Abstract:
An apparatus for automatically hybridizing nucleic acid samples is disclosed. The apparatus includes a fluid control module and a temperature control module for precisely controlling fluid contacting and temperature of a plurality of DNA samples. The DNA samples are typically arrayed on solid substrates (glass microscope slides), and the disclosed apparatus can process up to twelve slides at one time on a master unit; satellite units can be added to increase the number of slides. All slides can be processed in parallel, or may be addressed individually to undergo different hybridization protocols. Thermal control is typically by slide pairs, such that each slide pair undergoes the same temperature profile. Processes are carried out under software control by an embedded PC (personal computer). User input is by touchscreen or floppy disk drive.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention  
           [0002]    This invention relates to devices and processes for hybridizing nucleic acid samples, and more particularly, to an automated device for hybridizing DNA microarrays.  
           [0003]    2. Discussion  
           [0004]    Use of DNA (deoxyribonucleic acid) microarrays provides a powerful technique to analyze expression of thousands of genes simultaneously. The technique includes immobilizing DNA samples from large numbers of genes on a solid substrate, such as a glass microscope slide. The DNA samples appear as an array of spots on the substrate, and one can determine the origin of a particular DNA sample by knowing its position in the array. The technique typically provides contacting the DNA microarray with RNA (ribonucleic acid) probes to detect specific nucleotide sequences in the DNA samples. To distinguish between different RNA probes, each is labeled with a tag that fluoresces at a wavelength that is unique for the particular probe.  
           [0005]    Under proper conditions, the RNA probes will hybridize or bind to the immobilized DNA samples, resulting in hybrid DNA-RNA strands. For each of the immobilized DNA samples, and for a particular RNA probe, one can discern differences in hybridization among DNA samples by measuring the intensity and the wavelength dependence of fluorescence of each microarray element. In this way, one can determine whether gene expression levels vary among DNA samples. Thus, using DNA microarrays, one can learn much about expression of a large number of genes, and about comprehensive patterns of gene expression, using relatively small amounts of biological material.  
           [0006]    Although DNA microarrays are powerful tools, instruments currently available to hybridize DNA microarrays need improvement. Most instruments that can process DNA microarrays have rudimentary temperature control. But nucleic acid hybridization demands precise temperature control. Rates of hybridization and equilibrium concentrations of hybrid DNA-RNA strands depend strongly on temperature and therefore accurate comparisons among hybridization experiments require that the experiments be run at the same temperature. In addition, precise temperature programming during an experiment is often critical to minimizing spurious probe-sample binding. For example, rapidly decreasing temperature following hybridization—a process called step-wise probe annealing—reduces background binding.  
           [0007]    Generally, instruments that can process DNA microarrays also lack an adequate system for controlling fluid contacting. During hybridization, the DNA microarray is immersed in a fluid that contains the RNA probes. The rate at which the probes bind to the DNA samples will depend, in part, on the concentration of the probes. However, the concentration of the probes near the immobilized DNA samples may be much different than the bulk concentration of the probes. Although agitating the fluid helps minimize concentration gradients between the bulk fluid and fluid next to the substrate surface, excessive fluid mixing may create high shearing and normal forces that may dislodge the DNA samples.  
           [0008]    The present invention overcomes, or at least reduces, one or more of the problems set forth above.  
         SUMMARY OF THE INVENTION  
         [0009]    The present invention provides a DNA hybridization apparatus capable of precise thermal and fluid control. The present invention is particularly useful when used in conjunction with DNA spotted glass slides (DNA microarrays). The apparatus can also be used for hybridizing other materials on other substrates. Multiple slides can be processed at one time (in parallel) or in rapid serial fashion. A fluid manifold allows for control of multiple fluids across the surface of each slide. All slides can contact the same sequence of fluids or may undergo different fluid contacting protocols. Thermal control is by slide pair, so that each slide pair undergoes the same temperature profile or different pairs can have different temperature programming. Small volumes of liquids can be manually applied to each of the slides. Each slide pair is provided with separate clamping mechanisms to seal DNA sample areas of each slide. Fluids are moved under negative pressure throughout the instrument, ensuring that no dangerous chemicals can be ejected under pressure. The present invention also provides for software control of fluid contacting and temperature using software running on an embedded personal computer. User input is by touchscreen or a floppy disk drive. A system network distributes control signals and software between the master and satellite units and the thermal control module for each slide pair. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0010]    [0010]FIG. 1 shows a perspective front view of one embodiment of an automated DNA hybridization apparatus for use with DNA microarrays.  
         [0011]    [0011]FIG. 2 shows a perspective top view of one of a slide plate assembly.  
         [0012]    [0012]FIG. 3 shows a cross-sectional side view of a slide plate assembly and clamp.  
         [0013]    [0013]FIG. 4 shows a perspective top view of a slide carrier and the pair of glass slides (DNA microarrays).  
         [0014]    [0014]FIG. 5 shows a perspective bottom view of a slide cover.  
         [0015]    [0015]FIG. 6 shows a phantom top view of a master manifold.  
         [0016]    [0016]FIG. 7 shows a phantom bottom view of a satellite manifold.  
         [0017]    [0017]FIG. 8 is a schematic of a fluid control module.  
         [0018]    [0018]FIG. 9 illustrates fluid agitation within a slide cavity using valve actuation.  
         [0019]    [0019]FIG. 10 shows an exploded view of a temperature management module.  
         [0020]    [0020]FIG. 11 shows a schematic diagram of a control subsystem for each of the thermal management modules. 
     
    
     DETAILED DESCRIPTION  
     Overview  
       [0021]    [0021]FIG. 1 shows a perspective front view of one embodiment of an automated DNA hybridization apparatus  100  for use with DNA microarrays (glass microscope slides spotted with DNA). The apparatus  100  shown in FIG. 1 includes a housing  102  that contains six thermal management modules  104 , though the number of thermal management modules  104  can vary. Each of the thermal management modules  104  controls the temperature of one of six slide plate assemblies  106 . Each of the slide plate assemblies  106  includes a pair of glass microscope slides (not shown) spotted with DNA. During processing, each pair of glass microscope slides can undergo different temperature programming since the thermal management modules  104  can operate independently.  
         [0022]    During hybridization, a fluid control module  108  distributes various liquids (buffers, reagents, and the like) and various gases (air, for example) to each glass slide. The fluid control module  108  includes a master manifold  110 , which is in fluid communication with a first row  112  of slide plate assemblies  106 , and a satellite manifold  114 , which is in fluid communication with a second row  116  of slide plate assemblies  106 . The master manifold  110  and the satellite manifold  114  contain valves and conduits (not shown) that allow fluid flow from liquid reservoirs  118  to individual glass slides. In addition, the master manifold  110  and the satellite manifold  114  allow fluid flow from individual glass slides to waste containers  120 . Use of two waste containers  120  obviates the need to mix reactive wastes or to change collection vessels during processing. As described below, each of the DNA microarrays may contact the same or different fluids during hybridization. A pump (not shown) maintains vacuum within headspaces of the two waste containers  120 . Ambient pressure in the liquid reservoirs  118  and vacuum within the waste containers  120 , results in a pressure drop that drives fluid flow throughout the fluid control module  108 . Since all fluid contacting within the apparatus  100  occurs at below-ambient pressure, no dangerous chemicals can be ejected from the apparatus  100  under pressure.  
         [0023]    Thermal management and fluid contacting are under the control of software running on an embedded personal computer (PC) module  122 . User input is by touchscreen  124  or a floppy disk drive  126 . A proprietary system network distributes control signals and software instructions among the master manifold  110 , the satellite manifold  114 , and the thermal management modules  104  for each of the slide plate assemblies  106 . The user can program processing steps on the apparatus  100  via application software and either touchscreen  124  or floppy disk drive  126 . Process control programs entered on the touchscreen  124  can be stored on the embedded PC module  122  hard drive or downloaded to the floppy disk drive  126 .  
         [0024]    Though not shown in FIG. 1, the apparatus  100  also includes a power supply module. The power supply module, under the control of the embedded PC module  122 , provides current to actuate valves on the master  110  and satellite  114  manifolds, and provides energy to power each of the thermal management modules  104 . Because line voltage limits available current to about 10 amps, the power supply module cannot provide power to all of the thermal management modules  104  simultaneously without severely diminishing heating or cooling rate. Instead, the power supply module uses intelligent energy scheduling by first providing power to one or two of the slide plate assemblies  106 . After they attain their desired temperatures, the power supply module provides power to a second group of slide plate assemblies  106 . This process continues until all of the slide plate assemblies  106  reach their desired temperatures.  
       Fluid Control Module  
       [0025]    [0025]FIG. 2 and FIG. 3 show, respectively, a perspective front view and cross-sectional side view of one of the slide plate assemblies  106 . As shown in FIG. 2, the slide plate assembly  106  includes a slide cover  150  that is held in place with a clamp  152 . The clamp  152  is a generally rectangular frame  154  having a single, mid-span cross member  156 . The rectangular frame  154  is mounted on a pair of clamp arms  158 ,  160  using a cylindrical rod  162  that allows the rectangular frame  154  to pivot about the centerline of the cylindrical rod  162 . First ends of the clamp arms  158 ,  160  are pivotably mounted on hinges  164 ,  166 , which are fastened to the thermal management module  104 ; a rectangular bar  168  attached to second ends of the clamp arms  158 ,  160  prevents relative movement of the clamp arms  158 ,  160 . To secure the slide plate assembly  106 , a knob  170 , which is mounted on the rectangular bar  168 , is threaded into a clamp base  172  which is attached to the thermal management module  104 .  
         [0026]    As shown in FIG. 3, the slide plate assembly  106  includes a slide cover  150  disposed above a pair of glass slides  190  that are contained on a planar, stainless steel slide carrier  192 . During processing, the slide cover  150  is disposed on the glass slides  190 . The slide carrier  192  positions the glass slides  190  using a series of cut out tabs  194  that are bent upward at an angle of about 10 degrees. The cut out tabs  194  allow for slight variations in dimensions of the glass slides  190 . A U-shaped tab  196  located at one end of the slide carrier  192  engages a locator pin (not shown) on the manifolds  110 ,  114  shown in FIG. 1 to fix the position the glass slides  190  and the slide carrier  192  in the apparatus  100 .  
         [0027]    Further details of the slide plate assembly  106  are shown in FIG. 4 and FIG. 5. FIG. 4 shows a perspective top view of the slide carrier  192  and the pair of glass slides  190 . Each of the glass slides  192  is spotted with DNA in the form of an array  210 .  
         [0028]    [0028]FIG. 5 shows a perspective view of a bottom surface  220  of the slide cover  150 . The slide cover  150  is constructed from a high temperature plastic to prevent sagging or softening at the higher operating temperatures of the apparatus  100 . A suitable plastic includes polysulfone. Polysulfone possesses the requisite temperature characteristics and is transparent, which allows direct viewing of the glass slides  190  during processing. In addition, the absorption and attenuation characteristics of polysulfone help prevent photo bleaching of the DNA microarray, RNA probes, and the like during processing.  
         [0029]    A shim  222 , having a pair of rectangular cut outs of slightly smaller dimension than the glass slides  190 , is disposed on the bottom surface  220  of the slide cover  150 . The thickness of the shim  222  defines a standoff between the bottom surface  220  of the slide cover  150  and the glass slides  190 . Two perfluoroelastomer o-rings  224 , which are inert and will not bind to nucleic acids, are placed in grooves cut into the bottom surface  220  of the slide cover  150  around the inner periphery of the shim  222 . During processing, the bottom surface  220  of the slide cover  150  is disposed on the glass slides  190 , compressing the o-rings  224  and defining two slide cavities for fluid flow.  
         [0030]    Referring to FIG. 3 and FIG. 5, fluid enters and exits each of the slide cavities through ports  226  located at one end of the slide cover  150 . The ports  226  provide fluid connections with manifolds  110 ,  114  shown in FIG. 1, and are sealed with o-rings  228 . For each slide cavity, fluid enters one of the ports  226  into a first lateral diffusion channel  230  that is cut into the slide cover  150 . Next, fluid flows the length of the cavity along the surface of the slide  190  and dumps into in a second lateral diffusion channel  232 . From the second diffusion channel  232 , fluid flows within a return channel  234  bored in the slide cover  150  back towards the ports  226 , and exits the slide cavity through one of the ports  226 . Note that, in addition to diffusing flow, the diffusion channels  230 ,  232  act as small fluid reservoirs that empty and fill as the temperature within the slide cavity rises and falls.  
         [0031]    As shown in FIG. 3 and FIG. 5, the slide cover  150  contains two injection ports  236  for manually injecting small liquid volumes (a few microliters, say) directly into each of the slide cavities. The injection ports  236  are drilled with a taper that matches the profiles of an injection device—typically a pipette—and polyethylene plugs  238  that, as shown in FIG. 2, seal the ports  236  when not in use. The taper does not allow fluid to remain in the injection ports  236  once the plugs  238  are inserted, thus reducing the apparent volume of the slide cavity.  
         [0032]    During manual injection, liquid is drawn into the second diffusion channel  232  by capillary action and flows across the surface of the slide  190  within the slide cavity until the liquid reaches the first diffusion channel  230 . Because manual liquid injection occurs at the second diffusion channel  232 , and the slide plate assembly  106  tilts slightly upward (about 10 degrees or so) towards the first diffusion channel  230 , the liquid displaces and expels air within the slide cavity out of the ports  226  during injection. Liquid should not completely fill both diffusion channels  230 ,  232  since they are designed to compensate for thermal expansion and contraction of the fluid within the slide cavity.  
         [0033]    Thermal breaks  238 , such as the one shown in FIG. 3, are cut into the slide cover  150  to reduce the distortion resulting from thermal gradients in a direction parallel to the bottom surface  220  of the slide cover  150 . Distortion arising from thermal gradients in a direction perpendicular to the surface  220  of the slide cover  150  are reduced by making the slide cover  150  thinner and by reducing its thermal mass.  
         [0034]    Referring once again to FIG. 3, during processing, the clamp  152  presses the slide plate assembly  106  against elements of the thermal management module  104 —thermal plate  260  and thermal pad  262 —using spring  264  loaded contact ferrules  266  mounted in recesses  268  in the clamp frame  154 . The contact ferrules  196  are slidably mounted on screws  270  threaded into the clamp frame  154 . The contact ferrules  266  are arranged around the clamp frame  154  so they exert a downward force that is evenly distributed along the periphery of the slide cover  150 . The applied pressure is sufficient to ground out the shim  222  against the glass slides  190  and to prevent warping of the slide cover  150  due to thermal gradients.  
         [0035]    [0035]FIG. 6 and FIG. 7 show phantom top and bottom views, respectively, of the master manifold  110  and the satellite manifold  114 . Both manifolds  110 ,  114  are formed from multi-layer, diffusion bonded acrylic, in which channels  290 ,  292 ,  294 , and  296  are machined into planar surfaces of acrylic layers and the layers are bonded together under heat and pressure. The master manifold  110  is similar to the satellite manifold  114  except that the master manifold  110  provides fluid connections to the liquid reservoirs  118  and waste containers  120  via liquid ports  298  and waste ports  300 , respectively. In addition, the master manifold  110  provides fluid connections to a compressed air source and to ambient air via a gas port  302  and a vent port  304 . Locator pins  306  engage the slide cover  150  and the U-shaped tab  196  of the slide carrier  192 . The locator pins  306  serve to position the slide cover  150  and slide carrier  192  relative to the manifolds  110 ,  114 .  
         [0036]    Returning to FIG. 2, first and second conduits  320 ,  322  provide fluid communication between the liquid reservoirs  118  and the waste containers  120 , respectively, through first and second conduit ports  324 ,  326 . In addition, valves  328 , which are mounted to the underside of the master  110  and satellite  114  manifolds, selectively provide fluid communication between the slide cavities and the liquid reservoirs  118 , waste containers  120 , compressed air, or ambient air. The valves are under control of the embedded PC module  122 , and have zero dead volume to prevent retention of liquid when closed.  
         [0037]    [0037]FIG. 8 is a schematic of the fluid control  108  module, and illustrates how fluid moves from the liquid reservoirs  118 , through the master manifold  110 , the satellite manifold  114 , and slide cavities  350 , and into the waste containers  120 . Before fluid is introduced into the slide cavities  350 , a flow path between the liquid reservoirs  118  and the slide cavities  350  is preloaded or primed with liquid from an appropriate reservoir  118 . Priming purges any residual fluid remaining from a previous processing step that may contaminate the current processing step.  
         [0038]    To illustrate priming, suppose one desires to inject liquid from a first reservoir  352  into a first slide cavity  354  and then into a second slide cavity  356 . Initially, all valves  328  are closed. To begin priming, the embedded PC control module  122  (not shown) opens a first liquid input valve  358 , a primary primer valve  360 , and either a first  362  or a second  364  waste valve, which fills the conduit  290  with liquid from the first reservoir  352  since the waste containers  120  are under vacuum. Next, the control module  122  opens a first slide cavity output valve  366  and closes the primary primer valve  360 , which purges the first slide cavity  354  of any residual fluid from a previous processing step. Similarly, to prime the conduit  294  providing fluid communication between the first liquid reservoir  352  and the second slide cavity  356 , the embedded PC control module  122  opens the first liquid input valve  358 , a secondary primer valve  368 , and either the first  362  or the second  364  waste valves. This process fills the conduit  294  with liquid from the first reservoir  352 . Next, the control module  122  opens a second slide cavity output valve  370  and closes the secondary primer valve  368 , which purges the second slide cavity  356  of any residual fluid from a previous processing step.  
         [0039]    Once priming is complete, and all of the valves  328  are closed, the PC control module  122  injects liquid from the first reservoir  352  into the first slide cavity  354  by opening the first liquid input valve  358 , a first slide cavity input valve  372 , a first slide cavity pulse valve  374 , the first slide cavity output valve  366 , and either the first  362  or the second  364  waste valves. Similarly, the PC control module  122  injects liquid from the first reservoir  352  into the second slide cavity  356  by opening the first liquid input valve  358 , a second slide cavity input valve  376 , a second slide cavity pulse valve  378 , the second slide cavity output valve  370 , and either the first  362  or the second  364  waste valves.  
         [0040]    As described above, a vacuum pump  380  maintains vacuum within headspaces of the two waste containers  120 . Ambient pressure in the liquid reservoirs  118  and vacuum within the waste containers  120 , results in a pressure drop that drives fluid flow throughout the fluid control module  108 . As the waste containers  120  fill during processing, headspace within the two waste containers  120  decreases, which diminishes pumping capacity. As a result, the vacuum pump  380  is run continuously to maintain vacuum within the fluid control module under all operating conditions. When the waste container  120  headspace is large, it allows the fluid control module  108  to respond to peak or transient pumping demands. Typically, exhaust  382  from the vacuum pump is channeled to the rear of the apparatus  100 . When the exhaust  382  is hazardous, it is piped to a location for disposal. To aid in the handling of hazardous materials, the waste containers  120  can be preloaded with a neutralizing agent.  
         [0041]    [0041]FIG. 9 illustrates agitation of fluid  400  within one of the slide cavities  350  by valve actuation. FIG. 9 shows a cross sectional view of one of the slide plate assemblies  106  abutting the master manifold  110 . A pair of valves—a slide cavity input valve  402  and a slide cavity pulse valve  404 —provide fluid communication with the liquid reservoirs  118 . The embedded PC control module  122  (not shown) can agitate the fluid  400  by opening and closing the pulse valve  404 . This action draws air out of and into the first diffusion channel  230 , as shown by arrows  406 ,  408 . The diffusion channel  230  acts as a pressure reservoir that tends to dampen and distribute pressure forces within the slide cavity  350 , which minimizes shearing of any DNA adhering to the slide  190 .  
         [0042]    Fluid  400  within the slide cavities  350  often gases during heating forming bubbles that tend to collect in the first diffusion channel  230 . Gas collection in the first diffusion channel  230  is enhanced by agitation and by the slight incline of the slide plate assembly  106 . Intermittent venting of the slide cavity  350  through, for example, an output valve  366 ,  377  and venting valve  420  (FIG. 8), prevents the gas from pressurizing and displacing fluid  400 . Fluid loss by evaporation is minimized by a short vent period.  
       Temperature Control  
       [0043]    [0043]FIG. 10 shows an exploded view of the temperature management module  104 . The temperature management module  104  includes a thermal plate  260  that is designed and constructed to maximize heat transfer between peltier devices  440  and the glass slides  190 . The thermal plate  260  is designed to provide rapid temperature response and uniform temperature distribution across the surface of the glass slides  190 . To achieve these design goals, the thermal plate  260  has minimal thermal mass and a high degree of flatness to maximize thermal contact area. Where mechanical connections must be made to the thermal plate  260 , they are made in positions that do not cause substantial disruption to the temperature profile. The thermal plate  260  is disposed on a graphite-loaded thermal pad  262  that forms a thermal interface between the peltier devices  440  and an anodised surface of the thermal plate  260 . A thermal fuse (not shown) is bonded to the thermal plate  260  to prevent the module  104  from overheating. In addition, a PT100 temperature sensor  442  is embedded on the top of the thermal plate  260  in close proximity to the DNA sample (array)  210  to improve process control.  
         [0044]    Each thermal transfer plate  260  is serviced by four peltier devices  440  connected electrically in series and thermally in parallel to provide low thermal impedance between the thermal plate  260  and heat sink (source)  444 . The spatial configuration of the peltier devices  440  allows compression screws (not shown) to pass between them forming a compression assembly (sandwich) with the thermal plate  260  and the heat sink  444  forming opposing sides. The positions of the compression screws provide even compression force across the peltier faces when correct torque settings are applied to the compression screws. Graphite-loaded thermal pads  262  are used to connect the peltier devices  440  to the heat sink  444  and the thermal plate  260 .  
         [0045]    A mating face  446  of the heat sink (source)  444  has a high degree of flatness to maximize thermal contact area with the peltier devices  440 . Optimal thermal transfer to incident airflow is achieved using an efficient fin assembly (not shown) coupled to turbulent air flow preconditioned to have zero “dead zones.” Preconditioning is achieved by moving a fan  448  a selected distance from the heat sink&#39;s  444  fins, which disrupts dead zones created by the fan&#39;s  448  stator. A temperature sensor  450  is imbedded in the heat sink  444  to supply temperature data to the embedded PC control module  128 .  
         [0046]    Each thermal module is typically capable of temperature ramp rates of about 1° C./s, and can control temperature between about 1° C. and 100° C. Ramp rates are taken with the surface of a slide  190  in a dry condition measured on the top surface of the slide  190 .  
         [0047]    [0047]FIG. 11 shows a schematic diagram of the thermal management module  104  control subsystem  460 . Thermal control of the sample area (DNA array)  210  of the slides  190  depends on accurate and responsive control of the peltier  440  devices. The magnitude and direction of the electrical current input into each of the peltier devices  440  controls the amount and direction of heat transfer across the devices  440 . A switching power converter  470  coupled with an H-bridge reversing switch  472 , supplies the necessary current. Current is under control of a computer processor  474  via a digital to analog (D/A) converter  476 . The temperature of the thermal plate  260  and heat sink (source)  444  is monitored using PT100 sensors  442  and a temperature converter  476  makes the result available to the processor  474 . Electrical current polarity and flow are controlled using the computer processor  474  that in turn monitors temperatures on the thermal plate  260  and the heat sink (source)  444  to calculate applied current and polarity to achieve the demand temperature. A pulse output from the heat sink  444  fan  448  is monitored to provide warning of air flow failure.  
         [0048]    A solenoid valve driver  478  provides a link between the computer processor  474  and the valves  328 . In addition, a serial communication interface  480  provides a link between the computer processor  474  and the embedded PC control module  122 . The embedded PC control module  122  carries out scheduling of valve  328  operations and temperature changes.  
         [0049]    Valve state and temperature change commands are sent to the thermal management module  104  via the serial communications interface  480 . The processor  474  in the thermal management module  104  is responsible for direct valve  328  operation and temperature control. For optimum processing, the latter needs to apply rapid temperature changes, quickly stabilizing at the new temperature with no overshoot. This is achieved in the present embodiment using a modeling technique, rather than a traditional proportional-integral-differential (PID) control loop.  
         [0050]    The thermal module  104  runs a program that implements a model of the thermal characteristics of the combination of the heat sink  444 , peltier device  440 , thermal plate  260  and slides  190 . Heat pumping is modeled as a fixed transient response (of heat pump rate as a function of time), pumping efficiency (steady-state pump rate as a function of peltier current) and heat loss/gain from the thermal plate  260 , through the peltier device  440  to the heat sink  444 . The control algorithm predicts the expected thermal plate  260  temperature at a fixed time in the future (typically 5 secs) on the basis of the history of current through the peltier device  440 , thermal plate temperature  260  and heat sink  444  temperature. From this, the required (assumed constant) current to achieve the desired current is calculated. After ensuring that the calculated current will fall within the range for the power converter and peltier device  440  and that rate-of-change of temperature will not result in thermal shock damage to the peltier device  440 , the calculated current is applied to peltier device  440  by control of the power converter  470  and reversing switch  472 . This current is recalculated at a fixed period of around 1 second. Once the thermal plate  260  temperature is close to the target, fine temperature control is done by trimming the assumed thermal conductivity of the peltier device  440 , according to the temperature error.  
         [0051]    Three types of memory are built into the thermal module processor system different contents:  
                                       Flash 482:   A boot-loader program;       RAM 484:   Operating program and variables;       EEPROM 486:   Characteristics of a particular thermal management           module 104                  
 
         [0052]    (serial number, temperature calibration factors). The boot loader program runs at power-on, its purpose is to accept new program code that is sent to all of the controllers  474  in the thermal modules  104  by the embedded PC control module  122 . This is a convenience since the operating code for the thermal modules  104  is stored in the embedded PC control module  122 , allowing easy upgrade of instruments in the field.  
         [0053]    The six thermal management modules  104  sit on an internal network designed to pass information between the embedded PC control module  122  and the addressed thermal control module  104  (control processor  474 ).