Patent Publication Number: US-11655443-B2

Title: Thermochromic sensing devices, systems, and methods

Description:
RELATED APPLICATIONS 
     This application is a continuation of U.S. Ser. No. 14/984,719 filed Dec. 30, 2015, which is incorporated herein by reference in its entirety. 
    
    
     TECHNICAL FIELD 
     This disclosure relates generally devices for analyzing substances using thermochromic sensing and to related systems and methods. 
     BACKGROUND 
     Susceptibility testing is performed to determine the effectiveness of a substance to inhibit the growth or cause the death of a live substance, e.g., bacteria, fungi, etc. In some cases, the goal of susceptibility testing is to predict the success or failure of antibiotic or other drug therapy. Tests are performed in a test vessel to determine the growth or lack thereof of a particular microbe to various drug types, drug combinations, and/or drug concentrations. Susceptibility testing is generally performed under controlled conditions and may be used to identify the most effective drug type, combination, and/or dosage to treat an infection caused by a particular type of bacteria, for example. 
     Susceptibility testing for antibiotic testing can involve growing a secondary culture of bacteria from a primary culture obtained from a patient. Currently, culturing the bacteria involves many replication cycles before a measurable effect of the drug being tested can be detected. It is desirable to shorten the time required for susceptibility testing so that an appropriate therapy can be quickly delivered to a patient. 
     BRIEF SUMMARY 
     Some embodiments are directed to a device comprising a test vessel having one or more test locations configured to contain a medium suitable for culturing a live substance. A thermochromic material is thermally coupled to the one or more test locations. The thermochromic material is configured to exhibit a spectral shift in light emanating from the thermochromic material in response to an increase or decrease in energy conversion by the live substance that causes a change in temperature of the thermochromic material. 
     In some embodiments, a test vessel, e.g., a test plate, includes one or more test wells configured to contain a medium suitable for culturing a live substance. A coating is thermally coupled to the test wells. The coating comprises a thermochromic material configured to exhibit a spectral shift in light emanating from the thermochromic material in response to an increase or decrease in energy conversion by the live substance, which causes a change in temperature of the thermochromic material. 
     Some embodiments are directed to a test kit, comprising a sterile test vessel having multiple test locations. A medium suitable for culturing a live substance is contained by the test vessel at the test locations. A thermochromic material is thermally coupled to the test locations. The thermochromic material is configured to exhibit a spectral shift in light emanating from the thermochromic material in response to an increase or decrease in energy conversion by the live substance that causes a change in temperature of the thermochromic material. 
     According to some embodiments a method includes providing a test vessel having one or more test locations configured to contain a medium suitable for culturing a live substance. A thermochromic material is disposed so that it is thermally coupled to the one or more test locations. The thermochromic material configured to exhibit a spectral shift in light emanating from the thermochromic material in response to an increase or decrease in energy conversion by the live substance which causes a change in temperature of the thermochromic material. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG.  1    is a plan view of a thermochromic sensing test vessel in accordance with some embodiments; 
         FIG.  2    is a cross sectional view of the test vessel of  FIG.  1   . 
         FIG.  3    is a cross sectional view of a test plate that includes a thermochromic material disposed in a layer that extends across the test plate in x and y directions near the bottoms of several test wells in accordance with some embodiments; 
         FIG.  4    is a cross sectional view of a test vessel comprising a number of locations configured to contain a medium for culturing at least one live substance with thermochromic material disposed within the test medium in accordance with some embodiments; 
         FIGS.  5  and  6    depict cross sectional diagrams of a test vessels that include locations that contain a medium suitable for culturing a live substance within an area on a relatively flat substrate in accordance with some embodiments; 
         FIG.  7    is a flow diagram illustrating a process for making a thermochromic sensing test vessel in accordance with some embodiments; 
         FIG.  8    is a cross sectional view of a thermochromic sensing test vessel configured for identification of a live substance. 
         FIG.  9 A  shows a block diagram of a thermochromic temperature sensing test system in accordance with some embodiments; 
         FIG.  9 B  shows a diagram of a portion of a thermochromic temperature sensing test system that includes two color channels in accordance with some embodiments; 
         FIG.  10 A  conceptually illustrates a wavelength shift detector that can be used to determine the existence and/or amount of shift in the spectrum of light emanating from a thermochromic material in accordance with some embodiments; 
         FIG.  10 B  conceptually illustrates a wavelength shift detector that detects both reflected and transmitted light in accordance with some embodiments; 
         FIGS.  11 A and  11 B  are flow diagrams illustrating a thermochromic testing process in accordance with some embodiments; 
         FIGS.  12 A and  12 B  are flow diagrams illustrating processes for thermo-optical antimicrobial susceptibility testing (TOAST) using thermochromic sensing in accordance with some embodiments; 
         FIGS.  13 A through  13 C  are flow diagrams illustrating processes for bacteria identification and thermo-optical antimicrobial susceptibility testing using thermochromic sensing in accordance with some embodiments; 
         FIG.  14 A  shows graphs that illustrate the simulated change in temperature ΔT (K) with respect to time for a growing  E. coli  colony with no antibiotic and with a minimum inhibitory concentration of antibiotic over a the range of thermionic sensing using the wavelength shift detector discussed in  FIG.  10 A ; and 
         FIG.  14 B  shows a portion of the graphs of  FIG.  14 A  corresponding to the first 20 minutes of colony growth and indicating the measurement resolution achievable using the wavelength shift detector discussed in  FIG.  10 A . 
     
    
    
     The figures are not necessarily to scale. Like numbers used in the figures refer to like components. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. 
     DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 
     Thermochromism is the change in color of a material based on temperature. The color change of thermochromic materials can be relatively discrete and abrupt, or can vary gradually over a temperature range. The spectral changes may be evident in light that is scattered, reflected, absorbed, and/or fluoresces from the thermochromic material. Thermochromic materials may be organic or inorganic substances and/or may be monomers or polymers. Of particular interest for the approaches of the present disclosure are thermochromic liquid crystals, which exhibit thermochromism based on light reflectance. 
     The approaches described herein involve temperature sensing using thermochromic material to optically indicate temperature changes caused by energy conversion of a live substance. A non-limiting list of live substances that can be monitored using the thermochromic sensing techniques described herein include one or more of bacteria, archea, protists, fungi, plant cells, animal cells, viruses in appropriate host cells, phages in appropriate host cells, cancer cell cultures, and tissue cell cultures. The rate of energy conversion of the live substance can be related to the metabolism of the ensemble of live cells. In particular the number of cells increasing due to cell mitosis is a form of increase in ensemble metabolism. The metabolism of the individual cells combined comprises the ensemble metabolism. Metabolism often includes the oxidation of glucose or other carbohydrates to release energy and chemical byproducts. In this context metabolism is meant to be the mechanism through which chemical energy is converted into other forms of energy, including heat. Heat in turn can cause a temperature change of the substance or live matter that performs the metabolism. Temperature change of live matter will result in temperature change, normally a temperature increase, of the surrounding material including, cell culture medium, buffer material, vessel material and thermochromic material. The amount of heat transfer from one material to the next is dominated by material properties. Therefore, it is possible to control the heat transfer by material choice. It is desirable that the heat generated by metabolism is isolated from transfer into the vessel material and instead thermally connected or coupled to the thermochromic material. Heat generated by metabolism results preferably in a temperature change of the thermochromic material. An increase in ensemble metabolism can be described as “growth rate” of the live substance and/or to an increase or decrease in an amount of the live substance. A positive growth rate indicates healthy living condition for cells and it often corresponds to an increase in the number of live cells within the ensemble. The thermochromic sensing devices, systems, and methods disclosed herein can be used to detect and/or monitor growth of live substances and are particularly useful in determining the efficacy of various pharmaceutical agents, e.g., drug types, drug dosages, and drug combinations, such as anti-microbial, anti-viral, and/or anti-fungal drugs. 
     A spectral shift can occur in any kind of emission, absorption, fluorescence, reflection, or transmission, or any other light spectrum. A spectral shift in a light spectrum can be described as the difference between centroids of two light spectra. The wavelength shift may be determined by determining a measured centroid position with an implicit centroid position, determined in for example a calibration measurement or a nominal centroid position. The wavelength shift may be determined by comparing two different centroids of two different spectra effectively simultaneously to perform a referenced wavelength shift measurement. Light spectra, or light intensity spectra may be measured in various measurement unit. Commonly, the varying parameter of the spectrum (i.e. Abscissa) is the photon energy, often measured in wavelength. In such a measurement the wavelength shift can be measured in wavelength units, for example nanometers (nm). For certain emission spectra, in particular emission peaks or Gaussian emission profiles, the peak wavelength is a good approximation of the centroid position or the difference of peak positions relative to one another is a good approximation of wavelength shift. In practical measurements the centroid determination may be influenced by measurement parameters that may vary over the wavelength shift detection range so that there are additional measurement factors that are contributing to centroid measurements, for example wavelength dependent sensitivity of detectors. These measurement influences can be considered as systematic errors of the measurements and are often compensated for by calibration. Any such error, even if it is not compensated for, should be considered as part of the centroid, wavelength or wavelength shift measurement. It is noteworthy that emission spectra may consist of for example two relatively discrete emission distributions with two emission maxima. The centroid of these combined emission spectra can still be calculated and measured, a wavelength shift can still be calculated for such a spectrum. In particular, if two fluorescence emission spectra are used in such a way that one of the emission spectra changes the emission intensity with temperature then temperature changes result in a wavelength shift of the overall spectrum. 
       FIG.  1    and  FIG.  2    are plan and cross sectional views, respectively, of a thermochromic sensing test vessel  100  in accordance with some embodiments. The test vessel  100  which may be a substantially planar test plate, includes one or more locations  101  configured to contain a medium  140  for culturing at least one live substance  150 . In some embodiments, the test locations may be test wells which are recessed locations on a test plate, for example. Although the test vessel  100  may be any type of vessel or structure configured to contain a medium, in some implementations, the test vessel  100  is a MICROTITER test plate, such as a standard 24-well MICROTITER plate, a standard 96-well MICROTITER plate, a standard 384-well MICROTITER plate, or a standard 1536-well MICROTITER plate, etc. In implementations where the test vessel  100  is a test plate, the test wells of the test plate provide the locations  101  configured to contain the medium for culturing the live substance  150 . Each test well has walls  101   a  and a bottom  101   b  to contain the medium  140 . In some implementations, the test vessel  100  may include a cover  102  that covers the test wells and/or seals the medium within the test wells. 
     Although the test vessel  100  may be any type of vessel or structure configured to contain a medium, in some implementations, the test vessel  100  may maintain standard MICROTITER plate pitch distances for fluidic handling, such as a standard 24-well MICROTITER plate pitch distances, a standard 96-well MICROTITER plate pitch distances, a standard 384-well MICROTITER plate pitch distances, or a standard 1536-well MICROTITER plate pitch distances, etc. This could for example mean that test vessels could be loaded with standard MICROTITER fluidic handling tools (e.g. multiplexed pipettes) by using a compatible MICROTITER fluidic interface but the samples are afterwards routed into any other appropriate position that does not necessarily have to be compatible with the MICROTITER standard, for example a single row of test wells, for example 24, 96, 384, or 1536 wells. 
     At least one type of thermochromic material  110  is thermally coupled to the one or more test locations. In various embodiments, the thermochromic material may be disposed at the test locations, e.g., in, on, and/or about the test wells  101 . The thermochromic material  110  is thermally coupled to the medium  140  and/or live substance  150  contained within the test locations. The thermochromic material  110  is configured and arranged so that it exhibits a spectral shift in light, e.g., scattered, reflected, or fluorescent light, from the thermochromic material  110  in response to a temperature change of the live substance  150  and/or medium  140  due to energy conversion by the live substance  150 . The thermochromic material  110  is positioned to be sufficiently close and thermally coupled to the live substance  150  so as to be sensitive to changes in temperature due to energy conversion of the live substance  150 . 
     At least one type of thermochromic material  190  is disposed near test locations, such that the thermochromic material  190  is thermally coupled to the surrounding environment of test wells  101 . In some embodiments, the thermochromic material  190  is a coating of thermochromic material thermally coupled to the test locations, e.g., a thermochromic coating disposed at the bottom of the test wells. 
     At least one type of thermochromic material  191  is disposed on the test plate, in order to monitor a larger temperature range than the test wells. This temperature sensing region is not significantly influenced by the amount of energy conversion of the live substance in any of the test wells, rather it will track the temperature development of the test plate once the plate is moved into the incubator and the test plate temperature is approaching nominal temperature conditions. Additionally, proper incubator functioning can be tracked or controlled with this read out. 
     In individual locations  101  or the entirety of the test vessel  100  may be include a cover  102 , which may comprise a lid and/or seal, e.g., sealing film. One type of sealing film is a breathable sterile membrane (e.g. Corning microplate sealing tape white Rayon (with acrylic) or Thermo Scientific Gas Permeable Adhesive Seals). This sealing film is placed directly on the test vessel  100  to provide a sterile barrier over which the cover  102  (for example, non-sterile plastic) can then be placed. 
     Another embodiment uses a sterile non-breathable adhesive seal (e.g. E&amp;K Scientific SealPlate Adhesive Microplate Seals) as the lid  102  that covers the vessel. This type of film provides an air-tight seal and thus does not require another lid on top of the sealing film. For anaerobic bacteria, the cover  102  can be used to provide a barrier to exclude O2. Filling the test locations with media and using an air-tight seal will enable growth of anaerobic bacteria. 
     As illustrated in the cross sectional view of  FIG.  2   , the thermochromic material  110  may be a coating of thermochromic material  110  disposed along the walls  101   a  and/or bottom  101   b  of each individual test well  101 . The thermochromic material may be a layer  111 , e.g., a continuous layer, that extends across the test plate in x and y directions near the bottoms  101   b  of several test wells  101  as illustrated in the cross sectional view of  FIG.  3   . 
     The spectral shift in light emanating from the thermochromic material  110  (see  FIG.  2   ),  111  (see  FIG.  3   ), such as the spectral shift of the reflected, scattered, transmitted, and/or fluorescent light, can be detected using one or more optical detectors. The optical detectors may be located at any position relative to the test vessel where the light emanating from the thermochromic material is detectable. For example, in some embodiments, the detector may be positioned above, below, and/or along the walls  101   a  of the test wells  101 . 
     In some embodiments, the reflected, scattered, transmitted, and/or fluorescent light emanating from the thermochromic material  110 , is relayed onto the optical detector by appropriate optical components  180  such as lenses, objective lenses, lens combinations, imaging optics, plane-, concave-, convex-mirrors, fibers, gratings, prisms, and other elements. The optical components may maintain image information or not. 
     In some embodiments, the reflected, scattered, transmitted, and/or fluorescent light emanating from the thermochromic material  110  derives from measurement light that is ambient light, e.g., from sunlight, room light, etc., which encounters the thermochromic material  110 ,  111  and is scattered, transmitted, reflected or absorbed by the thermochromic material  110 ,  111 . In some embodiments, at least one light source  195 ,  196  is used to emit and to direct the measurement light  195   a ,  196   a  toward the test wells  101  such that the measurement light  195   a ,  196   a  encounters the thermochromic material  110 ,  111 . 
     In some embodiments, the thermochromic material  110 ,  111  reflects a portion of the measurement light  195   a ,  196   a .  FIGS.  2  and  3    show reflected light  198   a ,  199   a  that can be detected by detectors  198 ,  199  positioned above and/or below the bottom of the test wells  101 . In some embodiments, the thermochromic material  110 ,  111  absorbs a portion of the measurement light  195   a ,  196   a , the absorption of the measurement light  195   a ,  196   b  causing the thermochromic material  110 ,  111  to fluoresce.  FIGS.  2  and  3    indicate fluorescent light  198   b ,  199   b  that can be detected by one or more detectors  198 ,  199  positioned above and/or below the test wells  101 . In some embodiments, a portion of the measurement light  195   a ,  196   a  is scattered by the thermochromic material  110 ,  111 . The scattered light  198   c ,  199   c  can be detected by one or more detectors  198 ,  199  positioned above and/or below the test wells  101 . In some embodiments, a portion of the measurement light  195   a ,  196   a  is transmitted by the thermochromic material  110 ,  111 . The transmitted light  198   d ,  199   d  can be detected by one or more detectors  198 ,  199  positioned above and/or below the test wells  101 . In some embodiments, the measurement light may be transmitted to the thermochromic material by a waveguide, e.g., an optical fiber or polymer waveguide. In some embodiments the reflected, scattered, transmitted or fluorescent light emanating from the thermochromic material may be transmitted to the detector through a waveguide. In some embodiments the waveguides may be integrally formed in the test vessel for transmitting measurement light and/or light emanating from the thermochromic material. In some embodiments the reflected, scattered, transmitted or fluorescent light emanating from the thermochromic material may be transmitted to the detector through a lens that is integral to the well plate structure, for example formed during the injection molding of the test plate. 
     In some embodiments, at least a portion  100   a  of the test plate  100  in the region of the test wells  101  is substantially optically transmissive at the wavelengths of the measurement light  196   a  and/or at the wavelengths of the reflected  199   a , scattered  199   b , transmitted  199   d , and/or fluorescent light  198   c  emanating from the thermochromic material  110 . Substantially optically transmissive means that the transmittance of light at the wavelengths of the measurement light and/or the light emanating from the thermochromic material is greater than 50%. In some embodiments the reflected, scattered, transmitted or fluorescent light emanating from the thermochromic material may be transmitted to the detector through a flat transparent bottom, for example glass, polypropylene, polystyrene, polycarbonate or quartz. 
     The energy conversion of the live substance results in a temperature increase of the thermochromic material at the test location. These temperature increases are sub-Kelvin, and may be less than about 1 milliKelvin (mK). Temperature changes may depend on a variety of factors, such as test volume, number of live cells, ambient temperature, thermal insulation of test volume, buffer conditions etc. As discussed herein, thermochromic materials can be used to optically indicate the temperature of the test vessels. Thermochromic materials can show a variety of optical effects such as temperature dependent fluorescence intensity or temperature dependent reflection or scattering spectra. In particular, thermochromic liquid crystals show very strong temperature dependent reflection spectra. 
     The thermochromic material used for thermochromic temperature sensing of the live substances may comprise any suitable type of thermochromic material such as thermochromic liquid crystals, leuco dyes, fluorophores, Prodan bound to DPPC, and/or a fluorescent proteins. In thermochromic liquid crystals, the spectral changes result from temperature-dependent intermolecular spacing. For example, monitoring a specific selected reflectance from a thermochromic liquid crystal surface has shown up to a 13,000% change in intensity per K in a ratiometric color measurement or a wavelength shift of hundreds of nm/K up to about 1000 nm/K. 6-propionyl-2-(dimethylamino)naphthalene (Prodan) bound to ipalmitoylphosphatidylcholine (DPPC) shows a fluorescent emission shift of 6 nm/K between 40° C. and 50° C. Green fluorescence protein, which shows a shift in emission wavelength by about 0.3 nm/K, is an example of a thermochromic material that could be optimized genetically/biologically for thermochromic temperature sensing, e.g., optimized for pharmaceutical susceptibility testing and/or other monitoring of the growth/decline of live substances. 
     Changes in fluorescence intensity of some thermochromic materials can be particularly sensitive to temperature (over 100% per degree in some cases). In some scenarios, thermochromic temperature sensing can be further enhanced by comparing the response of two different types of thermochromic materials with differing temperature responses and monitoring the change in intensity ratio between the two emission peaks from the two thermochromic materials. In some cases the two thermochromic materials are chosen such that one material shows a temperature dependent fluorescence intensity change, and the other is either independent of temperature, or has a change that is opposite to the first material. 
     As a non-limiting example, a thermochromic liquid crystal having a wavelength shift of about 1000 nm/K would exhibit a wavelength shift of about 10 picometer (pm) when subjected to a temperature change of about 10 pK due to energy conversion by a live substance. In some implementations, a 1.6×10 −6  K−1.6×10 −5  K change in temperature due to energy conversion would result in a 1.6-16 picometer (pm) wavelength shift. In some embodiments, the thermochromic material may be configured to exhibit a spectral shift in the fluorescence, reflectance, or scattering spectrum with temperature in a range of about 0.5 nm/K to about 1000 nm/K. 
     In some configurations, one or more optional additional layers or coatings can be disposed along one or both major sides of the thermochromic material layer. In some embodiments, the optional additional layers may extend along the bottom  101   b  and/or walls  101   a  of the test wells  101 . For example, one or more optional additional layers  120 ,  121 ,  130 ,  131  can be positioned between the thermochromic material coating  110 ,  111  and the medium  140  and/or live substance  150  within each test well  101 , as shown in  FIGS.  2  and  3   . In some implementations, at least one of the optional additional layers  130  may be a light absorbing layer. The use of a light absorbing layer positioned between the thermochromic material  110 ,  111  and the medium  140  and/or live substance  150  can enhance sensitivity of the thermochromic sensing due to the absorbing properties of the layer. Light that is not reflected, scattered, absorbed by the thermochromic material coating  110 ,  111  now does not contribute to the reflected, scattered or fluorescence light detection. The use of a light blocking layer may enhance the signal to noise ratio of thermochromic sensing by reducing the component of the detector signal produced by non-signal light detected by the detector, wherein non-signal light is light other than light emanating from the thermochromic material. 
     In some implementations, at least one of the optional additional layers  130 ,  121  may be a heat conducting layer. The use of a heat conducting layer positioned between the thermochromic material  110 ,  111  and the medium  140  and/or live substance  150  can enhance sensitivity of the thermochromic sensing due to an improved heat conductivity from the medium  140  and/or live substance  150  to the thermochromic material coating  110 ,  111 . Energy converted by the live substance  150  results in heat generation within the medium  140  and thereby in a temperature increase of the medium and/or live substance  150 . A temperature difference between the medium and the ambient surrounding will result in a temperature gradient in the transition zone. As the thermochromic material is part of the transition zone, it is beneficial if a heat conducting layer ensures the heat transfer from the medium to the thermochromic layer so that both ideally have the same temperature. For example, the heat conducting layer may consist of indium tin oxide (ITO), metal, diamond, zinc oxide, graphene, graphite, and indium phosphide. 
     In some implementations, at least one of the optional additional layers  131 ,  120  may be a heat insulation layer. The use of a heat insulation layer positioned between the thermochromic material  110 ,  111  and the base material of the test vessel structure can enhance sensitivity of the thermochromic sensing due to reduced heat conductivity from the thermochromic material  110 ,  111  to the ambient equilibrium temperature. It is desirable to have the base material of the test vessel structure itself be made of low heat conductivity material. 
     In some embodiments, at least one of the optional additional layers  121 ,  130 , may be a sterile coating positioned to separate the thermochromic material  110 ,  111  from the medium  140 . For example, the thermochromic coating  110 ,  111  may be disposed along the bottom surface of the test wells with the sterile biocompatible coating disposed over the thermochromic coating so that the thermochromic coating is between the bottom surface of the test well and the sterile coating. For example, the sterile coating may comprise one or more of parylene, indium tin oxide (ITO), metal, polyethylene glycol (PEG), diamond, zinc oxide, graphene, graphite, and indium phosphide. Ideally these coatings are also biocompatible. 
       FIG.  4    illustrates a test vessel  400  comprising a number of locations  401  configured to contain a medium  140  for culturing at least one live substance  150 . In some embodiments, the thermochromic material, e.g., thermochromic particles or regions  410 , are disposed within the test medium  140  as depicted in the cross sectional diagram of  FIG.  4   . 
     The spectral shift in light emanating from the thermochromic material  410 , such as the spectral shift of the reflected, scattered, transmitted, and/or fluorescent light, can be detected using one or more optical detectors. The optical detectors may be located at any position relative to the test vessel where the light emanating from the thermochromic material is detectable. For example, in some embodiments, the detector  198 , 199  may be positioned above and/or below the test wells  401  as illustrated in  FIG.  4   . 
     In some embodiments, the reflected, scattered, transmitted, and/or fluorescent light emanating from the thermochromic material derives from measurement light that is ambient light, e.g., from sunlight, room light, etc., which encounters the thermochromic material  410 . In some embodiments, at least one light source  195 ,  196  is used to emit and to direct the measurement light  195   a ,  196   a  towards the test wells  401  such that the measurement light  195   a ,  196   a  encounters the thermochromic material  410 . 
     In some embodiments, a portion of the measurement light  195   a ,  196   a , is reflected by the thermochromic material  410 . The reflected light  198   a ,  199   a  can be detected by photo sensing elements  198 ,  199  positioned above and/or below the bottom of the test wells  401 . 
     In some embodiments, a portion of the measurement light  196   a ,  196   a  is absorbed by the thermochromic material  410  and causes the thermochromic material  410  to fluoresce. The fluorescent light  198   b ,  199   b  can be detected by one or more photo sensing elements  198 ,  199  positioned above and/or below the test wells  401 . 
     In some embodiments, a portion of the measurement light  195   a ,  196   a  is scattered by the thermochromic material  410 . The scattered light  198   c ,  199   c  can be detected by one or more photo sensing elements  198 ,  199  positioned above and/or below the test wells  401 . 
     In some embodiments, at least a portion  400   a  of the test plate  400  in the region of the test wells  401  is substantially optically transmissive at the wavelengths of the measurement light  196   a  and at the wavelengths of the reflected  199   a , scattered  199   b , and/or fluorescent light  198   c.    
     In some configurations, one or more optional additional layers or coatings  420  can be disposed along the bottom of the test well  401  or elsewhere, e.g., along the walls  401   a  of the test well  401 . In some embodiments, the optional additional layers may extend both along the bottom  401   b  and walls  401   a  of the test wells  401 . In some implementations, at least one of the optional additional layers  420  may be a heat insulating layer. The heat insulating layer can be designed to enhance sensitivity of the thermochromic sensing due to the reduced heat transfer from the test location  401  to the sacrificial material of the test vessel  400  or the surrounding. 
     In some implementations, at least one of the optional additional layers  420  may be a light blocking layer. The use of a light blocking layer may enhance the signal to noise ratio of thermochromic sensing by reducing the component of the detector signal produced by non-signal light, wherein non-signal light is light other than light emanating from the thermochromic material. 
       FIGS.  5  and  6    depict cross sectional diagrams of a test vessel  500 ,  600  that includes locations  501 ,  601  configured to contain a medium  540  suitable for culturing one or more live substances  560 . In these embodiments, medium  540  may be contained within an area on a relatively flat substrate  500   a . In some embodiments, the locations  501 ,  601  may be defined by surface treatments or coatings,  505 , e.g., a hydrophobic surface treatment configured to contain the medium within the locations  501 ,  601 . As shown in  FIG.  5   , the thermochromic material may be a layer  510  disposed on the substrate  500   a  at the locations  501 . 
     In individual locations  501  or the entirety of the test vessel  500  may be covered with a cover  570 , e.g., comprising a seal and/or lid. In some embodiments, the test vessel is covered with a sealing film with or without an additional lid. Some embodiments use a protective lid with or without a seal. The cover  570  reduces heat loss due to evaporation and helps to maintain an appropriate environment within the test vessel  500  at the test locations  501 . For example, in some embodiments mammalian cells are disposed at the test locations which need a certain head volume that contains the appropriate gas atmosphere, e.g., 5% CO2. As another example, anaerobic bacteria are disposed at the test locations and the cover provides a barrier that helps to exclude O2 which is toxic to these bacteria. Thus, filling the test locations with media and using an air-tight seal enables the growth of anaerobic bacteria. 
     One type of seal is a breathable sterile membrane (e.g. Corning microplate sealing tape white Rayon (with acrylic) or Thermo Scientific Gas Permeable Adhesive Seals). This sealing film is placed directly on the test vessel to provide a sterile barrier over which the lid (for example, non-sterile plastic) can then be placed. 
     Another embodiment uses a sterile non-breathable adhesive seal (e.g. E&amp;K Scientific SealPlate Adhesive Microplate Seals) to cover the vessel. This type of film provides an air-tight seal and thus does not require another lid on top of the sealing film unless the lid is desired or needed for additional protection. 
     As shown in  FIG.  6   , the thermochromic material may be thermochromic regions  610  within the medium  540 , e.g., thermochromic particles embedded within the medium. 
     The test vessel  500 ,  600  may include one or more optional additional layers  520 ,  530 ,  620  disposed above and/or below the thermochromic material as discussed above. For example, the additional optional layers  520 ,  530 ,  620  may comprise one or more of a heat absorbing layer, a light blocking layer and a sterile biocompatible layer. Optionally, as discussed above, the test vessel includes a cover  570 , e.g. a seal and/or lid. 
       FIG.  7    is a flow diagram illustrating a process for making a thermochromic sensing test vessel in accordance with some embodiments. The process includes providing  710  a test structure and disposing  720  a thermochromic material thermally coupled to test locations of the test structure. For example, in some embodiments, where the test structure is a standard MICROTITER test plate, the thermochromic material may be disposed by coating the test wells of the standard test plate with one or more thermochromic materials. For example, the bottom and/or walls of all of the test wells of a standard plate could be coated. In some embodiments the bottom and/or walls of some test wells may be coated with thermochromic material, whereas other test wells are left uncoated. In some embodiments, the thermochromic material may be disposed at the test locations by placing thermochromic particles into a medium contained by the test wells and/or by placing a medium that contains thermochromic material within the test wells. 
     In some embodiments additional functional material layers may be disposed  730  on the test structure, for example, heat conducting layers, light blocking layers, thermal insulation layers. The additional functional layers may be disposed before or after the thermochromic material is disposed at the test locations. Subsequently sterilizing the test structure  740  may be accomplished by one or more of the following methods: heat, chemicals, or irradiation. 
     Heat sterilization may be achieved using either moist heat (steam) or dry heat. Chemicals may be used to sterilize heat-sensitive materials including many plastics. Either gases or liquids may be used. Gases used for chemical sterilization include ethylene oxide (EtO), nitrogen dioxide (NO 2 ) or ozone. Liquid chemical sterilization may be achieved using glutaraldehyde, formaldehyde, hydrogen peroxide (H 2 O 2 ), or peracetic acid. Radiation sterilization may be achieved using electron beams, X-rays, gamma rays, or irradiation by subatomic particles. 
     In some embodiments the sterilized test structure is packaged and sealed in such ways that the content of the package remains sterile until the mechanical integrity of the package is compromised, either inadvertently or deliberately. Normal deliberate opening maintains a sterile test plate and allows filling the test vessels exclusively with the live matter from the intended sample. 
     One or more test substances, e.g., a pharmaceutical, antimicrobial, antifungal substance, may be contained within the medium. Different locations of the test vessel, e.g., test wells  101  of the test vessel  100 , may include different types, combinations, and/or concentrations of test substances  160  wherein the live substance  150  is the same at each test location. This test set up can be used to monitor the effect of the different types, combinations, and concentrations of the test substance on a live substance. In some embodiments the type, combination, and/or concentration of the test substance  160  may be substantially the same at a number of the test locations, and the live substance may vary. This test set up can be used to test the effect of the same type, combination, and concentration of the test substance on different types of live substances. 
     In some implementations, the thermochromic sensing test vessel is used for pharmaceutical, e.g., antimicrobial susceptibility testing (AST). The test substance  160  comprises one or more types of antibiotic and the test locations contain different types, different combinations, and/or different concentrations of antibiotic. Examples of antibiotics and combinations of antibiotics suitable for use in AST include, but are not limited to: Amikacin, Amoxicillin/Clavulanic Acid, Ampicillin, Ampicillin/Sulbactam, Azithromycin, Aztreonam, Cefalotin, Cefazolin, Cefepime, Cefoxitin, Ceftazidime, Ceftriaxone, Cefuroxime, Cephalothin, Chloramphenicol, Ciprofloxacin, Clarithromycin, Clindamycin, Daptomycin, Doripenem, Ertapenem, Erythromycin, Gatifloxacin, Gentamicin, Imipenem, Levofloxacin, Meropenem, Moxiflaxacin, Nalidixic Acid, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Penicillin, Piperacillin, Piperacillin/Tazobactam, Rifampin, Sulfamethoxazole, Synercid, Tetracycline, Ticarcillin, Ticarcillin/Clavulanic Acid, Tigecycline, Tobramycin, Trimethoprim, Trimethoprim/Sulfamethoxazole and Vancomycin. 
     In some implementations, illustrated by  FIG.  8   , the thermochromic sensing test vessel  100  is used for identification of the live substance  150 . The test substance  860  comprises one or more types of substrates to measure carbon source utilization (e.g. mannitol, glucose, lactose, maltose, citrate, acetate, acetamide), enzymatic activity (e.g. catalase, oxidase, coagulase, pyrase, urease, decarboxylase, dihydrolase, phenylalanine deaminase, cysteine desulfurase (H2S production), tryptophanase (indole production)), or resistance (e.g. bacitracin, novobiocin, optochin). The growth medium may contain an indicator substance ( 861 ) in addition to the test substance  860 . Examples of indicator substances include, but are not limited to: bromothymol blue, ferric ammonium citrate, bromocresol purple, ferric chloride, ferrous sulfate, 4-Dimethylaminobenzaldehyde, and methyl red. In some embodiments, the test substance  860  used for the identification of the live substance directly produces a fluorescent or chromogenic compound when incubated in the presence of the appropriate live substance  150 . 
     In other embodiments, the indicator substance  861  produces a fluorescent or chromogenic compound when the test substance  860  is incubated in the presence of the appropriate live substance  150 . In addition to measuring the response of the thermochromic material to the growth of the live substance in the presence of the test compound, the fluorescence or absorbance resulting from incubation of the live substance  150  in the presence of the test compound  860  can be measured using one or both of light sources  195 , 196  and using the detectors  198 ,  199  and/or additional light sources and/or detectors positioned above or below the test locations. 
     Combinations of enzyme substrates, growth promotors and growth inhibitors as measured by the TOAST mechanism or other optical means gives a metabolic or other biochemical profile that may be used for identification of live matter. 
     In cases of bloodstream infections, for example, the AST may be performed following isolation and identification of the live substance from a positive blood culture. The identification step may be performed a using the thermochromic sensing test vessel as described above. In other implementations, the live substance may be identified using another method such as standard growth and biochemical characteristics or rapid identification methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In another implementation, the AST may be initiated prior to identification of the live substance, relying on the Gram stain results of the positive blood culture to select the appropriate panel of test compounds to use in the AST. 
     In some embodiments, the test vessel can be designed as a one-use disposable component. In some embodiments, the thermochromic sensing test vessel may be part of a kit that includes the sterile thermochromic sensing test vessel, e.g., as discussed in connection with  FIGS.  1 - 6   . In some implementations, the thermochromic sensing test vessel of the kit can be pre-loaded with medium and test substance. In some implementations the test substance comprises different types, amounts and/or combinations of pharmaceuticals or other agents that are preloaded at different test locations of a test vessel. In some scenarios, the test laboratory receiving the kit would insert the live substance at the test locations, test of the efficacy of the types, amounts and/or combinations of the test substance preloaded at each of the test locations, and then dispose of the kit after the testing is complete. In some cases, some of the test locations may be used as control locations, wherein the test substance and/or the live substance is not inserted at the control test locations. 
     The test vessel may be configured to be removably inserted into a compartment of a system that facilitates automatic testing of test substances using thermochromic sensing. In some implementations, the test vessel may be configured as a cartridge with mechanical holding features that engage with compatible features of the compartment.  FIG.  9 A  shows a block diagram of a thermochromic temperature sensing test system  900  in accordance with some embodiments. In some embodiments the test system  900  includes an incubator  905  configured to automatically control the ambient environment, e.g., light, temperature, humidity, gas composition, CO 2  concentration, etc. of the test vessel  902  and/or other components of the system during testing. In test systems where the environment of the test vessel is uncontrolled or where additional thermal compensation is needed or desired, circuitry  930  configured to account for variation in temperature may be used. Temperature compensation circuitry  930  may comprise a temperature sensor coupled to compensation circuitry configured to compensate for temperature effects and therefore spectral shift of the thermochromic material that are caused by factors other than energy conversion by the live substance. These temperature effects may be caused by room temperature fluctuation and they may be larger than the maximum temperature measurement range of the optical temperature measurement range used for individual test wells  101 . Therefore, the temperature of individual, groups of individual or all test wells can be adjusted with temperature compensation circuitry  930 . Temperature compensation circuitry  930  may contain heaters, coolers, resistive heaters, radiative heaters, heat exchangers, water supply, thermoelectric coolers, Peltier elements, evaporative coolers, temperature sensors, thermistors, thermo couplers and optical temperature read out sensors that are based on wavelength shift detection of thermochromic material (for example 191 and 190). 
     In some embodiments the test vessel  902  includes fluidic channels  185   b  (shown in  FIG.  1   ) fluidically coupled to the test locations  101  so that a thermally equilibrated liquid, e.g., predominantly water based, potentially with the addition of disinfectant agents, from the host incubation and read-out system  900  can be connected to the test vessel  902 . The fluidic channels  185   b  can allow a fluid to be introduced into heat exchange regions of the test vessel near the test locations and/or control locations. Mass flow of the thermally equilibrated liquid into the test vessel  902  will bring the device temperature, including the content of the test sites  101  to a thermal equilibrium in a faster and more stable way than for example heat exchange by circulating gas or purely radiative heat exchange. 
     In some embodiments the test vessel  902  includes fluidic channels  185   a  (shown in  FIG.  1   ) that fluidically coupled to the test locations  101  so that the test substance can be filled into several or all test location through these fluidic channels. 
     As indicated by  FIG.  9 A , the test vessel  902  can be configured as a cartridge that may be removably inserted into a compartment  901  of the test system  900 . The test vessel  902  includes mechanical features  902   a , e.g., protrusions, which engage with mechanical features  901   a , e.g., slots, of the compartment to mechanically position and retain the test vessel  802  within the compartment  901 . Additionally, the test vessel  902  can bear unique markings or identifiers that can be either read by humans, e.g. alphanumerical combinations, serial number or names, or by appropriate machines, e.g. bar codes or QR codes. The test vessel  902  can also be marked with alignment markings that define measurement test regions, for example a blue ring around each thermochromic material region  110  (see  FIG.  2   ),  111  (see  FIG.  3   ) for each test vessel  101 , lines forming a coordinate system, alignment crosses in several positions on the test vessel. Especially, if a camera systems (RGB, differential illumination, hyperspectral, dichroic mirror multiplexed cameras, etc) serve as the multiplexed readout of thermochromic materials, the markings on the test vessel may indicate the regions of interest (ROI), i.e. the test locations  101  for automated readout. The markings may also imply the regions of interest by providing a coordinate system. The regions of interest would then be located at known, pre-defined coordinates. 
     The system  900  can include a measurement light source  910  configured to generate and direct measurement light toward the test locations of the test vessel  902 . The light source  910  includes a light emitter, e.g., a light emitting diode (LED), a lamp, and/or laser, configured to emit the measurement light and components configured to cause the measurement light be directed to the test locations of the test vessel. In some implementations the measurement light is optically multiplexed or directed to the multiple test locations by scanning the measurement light across the test locations of the test vessel, for example by scanning mirrors or rotating mirrors or mirror arrays (digital light processing) or by acousto-optical modulators or by phased array optics. In some implementations, the measurement light scanning may be implemented by directing the light produced by a stationary measurement light emitter across multiple test locations, e.g., using a lens and/or mirror array. In some embodiments, scanning the measurement light across the test locations may be implemented by physically moving the light source and test vessel relative to each other. In some embodiments, the measurement light may be directed to the test locations through an optical waveguide. In some embodiments, the measurement light may reach a subset of test regions of interest or the measurement light may reach all test regions simultaneously, for example by illuminating the total area of all thermochromic material regions of the test vessel. 
     In some embodiments, the measurement light may include two or more distinct measurement light sources or measurement light characteristics that are individually addressable. For example two or more individually switchable LEDs that exhibit a different spectral emission characteristic could serve as measurement light sources. These light sources could alternatingly probe the reflectivity of a thermochromic liquid crystal in different spectral regimes. A light intensity detector, for example a monochrome camera, could then compare the intensity values of the reflected light spatially resolved for the light spectrum of the first LED and then for the light of the second LED. Thus, in some embodiments LEDs with very different spectral characteristics can be utilized with a monochrome light detector to measure wavelength shifts. Alternatively, a single broad light source (e.g. lamp, LED with phosphor coating, etc.) could be used to provide measurement light and the color discrimination of the reflected, transmitted, or scattered light could be performed with an RGB-camera. The spectral selectivity of RGB-cameras is aimed to represent the color selectivity of the human eye and the choices of color selectivity may therefore be limited when the RGB camera is used as the detector. In some embodiments color sensitive camera systems could be used that sequentially utilize light different filters (e.g. dielectric transmission filters, absorptive transmission filters), or camera systems could be used that use several image sensors and the incoming light is split by color selective elements such as dichroic mirrors as discussed below with reference to  FIG.  9 B . 
     The system  900  includes detector subsystem  920  including one or more optical detectors configured to detect changes in the spectrum of light emanating from the thermochromic material of the test vessel, e.g., reflected, scattered, and/or fluorescent light. The sensors may comprise one or more of a photodiode, a phototransistor, photomultiplier tube, avalanche photo diode, a wavelength shift detector, an RGB camera, a hyperspectral camera, a spectrometer, a spectrograph, a dichroic mirror segmented image sensor, a Fourier spectrometer, and a dichroic mirror segmented sensor. In some embodiments there may be a one-to-one correspondence between the sensors and the test locations. In other embodiments, there may be fewer sensors than test locations and light emanating from a plurality of the test locations is optically de-multiplexed to a single sensor. In some implementations, the optical de-multiplexing may be accomplished by selectively directing the emanating light from each of the plurality of test locations to the sensor during different time periods. e.g., de-multiplexing using moveable mirrors for example scanning mirrors or rotating mirrors or mirror arrays (digital light processing) or by acousto-optical modulators or by phased array optics. In some implementations, the optical multiplexing may be accomplished by physically moving the sensors relative to the test vessel and/or physically moving the test vessel relative to the sensors. The output of the detector subsystem  920  can be provided to an processor  940  configured to detect, analyze, and/or monitor changes in the spectrum of the emanating light. The processor  940  may be configured to analyze results of the testing, and/or to generate reports of the testing results into a format that can be displayed, sent or in any way transmitted to a user, e.g., via a computerized user interface  950 . In some embodiments, the processor  940  may send continuous updates to the user interface  950  as the testing is being performed wherein the user interface continuously updates its display, allowing a user to be quickly apprised of testing results. In some implementations, the processor  940  may be configured to generate an alert signal that is sent to the user interface  950 , wherein the user interface  950  produces an alert, e.g., an auditory and/or visual alert, based on the alert signal sent by the processor  940 . 
       FIG.  9 B  illustrates a portion  960  of the thermochromic temperature sensing test system  900  in accordance with some embodiments. The portion of the system illustrated in  FIG.  9 B  includes two color channels  971 ,  972  that facilitate automatic testing of test substances according to some embodiments. In  FIG.  9 B , a test vessel  962  comprising multiple test locations  962   a  having thermochromic material disposed at the test locations  962   a  is shown disposed within an incubator  961 . The incubator  961  controls the ambient environment of the test vessel  962 . Measurement light is provided to the system  960  by a measurement light source  965  e.g., comprising one or more light emitting devices that provide broad band measurement light. Light from a spatial region  962   b  of the test vessel  962  is separated into two channels by a dichroic mirror  970 . Imaging optics, e.g., lenses  981 - 983 , may be disposed in the path of the light from the spatial region  962   b , e.g., between the test vessel  962  and the dichroic mirror  970  and/or in one or both of the two color channels  971 ,  972 . Lens  981  images the light from the spatial region  962   b  onto the dichroic mirror  970 . The dichroic mirror  970  separates the light into two different color channels  971 ,  972 , each color channel associated with a camera  991 ,  992 . Each color channel  971 ,  972  provides an image  962   b - 1 ,  962   b - 2  of substantially the same spatial region  962   b  at different wavelengths. The dichroic mirror  970  has a center wavelength, λ center , such that light having wavelengths greater than λ center  are directed toward camera  991  in a first color channel  971  and light having wavelengths less than λ center  are directed toward camera  992  in a second color channel  972 . 
     If the images  962   b - 1 ,  962   b - 2  are not sufficiently identical, then translation, rotation or scaling transformations on the images  962   b - 1 ,  962   b - 2  can be used to overlay the images so that they represent substantially the same spatial region  962   b . The images  962   b - 1 ,  962   b - 2  usually contain one, several or all test locations  962   a  and may include the thermochromic material disposed at the test locations  962   a . In some embodiments the images  962   b - 1 ,  962   b - 2  include additional information such as markings. Additional markings may be identified by well-known techniques of computer vision and image processing and they may provide the system with operation parameters such as calibration data, patient data, mechanical alignment etc. Any relevant information contained in the markings can be processed in the system&#39;s processor  940  (see  FIG.  9 A ). The images  962   b - 1 ,  962   b - 2  contain a representation of the different light intensities in the different wavelength regions for each image pixel. 
     In some embodiments the light emanating from thermochromic material at the spatial region  962   b  is included in the images  962   b - 1 ,  962   b - 2 . In these embodiments, it is possible to generate temperature maps of the imaged region by calculating the wavelength shift for each pixel in the color images  962   b - 1 ,  962   b - 2 . Groups of pixel may be combined into a region of interest (ROI) in a given image. Within a ROI the combination of pixels may for example be performed by the processor  940  by calculating the average intensity of pixel in the ROI, the sum intensity in the ROI, the median intensity in the ROI or any other mathematical operation based on the pixel values in the ROI to represent the intensity of the ROI. In some embodiments a ROI on the images substantially overlaps with a test location  962   a . More than one ROI can be defined in each image, in particular each test location  962   a  in the image can be associated with at least one ROI. An ROI has at least two values associated with it. These two values represent the light intensities originating from the at least two color channels. A wavelength shift of an ROI may for example be calculated by subtracting the average values of the ROI in two color channels from another and dividing that value by the sum of the two ROI average values. In some embodiments a hyperspectral camera system is used to determine the wavelength shift of a ROI. In such a system, the peak intensity with respect to wavelength may be determined by finding the image of the wavelength region with the highest intensity in the ROI. It may be possible in such a scenario to extrapolate intensity values of the ROIs between color frames. In some embodiments a RGB camera system is used. The wavelength shift of an ROI may be calculated by omitting one of the three channels, for example the blue channel and treating the red and the green channels as the two channels described above. It may also be possible to add the red and green channel and treat this sum as a first color channel with the blue channel providing the second color channel as described above. 
     With camera based detection system there are several ways to determine a wavelength shift in a ROI as described above. In some embodiments the ROI can contain test locations  962   a  and thermochromic material is thermally coupled to the test locations. Therefore the wavelength shift of a ROI can be related to the temperature in that ROI and thereby the temperature of the test locations of the test vessel. Ideally, the camera system images all test locations and all positive and negative control locations in order to calculate the temperature development of the test locations over time and analyze it by comparing it to positive control location temperature developments over time. 
     Any of the above described system embodiments is suited to trace the temperature development of many locations on the test vessel by tracing local wavelength shifts on the vessel. Assuming that at least one thermochromic material is dispensed across the test plate at least in the relevant test locations and control locations, the local wavelength shift represents the local temperature on the plate. 
     Ambient temperature changes will affect the wavelength shift of the whole test plate, independent of temperature changes in/on the individual test locations. These ambient temperature changes are not necessarily homogeneous across the test plate. The temperature change of a test location can be referenced by the temperature change of an adjacent control location that does not contain any test substance and thereby serves as a test location for common mode rejection of ambient temperature fluctuations. It is noteworthy that the control location may surround the test location or may have a different size or shape than the test location. In particular the control location may not contain any live substance and therefore this control location serves as a negative control location that traces ambient temperature changes. By subtracting the temperature of the negative control location from the temperature of the test location at each measurement point in time, the temperature change of the test location is traced through time, to first order independent of ambient temperature changes. A positive control location contains living substance without any drug that could inhibit the metabolism of the live substance and its colony growth. In fact the system conditions such as nominal ambient temperature, ambient gas composition, etc. should be chosen to promote the growth of live substance. Positive control locations may be corrected for ambient temperature changes with readings from negative control locations in the same way that other test locations are corrected. 
       FIG.  10 A  conceptually illustrates a wavelength shift detector  1000  that can be used as the detector subsystem  920  discussed in  FIG.  9 A  to determine the center of a spectral distribution. Thereby the existence and/or amount of shift in spectrum of light, for example emanating from the thermochromic material, can be determined by comparing two or more center of spectral light distributions. Light  1010  emanating from the thermochromic material and characterized by a central wavelength λ i  is input light to a spectrally varying optical transmission structure  1020 . The transmission structure  1020  has a laterally varying transmission function such that the transmission function varies as a function of position along a lateral axis  1099  of its exit surface  1020   a . The variation in transmission function can, for example, comprise a variation in intensity with wavelength according to a gradient, which can be a constant transmission gradient if it varies continuously and uniformly along the lateral axis  1099 . The variation in transmission function can be spike-like transmission gradient if the intensity varies with wavelength in a step-like manner along the lateral axis  1099 . More generally, light is described herein as transmitted with lateral variation when, in response to input light, transmitted light or output light varies with lateral position as a function of wavelength, and the variation with lateral position was not present in the input light. Variation with lateral position is illustrated in  FIG.  10 A  by regions  1042  and  1044 . As shown, region  1042  of the transmission structure  1020  transmits a sub-band of light in a subrange centered about wavelength λ a . Similarly, region  1044  transmits a sub-band of light in a subrange centered about wavelength λ b . As a result, the light from regions  1042  and  1044 , represented respectively by rays  1046  and  1048 , is incident on the photosensing component  1060  at different positions. Light characterized by central wavelength λ a  is detected predominantly by the portion of the photosensing component  1060  at position  1062 . Light characterized by central wavelength λ b  is detected predominantly by the portion of the photosensing component  1060  at position  1064 . If the central wavelength characterizing the input light  1010  is initially λ a , a change in the wavelength of the input light to kb will causes a change in the position of light exiting the transmission structure  1020 . This change in position will be indicated by a change in the light detected at positions  1062  and  1064  of the photosensing component  1060 . More generally, a difference between the intensity of incident light at wavelengths λ a  and λ b  can be indicated by a difference in light detected at positions  1062  and  1064 . A wavelength shift between wavelengths λ a  and λ b  or another change in wavelength distribution at the surface  1020   a  of transmission structure  1020  can change relative quantities of photons provided at positions  1062  and  1064 , meaning that the quantities provided at the two positions have a different relation to each other after the change than they did before it. For example, the quantities could increase or decrease, but by amounts such that the quantity at one position becomes a larger or smaller fraction of the quantity at the other position; the quantity at one position could change from being less than the quantity at the other position to being greater; or one quantity could increase while the other decreases; etc. All of these types of changes could occur over time. 
       FIG.  10 A  shows the relationship between light intensity and position across the photosensing component  1060  in response to two different incident spectral patterns with light sub-bands having peak energy values. The first pattern, with peak intensity at wavelength λ a , results in a light spot on the photosensing component  1060  that has an intensity distribution represented by curve  1066 . The second distribution, with a peak intensity at wavelength λ b , similarly results in a light spot with an intensity distribution represented by curve  1068 . As will be understood, the first light spot, represented by curve  1066 , may follow a continuous series of positions over time until it reaches the position of the second light spot, represented by curve  1068 , if a light narrow band of input light  1010  from the transmission structure  1020  makes a continuous transition from λ a  to λ b . 
     The graph also shows quantities of photons sensed by positions  1062  and  1064  in response to the first and second light spots. When the first spot is provided on photosensing component  1060 , position  1062  of the photosensing component  1060  generates a measurement quantity I 1  approximately proportional to the quantity of photons sensed by position  1062 , namely I a1 , and generates a measurement quantity  12  approximately proportional to the quantity of photons sensed by position  1064 , namely I b1 . I 1  and I 2  can, for example, be photocurrents generated by a position sensitive photo detector. When the second spot is on photosensing component  1060 , position  1062  senses a quantity proportional to I a2  and position  1064  senses a quantity proportional to I b2 . As will be seen, the relative quantities sensed by positions  1062  and  1064  change, with the first spot&#39;s relative quantity (I a1 /I b1 ) being greater than unity and the second spot&#39;s relative quantity (I a2 /I b2 ) being less than unity. Similarly, the difference (I a1 −I b1 ) is a positive quantity whereas the difference (I a2 −I b2 ) is a negative quantity. Furthermore, if a similar comparison is made with other adjacent or nearby positions, the peak intensity position of each spot can be approximated by finding the position on the photosensing component having the highest sensed quantity. 
     In some embodiments, the intensity of adjacent or overlapping spectral regions is integrated and compared to determine a wavelength shift in the distribution. The photosensing component  1060  may comprise two detectors and the integration over spectral regions can be performed by measuring the two adjacent regions  1062 ,  1064  using the two detectors, for example, photodiodes, split photodiodes, or photomultiplier tubes (PMT). 
     The spectrally varying transmission structure  1020  can comprise linear variable filters or spectrally dispersive elements (e.g., prisms, grating, etc.). For flexible measurements, stacked or multi-anode PMTs can be used on a spectrograph. The measurements may be performed at a frequency of at least about 0.01 Hz, up to at least about 1 MHz or even more. The combination of a laterally varying transmission structure  1020  and the position-sensitive photosensing component  1060  may resolve wavelength shifts significantly smaller than 10 femtometer (fm) or even smaller than 5 fm, e.g., about 3 fm. The individual photodiodes of the photosensing component  1060  can generate photo currents I 1  and I 2  that are amplified with a transimpedance amplifier  1080 . Signal subtraction and addition may be performed with an analog circuit for superior noise performance prior to sampling by the analyzer. The center of the wavelength distribution can then be computed by λi˜(I 1 −I 2 )/(I 1 +I 2 ). In some embodiments, the total size of the wavelength shift detector  1000  can closely approach that of the photosensing component  1060 , which is beneficial for mounting and long-term stability. Additional information involving the measurement of wavelength shifts in input light that can be used in conjunction with the thermochromic temperature sensing approaches disclosed herein is described in commonly owned U.S. Pat. No. 7,701,590 which is incorporated herein by reference. 
       FIG.  10 B  illustrates another embodiment of a spectral detector  1070 . All wavelengths of light emanating  1071  from the thermochromic material (not shown in  FIG.  10 B ) in response to measurement light are directed through a dichroic mirror  1072 . The dichroic mirror  1072  reflects certain wavelength regions while transmitting other wavelength regions. For example the dichroic mirror  1072  could transmit all wavelength λ 1 &lt;λ center  and reflect all wavelength λ 2 &gt;λ center . Two different detectors, first detector  1081  and second detector  1082  are disposed to collect the transmitted and reflected light from the dichroic mirror  1072 . Detector  1081  may be used to measure the total light intensity contained in the wavelength region that is smaller than the mirror&#39;s center wavelength λ center  and detector  1082  may be used to measure the total light intensity contained in the wavelength region that is larger than the mirror&#39;s center wavelength λ center . For a spectral distribution centered around the center wavelength, both measured light intensities would be identical (curve  1071   a ). For a spectral distribution that is shifted to longer wavelengths (curve  1071   b ), detector  1082  would measure higher light intensities than detector  1081 . Therefore this detector used with the above-described method represents another way of detecting spectral light intensity distributions. 
     In some embodiments, additional optical elements  1075  may be introduced into the light detection path. For example, additional bandpass filters in front of the detectors  1081 ,  1082  may be used to limit the detected light to the spectral region that shows the largest shift for a given temperature change. In some embodiments, additional optical elements  1075  may include imaging lenses. Imaging may be particularly interesting, when the light detectors are image detectors, such as cameras. The full area of the complete test vessel may be illuminated and the measurement light from numerous test sites may be sensed simultaneously in a scheme as presented in  FIG.  10 B , by imaging the test vessel onto at least two cameras. For two simultaneously taken images the color distribution and therefore the temperature of all test locations can now be measured by measuring the recorded intensity of the appropriate pixels for each test location on both cameras. Additional markings on the test vessel may be used to identify the test locations in the images. 
     Thermochromic sensing may be used for a variety of testing protocols, such as testing the efficacy of various pharmaceuticals, e.g., antibiotics, antimicrobial agents, antifungal agents, cancer drugs, etc. The flow diagram of  FIG.  11 A  illustrates a thermochromic testing process in accordance with some embodiments. The live substance is inserted  1101  into a medium disposed at test locations of a thermochromic test vessel. In some embodiments a test substance is also disposed in the medium. The live substance is thermally coupled to a thermochromic material located at the test locations. A spectral shift of light emanating from the thermochromic material is detected  1102 . The spectral shift occurs in response to a temperature change caused by energy conversion by the live substance. The amount and/or rate of energy converted by the live substance may be determined  1103  based on the spectral shift. For example, in some implementations, live substance is a pathogen and the amount and/or rate of energy converted by the live substance indicates the susceptibility of the live substance to the test substance, e.g., an antibiotic. In some implementations, the live substance is a cell or tissue culture and the energy converted by the live substance can be related to mutations or other effects of the test substance on the cell or tissue culture. 
     As discussed above, thermochromic sensing is particularly useful for antibiotic or antimicrobial susceptibility testing. The goals of antimicrobial susceptibility testing are to detect possible drug resistance in pathogens and to assure susceptibility of the pathogens to drugs of choice for particular infections. Antimicrobial susceptibility testing may provide quantitative results, e.g., minimum inhibitory concentration of the antimicrobial test substance, and/or may provide qualitative assessment of efficacy of the test substance with respect to the pathogen. New and emerging mechanisms of resistance exhibited by many bacteria require vigilance regarding the ability of AST to accurately detect resistance. Particularly in view of these emerging mechanisms of resistance, it seems likely that phenotypic measures of the level of susceptibility of bacterial isolates to antimicrobial agents will continue to be clinically relevant for years to come. 
     AST measures the effect of drugs on the replication of microbes to determine which drug is best suited to kill the bacterium. AST may test many drugs in parallel in vitro to predict which drug works best in vivo. Thus, AST may test a broad sample of drugs so that the treatment choice can be targeted to the most effective antimicrobial drug for the particular bacteria. 
       FIG.  11 B  is a flow diagram that illustrates a testing process in accordance with some embodiments. Test locations of a thermochromic sensing test vessel are filled with a sample  1112 . The sample may include one or more of a medium, a live substance, a substance to be tested, e.g., antibiotic, and, in some embodiments, a thermochromic material. The sample and other structures near the sample, e.g. test locations and control locations, are thermally equilibrated  1114  in an incubation chamber. One or more regions of the test vessel that include test locations and control locations are illuminated  1116  with measurement light. Images of the regions are detected  1118  by a camera system. In some implementations, multiple images of the regions are detected and alignment features of the images are registered  1120  to align the images. A transformation between the images is calculated based on the alignment. Regions of interest within the images are identified  1122 . For example, the regions of interest may include test locations and/or positive and/or negative reference control locations. The light intensity  1124  and wavelength shift  1126  of the regions of interest in the images are determined. The temperatures for the regions of interest are determined  1128 , including determining the temperatures of the test locations and the positive and/or negative control locations. The process indicated by steps  1118  through  1128  is repeated until the conclusion of the test. The temperature changes in the region of interest are analyzed  1130 . Any significant findings based on the temperature changes are reported  1132 . In some embodiments, registration of the alignment features and calculation of the transformation between images may be performed once and used for the test, rather than being performed during each measurement loop. 
     Current testing of significant bacterial isolates takes between 12 and 24 hours to detect possible drug resistance in common pathogens. The thermochromic sensing approaches discussed herein use optical calorimetry to monitor the temperature of incubation vessels, e.g., incubation test wells, and thereby to determine the growth of pathogen cultures. The disclosed approaches can speed up AST by significantly increasing the detection sensitivity providing the ability to monitor bacterial growth (or its absence) in real time rather than by end-point measurements. In some embodiments, use of the thermochromic sensing techniques described herein can reduce the time needed to obtain the minimum inhibitory concentration of antibiotic by more than 60%, more than 70% or even more than 80% when compared to current approaches.  FIG.  12 A  is a flow diagram illustrating a process for thermo-optical antimicrobial susceptibility testing using thermochromic sensing in accordance with some embodiments. After a patient sample is flagged as positive for growth by a blood culture instrument ( 1210 ), an aliquot of the sample is subjected to Gram staining ( 1220 ) to identify the bacteria as Gram-negative or Gram-positive. After separation from the blood culture medium, and suspension at an appropriate concentration, the bacteria are introduced to test vessels for TOAST ( 1230 ). Bacteria is cultured  1240  in a medium at test locations of a test vessel, wherein different test locations contain different types, concentrations and/or combinations of antibiotic. A thermochromic material is disposed at the test locations and is thermally coupled to sense energy conversion of the bacteria. A spectral shift of the light emanating from the thermochromic material at each of the test locations is detected  1250 . The susceptibility of the bacteria to the different types, concentrations and/or combinations of antibiotic is determined  1260  based on the spectral shift. 
       FIG.  12 B  is a flow diagram illustrating a process for using thermochromic sensing in accordance with some embodiments. After a patient sample is flagged as positive for growth by a blood culture instrument ( 1210   b ), an aliquot of the sample is subjected to Gram staining ( 1220   b ) to identify the bacteria as Gram-negative or Gram-positive. The culture is subjected to identification testing ( 1230   b ) for example via standard biochemical tests or rapid mass spectrometry methods. Following identification of the bacteria, the bacteria are introduced to test vessels for TOAST ( 1240   b ). Bacteria are cultured  1250   b  in a medium at test locations of a test vessel, wherein different test locations contain different types, concentrations and/or combinations of antibiotic. A thermochromic material is disposed at the test locations and is thermally coupled to sense energy conversion of the bacteria. A spectral shift of the light emanating from the thermochromic material at each of the test locations is detected  1260   b . The susceptibility of the bacteria to the different types, concentrations and/or combinations of antibiotic is determined  1270   b  based on the spectral shift. 
       FIG.  13 A  is a flow diagram illustrating a process for bacteria identification and thermo-optical antimicrobial susceptibility testing using thermochromic sensing in accordance with some embodiments. After a patient sample is flagged as positive for growth by a blood culture instrument ( 1310 ), an aliquot of the sample is subjected to Gram staining ( 1320 ) to identify the bacteria as Gram-negative or Gram-positive. After separation from the blood culture medium, and suspension at an appropriate concentration, the bacteria are introduced to test vessels for identification ( 1330 ) and for TOAST ( 1350 ). For identification  1330 , bacteria is cultured  1335  in a medium at test locations of a test vessel, wherein different test locations contain different types, concentrations and/or combinations of test substance and indicator substance. A thermochromic material is disposed at the test locations and is thermally coupled to sense energy conversion of the bacteria. A spectral shift of the light emanating from the thermochromic material at each of the test locations is detected  1370 . The identification of the bacteria is determined  1375  based on the spectral shift for example as explained in the description of  FIG.  8   . In addition, the color change in the growth medium resulting from incubation of bacteria in the presence of the test substance and indicator substance is detected  1340 . The identification of the bacteria is determined  1345  based on the color change in the growth medium. For TOAST  1350 , bacteria are cultured  1355  in a medium at test locations of a test vessel, wherein different test locations contain different types, concentrations and/or combinations of antibiotic. A thermochromic material is disposed at the test locations and is thermally coupled to sense energy conversion of the bacteria. A spectral shift of the light emanating from the thermochromic material at each of the test locations is detected  1360 . The susceptibility of the bacteria to the different types, concentrations and/or combinations of antibiotic is determined  1365  based on the spectral shift. 
       FIG.  13 B  is a flow diagram illustrating a process for bacteria identification and thermo-optical antimicrobial susceptibility testing using thermochromic sensing in accordance with some embodiments. After a patient sample is flagged as positive for growth by a blood culture instrument ( 1310   b ), an aliquot of the sample is subjected to Gram staining ( 1320   b ) to identify the bacteria as Gram-negative or Gram-positive. After separation from the blood culture medium, and suspension at an appropriate concentration, the bacteria are introduced to test vessels for identification ( 1330   b ) and for antimicrobial susceptibility testing TOAST( 1350   b ). For identification, bacteria are cultured  1335   b  in a medium at test locations of a test vessel, wherein different test locations contain different types, concentrations and/or combinations of test substance and indicator substance. A thermochromic material is disposed at the test locations and is thermally coupled to sense energy conversion of the bacteria. A spectral shift of the light emanating from the thermochromic material at each of the test locations is detected  1340   b . The identification of the bacteria is determined  1345   b  based on the spectral shift. For TOAST, bacteria are cultured  1355   b  in a medium at test locations of a test vessel, wherein different test locations contain different types, concentrations and/or combinations of antibiotic. A thermochromic material is disposed at the test locations and is thermally coupled to sense energy conversion of the bacteria. A spectral shift of the light emanating from the thermochromic material at each of the test locations is detected  1360   b . The susceptibility of the bacteria to the different types, concentrations and/or combinations of antibiotic is determined  1365   b  based on the spectral shift. 
       FIG.  13 C  is a flow diagram illustrating a process for bacteria identification and thermo-optical antimicrobial susceptibility testing using thermochromic sensing in accordance with some embodiments. After a patient sample is flagged as positive for growth by a blood culture instrument ( 1310   c ), an aliquot of the sample is subjected to Gram staining ( 1320   c ) to identify the bacteria as Gram-negative or Gram-positive. After separation from the blood culture medium, and suspension at an appropriate concentration, the bacteria are introduced to test vessels for identification ( 1330   c ) and for TOAST ( 1350   c ). For identification, bacteria are cultured  1335   c  in a medium at test locations of a test vessel, wherein different test locations contain different types, concentrations and/or combinations of test substance and indicator substance. The color change in the growth medium resulting from incubation of bacteria in the presence of the test substance and indicator substance is detected  1340   c . The identification of the bacteria is determined  1345   c  based on the color change in the growth medium. For TOAST, bacteria are cultured  1355   c  in a medium at test locations of a test vessel, wherein different test locations contain different types, concentrations and/or combinations of antibiotic. A thermochromic material is disposed at the test locations and is thermally coupled to sense energy conversion of the bacteria. A spectral shift of the light emanating from the thermochromic material at each of the test locations is detected  1360   c . The susceptibility of the bacteria to the different types, concentrations and/or combinations of antibiotic is determined  1365   c  based on the spectral shift. 
     Bacteria generate on the order of 2 pW per cell when alive. Thriving pathogen cultures accordingly increase their energy conversion over time due to culture growth by mitosis or other replication mechanisms. Inhibited or declining energy conversion output of cultures indicates culture death. In antimicrobial susceptibility testing, inhibited or declining energy conversion output of culture is related to the efficacy of antimicrobial drugs. Thermochromic sensing using the thermochromic sensing test vessel described in connection test vessels and/or wavelength shift detectors described herein can resolve changes in wavelength of Δλ≈3 fm at a sampling rate of about 100 Hz which provides a resolution for temperature change of about 60 nanoKelvin (nK). The temperature measurement bandwidth when using thermochromic materials that exhibit spectral shifts of 50 nm/K sampled with 14 bit resolution is about 1 milliKelvin (mK).  FIG.  14 A  shows graphs that illustrate the change in temperature ΔT (K) with respect to time for a growing  E. coli  colony with no antibiotic (graph  1401 ) and with a minimum inhibitory concentration (MIC) of antibiotic (graph  1402 ) over a the range of 1 mK (about 290 minutes) of thermochromic sensing using the wavelength shift detectors discussed herein. The total test volume is assumed to be 0.1 ml and the initial bacteria concentration is assumed to be 500000 colony forming units per ml (cfu/ml). 
       FIG.  14 B  shows a portion of the graphs  1401 ,  1402  corresponding to the first 20 minutes of colony growth. The gridlines along the ΔT axis of  FIG.  14 B  indicate the  60  nK resolution of the wavelength shift detector. Thus, it will be appreciated from the graphs of  FIG.  14 B  about the first 10 to 20 minutes of testing using the approaches disclosed herein can indicate growth trends sufficient to identify the MIC for antimicrobial testing. Specifically, uninhibited colony growth results in an exponential increase in temperature of the test site. An increasing temperature in a test site therefore indicates a thriving bacteria colony and can be used as a temperature reference to compare inhibited growth to. Inhibited colony growth will result in smaller temperature increases or in constant temperature when no growth at all occurs. Test sites with inhibited and uninhibited growth of live substance can therefore be identified by their differential temperature development over time. Test wells with live substance but without growth inhibitors will therefore serve as positive control sites in some embodiments. During an antimicrobial susceptibility test, different test sites with different antimicrobial drugs may show effects of the drug at different times. One drug may act faster on the microbes than a second drug, although both drugs may be effective in inhibiting the bacterium colony of interest. Therefore, a continuous reporting of temperatures at different test sites may inform about the efficacy of a particular drug earlier than about the efficacy of a second drug or the efficacy of a second concentration of the first drug. Consequently, a continuous reporting of differential temperature of test sites may serve a continuous representation of cell viability at that test site. Unlike an end-point measurement, such a reporting system can continuously update any user for example about the currently best performing test substance. In clinical practice, a physician could be informed by a TOAST system in an automated manner, for example by email or text message, that the first effective drug for a particular patient has been identified, if the energy conversion in the corresponding well has been continuously low for a prolonged time. Simultaneously, ambient temperature, even within the temperature controlled environment of an incubator, may drift significantly compared to the temperature increase of the test site. In some embodiments negative control sites or wells without live substance will be used to assess the ambient temperature drift. 
     To assess the efficacy of a test substance inhibiting live substance growth, including but not limited to bacterial growth, the temperature development of test locations is monitored over time. After initial insertion of the test plate into the full incubation and read-out system it is expected that temperatures will fluctuate strongly and that initial temperature reading will be ignored. After sufficient temperature stabilization, positive control locations show temperature changes for example as the ones shown in  1401 . Test locations with test substances that effectively inhibit growth of the live substance show temperature developments as depicted in  1402 . Test substances that not only inhibit the growth but produce cytotoxic or germicidal effects so that necrosis or apoptosis or any other form of death or reduction in metabolism of the live substance is induced will result in a temperature decrease of the test location compared with the positive control location. The temperature change over time will generally fall between the ones of positive control locations and negative control locations. Several metrics for determining the response of the live matter to the test substance can be used. As a simple non-limiting example the absolute value of temperature difference between a positive control location and a test location remains below a certain threshold value, for example 10 μK during the course of the experimental duration to determine uninhibited growth of the test substance at a test location. Alternatively, the same thresholding calculation could be performed by averaging the temperature of several positive control locations. Another example for a metric of inhibited or uninhibited growth may be a temperature difference that is normalized by the absolute temperature of control locations. Another example for a metric may be the consideration of the temperature derivatives with respect to time. Another example for evaluation procedures may be a curve fit, for example an exponential growth fit to the temperature-time development data. Individual fit parameters for control and test location may then be used for evaluating the growth or the lack thereof of live substance in test locations. It will be appreciated that these are merely examples of possible data evaluation concepts that can be used in order to extract meaningful information from the fundamental data generated in a TOAST system. Depending on the live matter and the actual intent of a particular test these concepts or others may be utilized. 
     Unless otherwise indicated, all numbers expressing feature sizes, amounts, and physical properties used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the foregoing specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings disclosed herein. The use of numerical ranges by endpoints includes all numbers within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5) and any range within that range. 
     Various modifications and alterations of the embodiments discussed above will be apparent to those skilled in the art, and it should be understood that this disclosure is not limited to the illustrative embodiments set forth herein. The reader should assume that features of one disclosed embodiment can also be applied to all other disclosed embodiments unless otherwise indicated. It should also be understood that all U.S. patents, patent applications, patent application publications, and other patent and non-patent documents referred to herein are incorporated by reference, to the extent they do not contradict the foregoing disclosure.