WO2013033080A1 - Dispositif et procédé d'optimisation de processus photobiologiques - Google Patents

Dispositif et procédé d'optimisation de processus photobiologiques Download PDF

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Publication number
WO2013033080A1
WO2013033080A1 PCT/US2012/052640 US2012052640W WO2013033080A1 WO 2013033080 A1 WO2013033080 A1 WO 2013033080A1 US 2012052640 W US2012052640 W US 2012052640W WO 2013033080 A1 WO2013033080 A1 WO 2013033080A1
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Prior art keywords
light sources
light
optical
wells
sample
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PCT/US2012/052640
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English (en)
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Amar Basu
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Wayne State University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/255—Details, e.g. use of specially adapted sources, lighting or optical systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/274—Calibration, base line adjustment, drift correction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/56—Means for indicating position of a recipient or sample in an array

Definitions

  • the present invention relates to arrangements for biological testing, monitoring, and screening.
  • the present invention relates to an apparatus and a method for monitoring and optimizing photo-biological studies under controlled conditions.
  • Cells and biological systems respond not only to chemical signals (such as drugs, signaling molecules, pH), but also to physical stimuli such as light, heat, radiation, electrical potentials, and mechanical stress.
  • Traditional high throughput screening technology based on microplates and pipetting robots has generally focused on large-scale screening of chemical stimuli for application in drug discovery, proteomics, and cytotoxicity.
  • a cell or biomolecule is serially tested against a library of compounds using well plates containing 96, 384, or 1536 sample wells. Ligand binding, phenotypic, and morphological changes are observed and recorded in each well, and are used to identify active, inactive, and toxic compounds.
  • Another common screen is performed by serial dilution and systematically measuring the response to compound concentration.
  • One example of physical stimuli is light.
  • Other physical stiimuli are pressure, humidity, temperature, wind, shock, noise, vibration, just to name a few.
  • photobiology for example, the study of photosynthesis in algae is of interest.
  • Societal challenges in energy sustainability have renewed interest in how nature utilizes photon energy to produce biomass.
  • Producing lipid-based biodiesel from algae is regarded as one of the most efficient and environmentally sustainable methods of generating biofuels, and appears to be a renewable source of oil that could meet the long-term global demand for transport fuels.
  • the present application provides an optical microplate illumination assembly and a method for simultaneous stimulation of photo-biological processes under a variety of different lighting conditions.
  • a circuit board carries a number of light sources equal to the number of the at least two sample wells of the sample well plate. The light sources are optically separated from each other. Each of the light sources is associated with one of the sample wells.
  • a controller is configured for individually controlling each of the light sources. Because each light source can be individually controlled, different light conditions can be created at the same time while requiring little space.
  • the at least two sample wells are optically separated from each other to reduce cross-illumination between the at least two sample wells.
  • the at least two sample wells have transparent bottoms and the light sources are arranged underneath the bottoms. This arrangement leaves the open tops of the sample wells accessible for further pipetting and for measurements.
  • the light sources are arranged above the sample wells in a top-down arrangement. Measurements can be performed by removing the light source to access the sample well plate.
  • At least one optical detector is provided with a detection cross-section that does not exceed a horizontal cross-section of at least one of the sample wells.
  • the detection cross-section being no larger than the sample-well cross- section ensures that each measurement is substantially limited to one light source or one sample well, respectively.
  • the controller is configured to individually vary at least a light intensity among the light sources by varying an electric current.
  • different brightness conditions can be simultaneously evaluated regarding the photo- biological effect on the biological samples.
  • the controller configured to calibrate the individual light intensities among the light sources by adjusting individual electric supply currents according to a calibration measurement curve to adjust for variations among the light sources.
  • the calibration compensates for manufacturing variances among the individual light sources.
  • the controller is configured to individually vary at least a duty cycle among the light sources so that the effects of different settings of light and dark phases can be simultaneously evaluated.
  • the controller is configured to individually vary at least a pulse width among the light sources so that the effects of different timing illumination patterns can be investigated.
  • the light sources are light-emitting diodes (LEDs).
  • LEDs are available in numerous color bands and are an energy-efficient form of illumination. Thus, LEDs allow for an individual adaptation of the desired light wavelength and only generate little heat compared to other light sources.
  • the LEDs may be multi-color LEDs that are configured to selectively emit two or more different light colors. Thus, time-dependent illumination patterns with varying light colors can be created within a sample well.
  • each of the LEDs emits at least one wavelength of light overlapping with a wavelength range of high optical absorption of a test sample designated for placement in the sample well paired with the LED.
  • the selection of well- absorbed wavelengths reduces energy loss and heat production by reducing the emission of less effective wavelengths.
  • the sample wells and the light sources are arranged in identical patterns in a matrix.
  • the arrangement in a matrix allows for a space- saving arrangement, where, for example, columns can undergo different light variations among each other than rows.
  • the matrix is dimensioned to be filled by commercially available pipetting robots. This ensures compatibility with common instruments used for testing with chemical methods or other biological methods.
  • the sample well plate illumination docking area is accessible for commercially available automatic plate loading robots. This ensures compatibility with high-throughput screening techniques.
  • the controller comprises a constant- current driver for each of the light sources. This arrangement enables a process of dynamic intensity control and absolute intensity calibration with consistent results over time and among the individual light sources.
  • a method for optimizing photo-biological processes comprises the steps of providing a sample well plate with at least two sample wells for receiving test samples; providing a circuit board with a number of light sources equal to the number of the at least two sample wells, the light sources being optically separated from each other and each of the light sources being paired with one of the sample wells; providing a controller configured for individually controlling each of the light sources; placing a biological sample in at least two of the at least two sample wells; illuminating the at least two of the biological samples with differently controlled light sources; and performing an analysis on the biological samples.
  • the method allows for simultaneous experimentation on the results of different lighting conditions at the same time.
  • an indicator substance can be added to each of the biological samples, where the indicator substance exhibits a property dependent on a biological condition of the biological sample.
  • the property to be measured need not be measurable in the biological sample alone, but can be revealed by the added indicator substance.
  • the biological property may be an optical property, such as optical density or fluorescence at the same wavelength or a different wavelength than the wavelength of the illumination.
  • the entire microplate assembly is placed in a controlled environment configured for culturing cells and has a data communication line from the circuit board to an external control interface. This allows an operator to control the individual illumination patters without disturbing the controlled environment.
  • the method includes the further step of repeating the sample analysis measurements after defined time periods by temporarily removing only the sample well plate or the entire microplate assembly from the controlled environment. Thus developments over time can be observed for each of the samples in the microplate assembly.
  • the light sources may be differently controlled by setting a different light intensity or by setting different pulse-width modulated duty cycles, or by different color wavelength or by any combination of the above.
  • the light sources may be calibrated before illuminating the biological samples.
  • FIG. 1A shows an example of a sample well plate with 96 sample wells according to a first aspect of the invention.
  • Fig. 1 B shows an example of a light-emitting-diode (LED) matrix configured to be matched with the sample well plate of Fig. 1A and controlled by a microcontroller according to a further aspect of the invention.
  • LED light-emitting-diode
  • Fig. 2 shows a printed circuit board (PCB) configured to provide electrical power to the LED matrix of Fig. 1 B according to another aspect of the invention.
  • PCB printed circuit board
  • Fig. 3 shows a detail view of a single LED of the LED matrix of Fig. 1 B matched with a single sample well of the sample well plate of Fig. 1 A and connected to the PCB board of Fig 2.
  • Fig. 4 shows a microplate assembly comprising the sample well plate of Fig. 1A, the LED matrix of Fig. 1 B, and the PCB of Fig. 2.
  • Fig. 5 shows a first calibration curve depicting the electric current flowing through one LED over the light intensity resulting from that current.
  • Fig. 6 shows a variation of light intensity before a calibration of LEDs mounted on one LED matrix.
  • Fig. 7 shows the variation of light intensity after calibration of the same LEDs as in Fig. 6.
  • Fig. 8 shows a graph of a light absorption spectrum by example of dunaliella tertiolecta, a lipid-producing algal species.
  • Fig. 9 shows the growth of dunaliella tertiolecta microplate cell cultures by plotting optical density over a duration of 12 days at different settings of light intensities of associated LEDs.
  • Fig. 10 shows the growth of dunaliella tertiolecta microplate cell cultures by plotting optical density over a duration of 12 days in different duty cycles of pulse-width modulation with LEDs emitting the same light intensity.
  • Fig. 1 1 is a bar graph of the algal growth of Fig. 10 represented by optical density after 12 days for different duty cycles.
  • Fig. 12 is a bar graph of oil production of the dunaliella tertiolecta microplate cell cultures of Fig. 10 represented by fluorescence emitted from the microplate cell cultures of Fig. 10 after 12 days for different duty cycles.
  • Fig. 13 is a bar graph of the quotient of Figs 1 1 and 12 after 12 days for different duty cycles.
  • Fig. 14 is a bar graph of the quotient of the fluorescence shown in Fig. 12 divided by the total expended electric work for different duty cycles.
  • Fig. 15 shows an example of an LED matrix carrying two-color LEDs for emitting red light, green light, or both red and green light, resulting in yellow light., according to a further aspect of the invention.
  • Fig. 16A shows a detail view of a single two-color LED with three output pins suitable for the LED matrix of Fig. 15.
  • Fig. 16B shows a three-color LED with four output pins for individually controlling the current of each of three different light colors emitted by the three-color LED according to yet another aspect of the invention.
  • Figure 17 shows a PCB configured to provide electrical power to the LED matrix of Fig. 15 carrying the two-color LEDs of Fig. 16A.
  • Figure 18 shows various different light patterns enabled by the two-color LED matrix of Fig. 15.
  • Figure 19 illustrates an example of using the microplate assembly of Fig. 15 for analyzing a synthetic optogenetic pathway.
  • Figure 22 schematically shows a further embodiment of a microplate assembly with a top-down illumination by example of two-color LEDs.
  • a high-throughput system to evaluate tens or hundreds of light conditions simultaneously provides a valuable tool for photo-biological research.
  • This application presents an optical microplate configured for photonic high-throughput screening of photo-biological processes in general using programmable illumination by a multi-color, duty-cycle and intensity calibrated light source in particular.
  • a sample well plate 1 10 comprises a matrix of individual microwells 1 12.
  • twelve columns of microwells 1 12 are arranged in eight rows for a total of 96 microwells.
  • Each microwell 1 12 has a generally cylindrical shape with a vertical cylinder axis.
  • each sample microwell 1 12 has an opaque cylinder wall, preferably of black color. The opaque walls optically separate the individual sample wells 1 12 from each other and prevent that light can leak from one sample well to another and thus prevent cross-illumination.
  • the bottoms of all sample microwells 1 12 are transparent, at least for such wavelengths of light that are used for irradiating cell cultures placed in the sample microwells 1 12.
  • the sample microwell plate 1 10 may be a standard 96-well microplate available with flat, u-shaped or v-shaped well bottoms 112 and can be prepared with a variety of surface treatments to optimize cell-culture and other biochemical experiments.
  • the sample well plate 1 10 has a standard footprint so that it is compatible with robotic loading and sample filling arrangements used for screening chemical reactions. Plate readers for automated high-content image analysis and liquid chromatography coupled to mass spectrometry have all been adapted to work in 96-well formats for applications in life sciences, environmental analysis, clinical, forensic and industrial markets.
  • an LED matrix 120 comprises a number of LEDs 122 that corresponds to the number of microwells 1 12 arranged on the sample well plate 1 10.
  • the LEDs 122 counting 96 in this example, are arranged in the same pattern as the microwells 1 12 and emit light in a spectral band around 650 nm.
  • Each LED 122 is paired with one individual sample well 1 12.
  • the LED matrix 120 is encased with a black-wall well plate 124 that optically separates the LEDs 122 and prevents light spillover between adjacent LEDs 122.
  • the well plate 124 may be an inverted sample well plate in which the bottoms of the wells may have been removed to accommodate the LEDs 122.
  • the well plates 1 10 and 124 may have identical dimensions or may have different well depths. Additional lenses or spacers may be inserted between well plates 1 10 and 124 to adjust the light emitted by the LEDs relative to the sample wells.
  • the LEDs 122 are supplied with electric power by a constant-current drivers 126 that are individually controllable for each LED 122 through a serial microcontroller 128 so that 96 different lighting conditions can be programmed simultaneously.
  • the microcontroller 128 can control intensity and pulsed duty cycles of the LED light emission in each sample well 1 12. In the shown example, the intensity control provides about 128 analog light intensity levels (for example with a 7-bit resolution), with a maximum of about 120 imE/cm 2 .
  • the temporal resolution of the duty cycles useful for studying the regulation and dynamics of photosynthesis, can be set to provide time increments as little as about 10 ⁇ .
  • the LEDs 122 and the constant- current drivers 126 are placed on a printed circuit board (PCB) 130.
  • the PCB 130 is shown in greater detail in Fig. 2.
  • Fig. 2 shows the PCB 130 carrying the control electronics for controlling the LEDs 122 of Fig. 2B.
  • the PCB 130 carries six serial LED drivers 132, each accommodating 16 programmable constant-current drivers 126, for a total of 96 constant-current drivers with up to 96 different outputs. Suitable serial LED drivers are, for example, available from Texas Instruments.
  • Each output can provide 128 discrete current levels (7-bit resolution) to its respective LED.
  • the range of current can be selected by choosing an appropriate bias resistor.
  • the maximum current is about 80 mA, which is suitable for intermediate power LEDs which can provide up to about 1000 ⁇ /cm 2 .
  • the light intensity and on/off state in each sample well 1 12 can be set using a high-speed serial peripheral interface (SPI) operating at up to about 12 MHz.
  • SPI serial peripheral interface
  • refreshing the state of entire array requires n clock cycles, where n is the number of LEDs 122, in the shown example n being 96.
  • the hardware-limited refresh rate of the array is about 100 KHz, or about 10 ⁇ sec. This time resolution is sufficient to study light/dark reaction pathways in photosynthesis.
  • the PCB 130 may be, for example, a standard FR4 PCB and fabricated at a commercial foundry.
  • the LEDs 122 and drivers are assembled using through-hole and surface mount connections 134.
  • Passive aluminum heat sinks (not shown) may be attached to the PCB 130 using a thermal adhesive compound in order to reduce the system temperature.
  • a 10 pin ribbon cable may connect the cascaded drivers to a USB serial interface card.
  • a USB serial interface card suitable for this purpose is, for example, available under the name USB-8451 from National Instruments.
  • a microcontroller can control the light intensities via a digital interface through the USB serial interface card.
  • the card may, for example, be controlled via Labview software by National Instruments run on a desktop computer.
  • Light levels may be measured using quantum light meters (138, see Fig. 3), for example available under the name LI-1400 from LI-COR Biosciences. The quantum light meters can be aligned manually above each sample well.
  • Fig. 3 shows a schematic view of an LED 122 paired with a microwell 1 12, in which a biological culture 136 is placed.
  • the LED 122 is powered through the constant-current driver 126 to provide a specific illumination characteristic to the paired microwell 1 12. Due to the black walls surrounding the LED 122 and the microwell 1 12, other microwells remain unaffected by the light emitted by the LED 122.
  • optical properties of a culture 136 can be measured with a quantum light meter 138 placed directly over the microwell 1 12 containing the biological culture 136.
  • the measurement may be performed intermittently so that the light meter is only placed above the sample well 1 12 at specified time intervals.
  • the light meter has an optically sensitive cross- section that does not exceed the cross-section of the sample well 1 12, and the light meter is positioned to receive only optical information from the one sample well 112.
  • the optical property may be an optical density at a specific wavelength that may be identical to or different than the wavelength or wavelengths selected for the illumination.
  • the optical property may also be a property of an indicator substance reacting to a biological property of the biological culture.
  • the optical property of individual sample microwells 1 12 can be measured by removing sample well plate 1 10 from the assembly and using a fluorescence microplate reader, a high content microscope or any standardized microwell analysis system.
  • an assembled optical microplate assembly 100 comprises the sample well plate 1 10 carrying the cell cultures on top of the LED matrix 120 mounted on the PCB 130.
  • the PCB 130 is provided with an optional cover 140 protecting the electronic circuitry from damage and contamination.
  • the LED drivers and the USB interface may also be arranged underneath the PCB 130 so that the PCB 130 may have substantially the same footprint as the well plate 1 10.
  • a schematically shown cable 142 includes data connections to the drivers 126 and a power supply for the LCDs 122.
  • the cable 142 may be an extended ribbon cable containing a multitude of wires. It connects the microplate assembly 100 with a user interface (not shown) that provides for external control of the individual illumination patterns.
  • the LEDs 122 of the example shown are configured for analog control and have a substantially linear correlation between the electric current supplied by the constant-current drivers 126 and the emitted light intensity.
  • doubling the flow of current will generally double the light intensity of each LED 122.
  • the LED drivers 132 use a current mirror configuration which provides a high degree of linearity and precision in current control.
  • the LED current vs. intensity setting shows an R 2 value of about 0.9992.
  • Fig. 6 shows variations among the individual LEDs 122 in the absolute light intensity when supplied with an identical supply current.
  • a well-to-well variation in current was characterized by measuring LED currents for all 96 wells on an intensity setting of 20.
  • Well-to-well variation in light intensity was measured by manually aligning the light meter each well in the plate. The variation in light levels is about 17%, for example due to manufacturing tolerances in the LEDs.
  • Fig. 7 illustrates, the variation in light levels can be reduced to less than about 10% through a one-time compensation procedure, where the intensity settings can be scaled by an appropriate calibration factor. After a one-time calibration, the standard deviation among all 96 microwells has been reduced to about 1.8%.
  • One illustrative example of using the optical microplate assembly 100 of Figs. 1A and 1 B is presented in the following by experimental research on the photosynthesis of dunaliella tertiolecta. Although basic photosynthetic pathways have been well documented, the dynamics and regulation of light-to-biomass energy conversion pathways and their impact on conversion efficiency are still not comprehensively understood. For example, one may expect that higher light intensity leads to higher lipid production in oil-producing plants.
  • dunaliella tertiolecta a member of the dunaliella algal genus.
  • the conversion efficiency (lipid produced divided by the input power), however, is relatively low in comparison with other light-dependent processes. Even at moderate light levels, most of the light energy is wasted as heat or in non-photosynthetic processes. This is largely because chlorophyll absorbs energy at a faster rate than what can be utilized by the slower downstream processes of photosynthesis. Temporal variation in light can significantly affect energy conversion efficiency.
  • FIG 8 illustrates the light absorption spectrum 102 of dunaliella tertiolecta in a broken-line curve and an emission spectrum 104 of a light-emitting diode (LED) selected to largely overlap with a peak 106 of the absorption spectrum 102. Both the peak 106 and the emission spectrum reside in a wavelength range between about 650 nm and about 700 nm.
  • LED light-emitting diode
  • the LED emission spectrum 104 does not exactly coincide with the peak 106, but it is evident that the emission spectrum 104 is absorbed to a much higher degree than an emission spectrum that, for example, is located around about 550 nm, where the absorption spectrum has a minimum.
  • LEDs are efficient light sources for horticulture.
  • a wavelength of about 650 nm was chosen because it provides photosynthetic rates comparable to white light. It should be noted that different wavelengths may be selected for the optimization of different photo-biological processes because different cultures or processes may exhibit a better reactivity at different wavelength ranges.
  • the optical microplate 100 is used to perform a high throughput study of growth rates and photosynthetic efficiency in the model organism Dunaliella tertiolecta, a lipid-producing algae of interest in 2nd generation biofuels. Due to the ability to conduct 96 studies in parallel, experiments that would require about 2 years using conventional tools can be completed in about one week. This system can enable novel high-throughput protocols for photobiology and the growing field of systems biology.
  • each microwell 1 12 received about 200 ⁇ _ of dunaliella tertiolecta.
  • the microplate assembly 100 containing the cell cultures was placed in a laboratory incubator at about 37°C and about 5% C0 2 .
  • the microplate assembly 100 was placed on an aluminum block which served as a heat sink.
  • An extended ribbon cable provided a data connection and a power supply from outside the incubator.
  • Nile red stain was added to the culture at the end of the experiment to quantify lipid production. Nile red is a selective indicator for intracellular lipids, and fluoresces only in a lipid environment. Fluorescence intensity was quantified daily using a customary microplate reader at 450 nm excitation/530 nm emission.
  • Figs 9 through 14 show examples of findings that allow for optimization of the growth process.
  • Fig. 9 is a photo-irradiance curve which characterizes the growth rate of algae under 6-fold variation in light intensity.
  • algae were cultured in 6 groups of 8 wells. Each group was assigned a light intensity ranging from about 10 ⁇ /cm 2 to about 60 ⁇ /cm 2 . Each experiment was thus replicated in 8 wells to compensate for well-to-well experimental error.
  • daily averages of optical density are plotted over time. As expected, cultures receiving higher light have a faster doubling time and reach a stationary phase faster than those receiving less light. This is due to the dependence of metabolic rates and reproduction on light intensity.
  • Microscope observation reveals that cells under strong light conditions (for example greater than about 40 ⁇ /cm 2 ) reach 100% confluence in up to about eight days. Subsequently, these cells begin to die in days 8-12 due to the depletion of nutrients in the well. Upon death, their color changes from green to white, causing the optical density to decrease as shown in Fig. 9. By contrast, cultures receiving less light tend to remain in a low metabolic state where little replication occurs. Due to the reduced nutrient consumption compared to cultures receiving a high light intensity, these cells appear to remain viable and to continue to grow well beyond day 8.
  • strong light conditions for example greater than about 40 ⁇ /cm 2
  • Fig. 10 shows that a similar result can be obtained when a pulse-width modulated duty cycle is varied at a fixed frequency.
  • the wells were again split into 6 groups of 8. This time, the intensity was fixed at about 60 ⁇ /cm 2 , the highest intensity used in the graph of Fig. 9.
  • the frequency of the duty cycles was fixed at about 10 Hz, and the pulse width of the duty cycles was set between about 10% and 100%.
  • the curve indicating a 100% pulse width is substantially identical to the curve of Fig. 9 showing the highest intensity.
  • the experimental results of the pulsed irradiation support the expected relationship that algal growth rate scales with the total photon flux, i.e. the light intensity integrated over the time of the duty cycles.
  • Fig. 1 1 shows a bar graph reflecting the optical density, i.e. the optical absorbance, of the curves at about 405 nm wavelength depicted in Fig. 10 on the twelfth day.
  • the optical density at this wavelength represents the algae growth in the individual wells 1 12.
  • the optical densities of cultures exposed to duty cycles of about 100%, 80%, and 60% pulse width all range about the same value of about 1 on an arbitrary scale.
  • the optical densities of cultures exposed to smaller pulse widths of about 40%, 20%, and 10% are smaller and substantially proportional to each other.
  • Fig. 12 is a bar graph based on the same samples as in Figs. 10 and 1 1 .
  • Fig 12 measures the fluorescence of the algae cultures after the addition of Nile red as indicator of lipid synthesis.
  • the excitation wavelength was about 450 nm
  • the measured emission wavelength was about 530 nm.
  • the bar graph of Fig. 12 indicates that a minor saturation effect is observable where the duty cycle was set to 100%.
  • the lipid production behaved substantially linear to the total photon flux in the range of 100% to 20% pulse width.
  • the fluorescence observed at a duty cycle of 10 pulse width is nearly equal to the fluorescence at a 20% pulse width.
  • Fig. 13 depicts a bar graph showing the quotient of fluorescence and optical density.
  • the values of Fig. 13 represent the lipid synthesis per algal unit, where algae exposed to a high photon flux of 100% or 80% pulse width also produce a comparably high amount of oil.
  • the ration of lipid per algae is somewhat reduced in the range of 60% to 20% pulse width.
  • the ratio rises to about the same levels as at duty cycles of 100% and 80% pulse width, respectively.
  • the high level of lipid production at a 10% pulse width is a notable deviation from the quasi-linear behavior outside the observed saturation levels.
  • Fig. 14 shows a bar graph of the lipid conversion efficiency of the photon flux.
  • the conversion efficiency is calculated by dividing the fluorescence intensity reflecting the lipid content by the total integrated photon flux provided to the cell culture.
  • the total integrated photon flux can be represented by the total integrated electrical current, possibly with a reverse calibration factor for consistency among the individual wells.
  • Figs. 1 1 through 14 depict results obtained with a constant intensity of LED light at a light intensity of about 60 ⁇ /cm 2 undergoing a pulse width modulation, corresponding results were obtained with a variation of the light intensity of the LEDs without pulse width modulation.
  • the smallest light intensity of about 10 ⁇ /cm 2 achieved a significantly higher lipid conversion efficiency that the next higher light intensity of about 20 ⁇ /cm 2 .
  • This phenomenon is caused by the presence of two reaction cycles involved in photosynthesis light-dependent reactions and dark reactions.
  • the light-dependent reactions generate energy and reduce power via ATP and NADPH intermediates while the dark reactions, called the Calvin cycle, consume these molecules along with C0 2 to produce glucose.
  • the light-dependent reactions occur on a timescale of less than about 1 ms, while the dark reactions occur over seconds.
  • the dark reactions are therefore rate limiting, causing photosynthesis to saturate at high light intensity.
  • the majority of light can be fully utilized, resulting in improved efficiency.
  • Pulsed illumination produces a similar effect by allowing the intermediates produced by light-dependent reactions to be fully consumed by the dark reactions before the next pulse arrives.
  • Fig. 15 illustrates a further embodiment of the invention, in which LEDs 222 installed in the optical microplate assembly 200 may also emit different wavelengths of light if so desired. More than one LED may be allocated per sample well 1 12, or individual LED bulbs may be capable of emitting two or more light colors. Not all sample wells 1 12 of the sample well plate 110 need to be exposed to the same type of light. For example, different light colors open possibilities for further comparative studies. Also, portions of the LEDs 222 may be pulsed while another portion emits varying light intensities, where these portions of varying duty cycles and varying intensities may overlap so that duty cycles may not only differ in pulse width, but also in frequency, in color, in intensity or a combination of one or more of these parameters. Examples of such variations will be described in connection with Fig. 18.
  • microplate assembly 200 bears different hatchings for different colors of light emission.
  • wells without any illumination are shown as white and are designated with reference numeral 258.
  • Fields illuminated with a red light color are shown with a horizontal hatching and are designated with reference numeral 252.
  • Illumination by green light is shown with a vertical hatching and is designated with reference numeral 254.
  • a combination of both red and green is shown as a cross-hatching and is designated with reference numeral 256.
  • the simultaneous illumination 256 with red and green light results in a light color that appears to be yellow.
  • Figs. 16A and 16B show examples of multi-color LEDs. Multi-color LEDs that include combined light sources under a single cover are available from several manufacturers
  • Fig. 16A is a schematic view of the two-color LED 222 powered through a constant- current driver 226. Because the LED 222 is configured for independent illumination in two colors, here red and green, it has three pins 244, 246, and 248 to provide specific illumination characteristics. Pin 244 is the neutral or common ground pin for both colors. Pin 246 is the power supply for the first color, for example red, and pin 248 is the power supply for the second color, for example green.
  • the LED 222 is one example of a two-color LED suitable for the arrangement of Fig. 15.
  • Fig. 16B is a schematic view of a three-color LED 322 powered through a constant- current driver 326. Because the LED 322 is configured for independent illumination in three colors, for example red, green, and blue, it has four pins 344, 346, and 348 to provide specific illumination characteristics. Pin 344 is the neutral or common ground pin for all colors. Pin 346 is the power supply for the first color, for example red; pin 348 is the power supply for the second color, for example green; and pin 350 is the power supply for the third color, for example blue.
  • the LED 322 is one example of a three-color LED suitable for the arrangement of Fig. 15.
  • Fig. 17 shows a PCB 230 for controlling two-color LEDs 222 as shown in Fig. 16A for illuminating a sample well plate 1 10 as shown in Fig. 15.
  • the PCB 230 carrying the control electronics for controlling the LEDs 222 of Fig. 16A.
  • the PCB 230 carries twelve serial LED drivers 332, each accommodating 16 programmable constant-current drivers 226, for a total of 192 constant-current drivers with up to 192 different outputs. Suitable serial LED drivers are, for example, available from Texas Instruments.
  • Each output can provide 128 discrete current levels (7-bit resolution) to its respective LED. The range of current can be selected by choosing an appropriate bias resistor.
  • the maximum current is about 80 mA, which is suitable for intermediate power LEDs which can provide up to about 1000 ⁇ /cm 2 .
  • the light intensity and on/off state of each LED 222 can be set using a high-speed serial peripheral interface (SPI) operating at up to about 12 MHz.
  • SPI serial peripheral interface
  • refreshing the state of entire array requires n clock cycles, where n is the number of LEDs 122, in the shown example n being 192.
  • the hardware-limited refresh rate of the array is about 50 KHz, or about 20 sec
  • the PCB of Fig 17 provides two power lines to each location of the LEDs 222, one line for pin 246, and one line for pin 248.
  • the neutral or ground poles for both colors are combined in pin 244. Because twice as many power lines lead to the LEDs, the PCB 230 also contains twice as many constant-current drivers 226 as the PCB 130 of Fig. 2, thus 192 instead of 96.
  • a PCB for controlling the three-color LEDs 322 of Fig. 16B includes 288 constant-current drivers and 288 power lines for a 96-well microplate.
  • the architecture of the PCB for the three-color LEDs 322 can be derived from the examples shown and is not shown.
  • Fig. 18 shows different illumination patterns realizable within one LED matrix by the example of five two-color LCDs 222A through 222E that may be mounted on the PCB 230 of Fig. 17..
  • the five two-color LEDs 222A, 222B, 222C, 222D, and 222E are powered with varying intensity, duty cycle and pulse width control.
  • An illumination in a first color is shown as a solid line, and an illumination with a second color is shown as a broken line over time.
  • LEDs 222A, 222B, and 222C shows the two colors with a 50% duty cycle at different relative intensities.
  • the pattern of LED 222A shows the two colors having a simultaneous illumination scheme, in which the second color is operated at half the intensity of the first color.
  • LED 222B shows an alternating pattern of 50% duty cycles, in which the two colors operate at the same frequency, but alternate their maxima and minima.
  • the first color operates between zero and full brightness, while the second color is dimmed at different intensity levels.
  • the two colors of LED 222C are operated different pulse widths at a 50% duty cycle, in which the second color is operated at twice the frequency as the first color.
  • the maxima of the second color additionally have half the intensity of the maxima of the first color.
  • LED 222D shows a more complex time- varying pattern, in which the first color (solid line) varies in both intensity and pulse width in time and the second color (dotted line) operates with a fixed intensity, pulse width and duty cycle.
  • LED 222E is operated in one color with an arbitrary intensity pattern varying over time.
  • the foregoing light patterns present only a small number of examples for possible variations of the illumination of samples wells 1 12. Many further variations and combinations of illumination patterns are well within the scope of the present invention.
  • optogenetics represent an area of photo-biology, in which a multi-color stimulation may be particularly useful in driving photo-systems with coupled light processes.
  • Figure 19 shows an example, in which the optical microplate assembly 210 is used for optogenetic control.
  • the green light activates a protein pathway, and the red light suppresses it.
  • red and green pulses By alternating red and green pulses in various ratios a quasi-linear control of this pathway can be achieved.
  • Biological test samples have been modified with light sensitive proteins to enable optogenetic control.
  • Fig. 20 schematically shows a further embodiment of a microplate assembly according to the present invention.
  • Two-color LEDS 222 individually controlled by constant-current drivers 226, are arranged above sample wells 1 12, while optical detectors are arranged underneath the sample wells 1 12.
  • the arrangement of Fig. 20 operates in analogy to the arrangements of Figs. 3 or 15.
  • the optical detectors 238 are optional. Instead, the sample wells that are part of a sample well plate (not shown), may be removed from the LEDs 222 for any measurements through the open tops of the sample wells 1 12. These measurements may be optical, but may also involve testing of chemical or physical properties other than light absorption or emission.
  • Optogenetic methods have expanded to include a wide range of signaling currencies. Light-gated protein interactions may occur reversibly within milliseconds.
  • the presented description provides multiplexed light induced assay.
  • Conventional light induced reactions are controlled through lamps, fiber coupled lights, steerable laser beams or other light sources compatible as illumination sources for multiwell plates. These systems do not easily permit automated individual high-throughput well control.
  • LED arrays for use with well plates are typically designed to serve as a large uniform light source for a single purpose - such as plant growth lamps or UV sterilization.

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Abstract

Un ensemble microplaque optique destiné à l'optimisation de processus photobiologiques comprend une plaque à cupules à échantillon comportant au moins deux cupules destinées à accueillir des échantillons d'essai. Une carte de circuits imprimés comporte un nombre de sources lumineuses optiquement séparées égal au nombre desdites cupules à échantillon. Chaque source lumineuse est appariée à l'une des cupules. Un dispositif de commande contrôle indépendamment l'intensité et/ou le cycle opératoire de chacune des sources lumineuses. Les cupules ont un fond transparent et les sources lumineuses, qui sont, de préférence, des LED, sont disposées sous les cupules. Les échantillons biologiques sont placés dans les cupules et exposés aux sources lumineuses indépendamment commandées. Par la suite, on mesure au moins une propriété optique de chacun des échantillons biologiques présents dans les cupules.
PCT/US2012/052640 2011-08-29 2012-08-28 Dispositif et procédé d'optimisation de processus photobiologiques WO2013033080A1 (fr)

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EP3938184A4 (fr) * 2019-04-11 2023-02-01 Allegro 3D, Inc. Procédé et appareil pour la fabrication sans masque à haut débit d'échafaudages en polymère et de tissus biologiques dans des plaques à plusieurs puits

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