Classifications

• • 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
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/76—Chemiluminescence; Bioluminescence
• • 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
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
• • 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
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N2021/6491—Measuring fluorescence and transmission; Correcting inner filter effect
  • G01N2021/6493—Measuring fluorescence and transmission; Correcting inner filter effect by alternating fluorescence/transmission or fluorescence/reflection
• • 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
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/76—Chemiluminescence; Bioluminescence
  • G01N21/763—Bioluminescence

Definitions

• the present invention relates to luminescence imaging devices and methods. BACKGROUND OF THE INVENTION
• the invention relates principally to a luminescence imaging device comprising: a light-tight enclosure comprising a scene adapted to receive a sample to be imaged emitting a first light signal carrying a. luminescence information of the sample, a light source " adapted to generate incident light in the direction of the scene, the interaction of said incident light and the sample forming a second light signal, a detection device adapted to one detects light signals having a luminescence spectrum, and for storing a first image on the basis of light signals having a luminescence spectrum, and secondly for detecting light signals corresponding to the reflection of incident light from of said light source on the sample, and for storing a second image on the basis of the light signals corresponding to the reflection of incident light from said light source on the sample, an electronic control unit adapted to define a plurality of time frames, each time frame during a time corresponding to the acquisition and the tockage the second image, said electronic control unit being also adapted to control the light source to generate the incident illumination during each time frame,
• the document WO 01/37, 1955 describes an example of such a device.
• This device has a "live" mode for taking a plurality of photographic representations of the sample. Then, when the sample emits light due to a chemical reaction taking place inside the sample (luminescence phenomenon), this device can take luminescence images of the sample, thus detecting the amount of light emitted by the sample due to the chemical reactions in question.
• this device does not make it possible to follow the rapid temporal evolution of the information relating to the luminescence. If the sample moves during measurement, (especially if it is necessary for the sample to move during the measurement, because the measurement corresponds to muscle activity that can not be recorded for an anesthetized sample), such installation will not be suitable.
• the present invention is intended to provide a device for overcoming these disadvantages.
• a device of the kind in question is characterized in that it comprises separation means adapted so that during each time frame, the detection device acquires both a first data relating to the luminescence information and a second data relating to the second light signal.
• the detection device has a plurality of pixels each adapted to detect light signals coming from a given region of the enclosure; the detection device is adapted to store the first image according to a first sampling of time frames and to store the second image according to a second sampling of time frames, the first sampling having a frequency different from that of the second sampling; the frequency of the first sampling is lower than that of the second sampling; the second light signal comprises a light signal relating to the reflection of incident light from said light source on the sample, and a light signal of autofluorescence of the sample subjected to said incident light, and the imaging device; is adapted to separate the light signals having a luminescence spectrum from the autofluorescence and reflection light signals; the detection device comprises: a first detector adapted to detect light signals carrying a luminescence information, and - a second detector adapted to detect light signals corresponding to the reflection of incident light from said light source on the sample ; the separation means comprise a filter arranged at the input
• the invention relates to a luminescence imaging method comprising the following steps: having a light-tight enclosure comprising a scene comprising a sample to be imaged emitting a first light signal carrying a luminescence information of the sample,
• an electronic control unit defines a plurality of time frames, and controls a light source to generate incident light in the direction of the scene during each time frame, the interaction of said incident light and sample forming a second light signal, a combined light signal corresponding to a combination of the first and second light signals arriving at a detection device during each time frame,
• the detection device which is adapted for firstly detecting light signals having a luminescence spectrum and for storing a first image on the base of the light signals having a luminescence spectrum, and secondly for detecting light signals corresponding to the reflection of incident light from said light source on the sample, and for storing a second image on the base light signals corresponding to the reflection of the incident light from said light source on the sample, acquires both a first data relating to the luminescence information and a second data relating to the second light signal, each frame of time during a time corresponding to the acquisition and storage of a second image.
• each time frame is divided into a time light during which the light source emits, and a dark instant during which the light source does not emit, the combined light signal being a temporal combination of the first and second light signals, and, in step (b), controlling a first detector adapted to detect light signals having a luminescence spectrum from being in a detection state during the dark instant, from being in a state of non-detection during the clear state, and to a second detector adapted to detect light signals corresponding to the reflection of the incident light on the sample to be in a detection state at least during the clear moment; during step (b), a first detector detects the first light signal carrying the luminescence information, and a second detector detects the light signal corresponding to the reflection of incident light from said light source on the sample, preventing light signals corresponding to the reflection of incident light from said light source on the sample to reach the first detector; the detection device comprises a plurality of pixels, and during each
• the invention relates to a luminescence imaging method comprising the following steps:
• a sample to be imaged on a stage of a light-tight enclosure comprising an outer surface including an interior, the interior of said sample emitting, through the outer surface, a light signal in the chamber, the sample also emitting a second signal of a different nature of the light signal,
• one or more of the following arrangements may be implemented: for each time frame, a luminescence image of the sample is formed directly by detection of the light signal, and for each time frame, a cinematic image of the sample is formed directly by detecting the second signal; for each time frame, a cinematic image of the sample is formed directly by detecting the second signal; during a detection subperiod, included in the detection period, and comprising at least the first and second time frames, an accumulated light signal is detected, and forming said luminescence image for each time frame by processing said signal luminous on the basis of said corresponding cinematographic images; for at least one time frame, and preferably for each time frame:
• each image comprises a plurality of pixels (x, y) each corresponding to a zone (Du, Dv, Dw) of the enclosure;
• the luminescence signal has a spectrum, and the second signal is a light signal having a spectrum remote from the spectrum of the luminescence signal;
• each time frame has a clear time during which the sample is illuminated and during which the second signal is detected, and a dark time during which the sample is not illuminated and during which the first
• FIG. 1 is a schematic perspective view of an imaging device
• FIG. 2 is a schematic plan view of the inside of the enclosure of the device of FIG. 1 according to a first embodiment
• FIG. a schematic diagram of an example of data processing
• FIG. 4 is a diagram showing an example of processing carried out by the processing unit of FIG. 3, FIGS.
• FIG. 5a, 5b and 5c are graphs showing the states of the light source, the second and the first detector, respectively, according to an alternative embodiment of the invention
• - Figure 6 is a view corresponding to Figure 2 for a second embodiment of the invention
• Figure 7 is a view corresponding to Figure 2 for a third embodiment.
• FIG. 1 schematically represents an imaging device 1 intended to take an image of a sample 2 and a display screen 3 comprising a display 4 presenting an image of the sample 2.
• the imaging device described here is a luminescence imaging device, for example in bioluminescence, that is to say intended to take an image of a sample 2, such as in particular a small laboratory animal, for example mammal, emitting a light from inside its body.
• This light is for example produced following a chemical reaction inside the body of the small animal.
• a chemical reaction one can for example having beforehand a small genetically modified laboratory animal to include a gene coding for a protein which has the particularity of emitting light when it reacts chemically with a given complementary chemical entity, such as a molecule, an atom or an ion.
• said complementary molecule Before placing the small laboratory animal 2 in the imaging device 1, it is provided to it, for example by inoculation, said complementary molecule and, optionally, the time is allowed for it to reach the possible reaction site with the protein.
• the quantity of light released locally by the chemical reaction is a factor of the quantity of the protein produced, and thus makes it possible to locally measure the level of expression of the gene.
• the experiment in question may for example consist of measuring the muscular activity generated by an event in a laboratory animal, by detecting the amount of light emitted by the coelentherazine-Aequorin substrate-photoprotein pair which reacts with a complementary complementary chemical entity.
• the entity in question is for example the calcium arriving in the vicinity of the photoprotein at the level of the axons.
• the device described here can also be used to implement a phosphorescence or delayed luminescence imaging method.
• a molecule adapted to emit light for a sufficiently long time, of the order of a few minutes, by phosphorescence is illuminated ex-vivo to trigger this phosphorescence.
• the molecule is then introduced inside the small laboratory animal and can be used as a light tracer.
• the concentration of the molecule in a location of the organism, for example because a certain reaction takes place at this location, and that the molecule in question participates in this reaction is detectable by the device described below and makes it possible to characterize the reaction in question quantitatively or qualitatively.
• the small laboratory animal 2 is placed in a light-tight enclosure, for example by closing a door 6 or the like.
• the chamber comprises, as represented in FIG. 2, a scene 7, for example forming the floor of the enclosure, on which the small laboratory animal 2 is placed, and a light source 8 generating incident lighting in the direction of scene 7 (for example transmitted by an optical fiber). Due to the reaction described above, the small laboratory animal 2 naturally emits a first light signal which carries luminescence information of the small animal. In addition, because of the light generated by the light source 8, a second light signal corresponding substantially to the reflection by the small laboratory animal 2 of the incident light 8 is also emitted in the enclosure 5.
• This second light signal may also include a portion corresponding to the auto-fluorescence of the sample 2 due to the illumination by the light source 8.
• the detection device comprises a first detector 10 adapted to detect light signals from sample 2 having a luminescence spectrum.
• a first detector 10 is for example an ICCD, an EMCCD (internal multiplication CCD) or other, having a matrix of pixels arranged in rows and columns.
• the detection device 9 also comprises a second detector 11, which is for example a conventional CCD camera or intensified having a large number of pixels arranged in columns and rows.
• the first and second detectors 10, 11 are each disposed on a face distinct from the enclosure 5.
• the scene 7 carrying the sample forming the floor of the enclosure, the scene is immobile with respect to the detection device at least during the entire observation period.
• the light source 8 continuously emits incident light towards the scene so that the combined light signal corresponds to a spectral combination of the first (carrying the luminescence information) and the second light signal.
• the combined light signal is separated by a splitter plate 12, which separates the signals on the basis of their wavelength.
• a splitter plate 12 which separates the signals on the basis of their wavelength.
• Such a separating blade is for example a dichroic mirror or "hot mirror” type mirror separating the visible from the infra-red.
• the signal The illuminated luminescence information light is substantially completely transmitted to the first detector 10, while the second light signal is almost entirely transmitted to the second detector 11.
• a filter 13 at the input of the first detector 10, adapted to prevent wavelengths that do not correspond to this signal from reaching the first detector 10.
• the signal reaching the first detector 10 only corresponds to the luminescence from inside the sample 2
• the auto-fluorescence signal emitted by the sample 2 under the effect of the light source 8 has a wavelength different from the signal in question.
• a light source 8 which emits an incident light having a suitable spectrum, distributed beyond the range of wavelengths emitted by luminescence.
• infrared illumination centered at a wavelength substantially equal to 800 nm may be used when the luminescence spectrum has a maximum wavelength of 700 nm or less. As shown in FIG.
• an electronic control unit 14 which defines a plurality of time frames each lasting a few milliseconds, corresponding substantially to the time required to acquire and store a cinematographic representation of the scene 7 using the second detector 11.
• This cinematographic representation comprises a plurality of coordinated data pairs, luminous property (intensity, ).
• These time frames can be set to have a duration determined by the user, if the latter wishes a given acquisition rate, such as 24 images per second, for example or other.
• the signal previously produced in the second detector 11 is read and stored in a second memory 21, as well as coordinate information relating to each pixel and a new acquisition starts at the second detector 11.
• the signal produced by the first detector 10 is stored in a first memory 20 as well as coordinate information relating to each pixel.
• a processing unit 15 is adapted to read the data stored in the first and second memory 20, 21, to store them and / or to display on the screen 4 the corresponding images.
• the first detector 10 will be triggered to be read only approximately every 0.3 seconds, which remains a relatively short time in the dynamics of the phenomena observed.
• the processing unit 15 is adapted to recalculate for each photographic representation acquired by the second detector 11 a significant value of the luminescence information for each of these representations, for example in the manner schematically shown in FIG.
• FIG. 4 shows at the top four images of the sample 2 successively acquired by the second detector 11, at successive times T1, T2, T3 and T4. As shown roughly in Figure 4, sample 2 may have moved forward of the instant T1 at time T4, of a given displacement D, which is intentionally exaggerated in FIG. 4 for explanatory purposes.
• the embodiment presented with reference to FIG. 2 implies a certain constraint at the level of the light source 8, because it must illuminate the sample 2 in a range of wavelengths such that the This fluorescence of the sample 2 has a spectrum that is far from the emission spectrum of the sample 2.
• a pulsed lighting of the order of the video frequency is for example provided from a laser diode, or other.
• the electronic control unit comprises a sequencer 17 which controls the light source 8 to generate the incident illumination during a clear time t c of the time frame T. This incident illumination is for example synchronized with the acquisition of the luminescence signal detector.
• the electronic control unit controls the light source to generate incident light continuously, and therefore during each time frame.
• incident lighting is emitted in the direction of the scene, so that a light signal mainly comprising a reflection of the incident light by sample 2 reaches the detection device 9.
• the first detector 10 is then blinded, so that it can not detect any signal.
• a mechanical shutter located at the input of the first detector 10 is used, or an electrical shutter obtained for example by a voltage inversion exerted at the terminals of the first detector 10 is used. detector.
• the electronic control unit commands to cut off the incident lighting, so that a few moments after t c , only the luminescence coming from the sample 2 is detectable in the enclosure 5.
• Au During this dark instant t o , the first detector 10 is again in the detection state, and detects the light signal carrying the luminescence information from the sample 2.
• the combined light signal therefore substantially corresponds to a temporal combination of the first and second signals, the first light signal (luminescent) being predominant during the dark time, and the second light signal corresponding to the photographic representation of the light. sample being the majority during the clear moment t c .
• the sample 2 also emits a signal carrying the luminescence information has no influence on the signal detected by the signal.
• second detector 11 It can also remain in acquisition mode during dark time t o , as shown in Figure 5b, without significant influence on the measurement made by this detector.
• FIG. 6 now presents a second embodiment of the invention, which applies both to case of a continuous light source in the case of a pulsed light source as presented above in relation to Figures 1 to 5c.
• a splitter plate 12 is not necessarily used, and the separation of the combined light signal is carried out integrally by the filter 13.
• a reconstruction unit of the processing unit 15 has been previously calibrated to report the images obtained by these two detectors in a common repository, which may be a reference linked to one or the other another of these detectors, or another repository.
• the detection is performed by the first and second detectors 10, 11 during an observation period during which the enclosure is kept closed, containing the sample 2, which can move in the enclosure. During this observation period, a plurality of time frames are defined during which the detection takes place. The signal detected by each detector during each time frame can be converted directly into a luminescence image
• each detector may comprise a plurality of pixels organized in rows and columns in a plane of the detector, each pixel being identifiable by its coordinates.
• Each pixel of coordinates (x; y) detects a signal from a coordinate region (Du, Dv, Dw) of the enclosure corresponding for example to a cone whose base is formed by the pixel (x; y).
• the device described in FIG. 6 makes it possible to implement the method described for the first embodiment, in its first variant, with reference to FIG. 2.
• filter 13 may be dispensed with.
• a luminous signal emitted by sample 2 has been used to obtain the cinematographic image, as in FIG. particularly the reflection by the sample of a light signal emitted by a light source 8.
• the signal making it possible to obtain information on the position of the sample in the enclosure n ' is not necessarily an optical signal.
• the first detector 10 any type of detector to obtain information on the position of the sample 2 in the chamber.
• a detector may for example be constituted by a thermal detector adapted to detect a release of heat from the mammal 2. Therefore, in such an embodiment, it is no longer necessary to illuminate the sample 2 at the same time.
• the luminescence signal detected by the second detector 11 and the thermal signal detected by the first detector 10 are of such a different nature that the separation of these signals is done naturally by the use of detectors of a very different nature. The thermal detector is not disturbed by the luminescence signal emitted by the sample and the detector of the luminescence signal is not disturbed by the signal thermal emitted by the sample.

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• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

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• 2006
  • 2006-10-04 WO PCT/FR2006/002234 patent/WO2007042641A1/fr active Application Filing
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