module (i.e., including the array  24  and the driver  26 ) can further include thermal pads  50  below each of the array  24  and the driver  26  and a heat spreader  52  below the driver  26 . These components can transfer heat through the array driver  26  to the heat sink  32  or other thermal control devices. 
       FIGS. 5 and 6  illustrate an electric field array  24  according to one embodiment of the disclosure. The electric field array  24  comprises an electric field emitter board  46  driven by an electric field driver board  26 . The electric field emitter board  46  can be a printed circuit board with rows and columns of individual electric field emitters  48 . The electric field driver board  26  can be a separate printed circuit board that drives the electric field emitter board  46  to create an electric field flux, as shown in  FIG. 6 . In one embodiment, for a driving voltage of 5 volts, peak-to-peak (Vp-p), the peak field strength can be between 1.5 volts per centimeter (V/cm) and 2.5 V/cm. In some embodiments, if a higher electric field strength is desired, the array driver voltage can be increased to 190 Vp-p up to 100 kiloHertz (kHz) using high voltage amplifiers. In some embodiments, the emitters  48  can deliver a peak electric field in a culture well  42  between about 60 V/cm and about 100V/cm. In such embodiments, the electric field array driver  26  also includes a voltage boost converter to generate the 200-volt bias for the amplifiers. Additionally, as shown in  FIG. 6 , the energy field module (i.e., including the array  24  and the driver  26 ) can further include thermal pads  50  below each of the array  24  and the driver  26  and a heat spreader  52  below the driver  26 . These components can transfer heat through the array driver  26  to the heat sink  32  or other thermal control devices. 
       FIG. 7  illustrates a magnetic field array  24  according to one embodiment of the disclosure. The magnetic field array  24  includes a magnetic field emitter board  46  driven by a magnetic field driver board  26 . The magnetic field emitter board  46  can be a printed circuit board, functioning mainly to hold the tops of magnetic field emitters  48 . More specifically, a magnetic field emitter  48  consists of a ferrite bar  54 , which in a preferred embodiment has a length of 7.5 mm and diameter of 0.8 mm. Coil winding  56  around the bar  54  can include 200 turns of #36 wire, presenting about 100 uH of inductance. The moderate permeability (ui=125) of the ferrite bar  24  will tend to efficiently couple higher harmonics generated by the driving waveform. 
     The magnetic field array driver  26  can be a separate printed circuit board that drives the magnetic field array  24 . In some embodiments, the duty cycle of the driving waveform can be adjusted to produce a DC bias of 2 milliamperes (mA) at 5 V. A circuit (not shown) for monitoring DC bias can be connected to a single element  48 , on the assumption that all elements  48  will have a similar bias. A calibration routine can be used to determine the required duty cycle for a given frequency. Additionally, as shown in  FIG. 7 , the magnetic field module (i.e., including the array  24  and the driver  26 ) can further include thermal pads  50  between the emitters  48  and a heat spread  52  below the magnetic field driver  26 , which can conduct heat past the control module  28  over to the TECs  30  (e.g., via aluminum blocks, not shown). In one embodiment, the heat spreader  52  can include aluminum nitride, which will keep metal farther away from the ends of the inductors. Also, in one embodiment, the driver PCB  26  can be positioned to maintain an area around the ferrite bars  54  with no metal for a diameter of about 0.25 inches. 
       FIG. 8  illustrates an optical array  24  according to one embodiment of the disclosure. The optical array  24  includes an optical emitter board  46  driven by an optical driver board  26 . The optical emitter board  46  can be a printed circuit board with light emitting diodes (LEDs)  48  mounted on it. LEDs  48  with a wide variety of wavelengths may be used in certain embodiments, for example from near infrared (NIR, around 850 nanometers (nm)) to ultraviolet (UV, around 395 nm). In one embodiment, the LEDs  48  can be in a  1206  package with lens. 
     The optical driver board  26  can be a separate printed circuit board that drives the optical array  24 . The LED drivers  26  can be designed to run at a fixed current, but the average power can be reduced by adjusting the duty cycle. In some embodiments, the LEDs  48  can be mounted upside down into the driver PCB  26  to create a flush surface for thermal contact with the culture plate  44 . Additionally, as shown in  FIG. 8 , the optical module (i.e., including the array  24  and the driver  26 ) can further include thermal pads  50  around the LEDs  48  to improve thermal transfer and a heat spreader  52  below the driver  26 . These components can transfer heat through the array driver  26  to the heat sink  32  or other thermal control devices. 
       FIGS. 9 and 10  illustrate a plasma array  24  according to one embodiment of the disclosure. The plasma array  24  comprises a plasma emitter board  46  driven by a plasma driver board  26 . The plasma emitter board  46  can be a printed circuit board with rows and columns of individual plasma emitters  48 . In one embodiment, the plasma emitters  48  can be those described in co-pending U.S. patent application Ser. No. 15/055,028, which is incorporated herein by reference in its entirety. The distance between the plasma dielectric and the base of the culture plate  44  may be defined by a copper and solder mask thickness of about 2 mils. To maintain constant plasma characteristics, ventilation holes  58  may be included outside the plasma areas. 
     The plasma array driver  26  can be a separate printed circuit board that drives the plasma array  24 . In one embodiment, the plasma array driver  26  can require multiple lines from the control module  28  to control the plasma driver  26  and high voltage transformers  60  to create a total of twenty-four drivers  26 . Each driver  26  can then be connected to a set of 4 micro-plasma arrays  24 , which sit underneath the culture wells  42 . Each set of plasma arrays  24  can present a capacitance of about 20 picofarads (pF). This capacitance resonates with the plasma drive transformer  60 , and the plasma drive frequency can be about 420 kHz. In one embodiment, the plasma modulation frequency can be adjusted by the control module  28  up to 10 kHz. The plasma tends to run at a fixed current, and a duty cycle of 10% may be used to reduce average power and extend the array life. Additionally, as shown in  FIG. 10 , the plasma module (i.e., including the array  24  and the driver  26 ) can further include thermal pads  50  below each of the array  24  and the driver  26  and a heat spreader  52  below the driver  26 . These components can transfer heat through the array driver  26  to the heat sink  32  or other thermal control devices. 
     In light of the above, embodiments of the present disclosure provide a device  10  that enables treating multiple wells  42  of a culture plate  44  simultaneously with different pulse frequencies of energy, such as RF, electric field, magnetic field, light, or plasma.  FIG. 11  illustrates a method for using the device  10  according to one embodiment of the disclosure. Generally, as shown in  FIG. 11 , at step  70 , a sample culture of interest, such as cells, tissues, bacteria, fungus or virus, is deposited in each well  42  of the culture plate  44 . Next, at step  72 , the culture plate  44  is inserted into the socket  14  of the device  10 . A treatment program is then selected and run, at step  74 , and energy at different frequencies is applied to each of the wells simultaneously for a time period, at step  76 . During or after the time period in step  76 , the culture wells are incubated for a second time period, at step  78 . After the time period in step  78 , the cultures in each culture well may be analyzed, at step  80 , to determine the effects of the applied energy to each well, for example to determine an optimal frequency for a desired outcome. 
     More specifically, at step  70 , a sample culture of interest, such as cells, tissues, bacteria, fungus or virus, is deposited in each well  42  of the culture plate  44 . For example, each well may include the same type of sample, or different wells may include different types of samples. Next, at step  72 , the culture plate  44  is inserted into the socket  14  of the device  10 . As described above, the socket  14  can be sized so that the culture plate  44  rests within the socket  14  atop the array  24 . Additionally, in some embodiments, steps  70  and  72  may be reversed (i.e., the culture plate  44  can be inserted into the socket  14  and then the samples can be deposited in each well  42 ). 
     When the culture plate  44  is received within the socket  14 , a treatment program may be run on the device  10  at step  74 . Generally, a treatment program can include specific frequencies to apply, the first set time period, the second set time period, temperature set points or thresholds, and/or other testing parameters. For example, the device  10  may be powered on and a memory card may be inserted in the memory card slot  38  so that the control module  28  automatically executes a treatment program stored on the memory card. In another example, the device  10  may display multiple treatment program options on the display  20  and a user may select a desired treatment program for the control module  28  to run via the push buttons  18 . In yet another example, the device  10  may be connected to an external computer via the RS-485 interface  40  (or another input/output interface  22 ) and a user may select a desired treatment program for the control module  28  to run via the external computer. 
     According to the selected treatment program, the control module  28  can control the array  26  to apply energy to the culture plate  44  at step  76 . More specifically, the control module  28  can generate a set of separate frequencies (i.e., more than one frequency) for each emitter  48  or for groups of emitters  48  to emit toward individual wells  42  of the culture plate  44  simultaneously. As discussed above, the array  24  can be configured so that a single emitter  48  is aligned under each well  42 , or so that groups of emitters  48  are aligned under each well  42 . Thus, each emitter  48  (or each group of emitters  48 ) can output a different frequency simultaneously in order to test the effects of multiple frequencies on separate samples (i.e., in the separate culture wells  42 ) at the same time. In one example, the control module  28  can drive at least two emitters  48  at different frequencies simultaneously. In another example, the control module  28  can generate a set of ninety-six separate frequencies simultaneously to individual control ninety-six emitters  48 . The control module  28  can apply the energy to the culture wells  42  for a first set time period according to the selected treatment program. 
     The control module  28  can also operate the thermal control system to incubate the culture wells  42  for a second set time period, at step  78 , according to the selected treatment program. For example, in one embodiment, the control module  28  can thermally control the temperature of the array  24  in order to incubate the wells. In some embodiments, the second set time period may be equal to the first set time period of step  76 . Additionally, in some embodiments, the second set time period may partially or completely overlap with the first set time period. 
     Once the second set time period is completed, the culture wells  42  may be analyzed to determine the effects of the applied energies to the samples, at step  78 . In some embodiments, the device  10  may automatically turn off once the second set time period is completed, may automatically save treatment data to memory (e.g., store frequency and temperature control data to internal memory or the memory device), or may display an alert via the display  20  to notify a user that the treatment program is complete. 
     In accordance with the above method, multiple samples can be simultaneously treated in vitro with different frequencies and examined to determine which sample had a desired or optimal outcome, thus allowing a determination of the optimal frequency for a specific treatment. Furthermore, the method may be repeated, for example, to see if the energy effects are repeatable. For example, the control module  28  can apply the same treatment program, but shuffle the modulation frequencies to different emitters  48 . In particular, repeating the methods and shuffling the frequencies can compensate for signal overlap between adjacent array elements. Additionally, the present methods can be applied to multiple devices  10  simultaneously using a daisy-chain configuration, as described above. Multiple tests can thus be completed quickly and accurately and both frequency and temperature data may be stored, for example, to memory devices or a database as part of a research program and to track tested frequencies. 
     In light of the above, embodiments of the disclosure provide a device and method for studying the application of energy at different frequencies to samples in vitro. By allowing multiple samples to be tested at different frequencies simultaneously and then incubated, the present device and method provide a standalone low-cost, quick, and accurate way to study the biological effects of samples based on the modulation frequency of RF, electric fields, magnetic fields, light, and plasma. 
     It will be appreciated by those skilled in the art that while the invention has been described above in connection with particular embodiments and examples, the invention is not necessarily so limited, and that numerous other embodiments, examples, uses, modifications and departures from the embodiments, examples and uses are intended to be encompassed by the claims attached hereto. The entire disclosure of each patent and publication cited herein is incorporated by reference, as if each such patent or publication were individually incorporated by reference herein. Various features and advantages of the invention are set forth in the following claims.