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
  • G01N21/62—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
  • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell 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
  • G01N21/62—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
  • G01N2021/6417—Spectrofluorimetric devices
  • G01N2021/6419—Excitation at two or more wavelengths
• • 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
  • G01N21/6458—Fluorescence microscopy
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/06—Illumination; Optics
  • G01N2201/062—LED's
  • G01N2201/0627—Use of several LED's for spectral resolution

Definitions

• the present application relates to a reading device for slides carrying fluorescent deposits, as used in serology or in molecular biology analysis.
• test devices have appeared on microscope slides, or more generally on a flat support, where a series of deposits are aligned, which are the supports of a biochemical reaction during contact with a biological sample. After a possible reaction with fluorescent developers, the device must be read, that is to say the quantification of the reaction on each spot.
• the blades can be made of glass or transparent plastic.
• the number of spots on a blade can range from a few units to several thousand.
• the diameter of the spots is generally between 50 and 250 microns. These deposits are generally referred to as microarrays, an American term that has become internationally recognized.
• the deposits consist of nucleic acid sequences (DNA, deoxyribonucleic acid) and the biological sample to be tested contains a mixture of nucleic acid sequences, for example the amplified forms of its RNA (ribonucleic acid). messengers called complementary DNA (cDNA). Each hybrid V cDNA deposit corresponding to it.
• the hybridization reaction can be visualized and quantified by fluorescence, either that the cDNAs themselves are labeled, or that the hybridization zones are labeled with a specific dye.
• the biological sample to be tested contains a serum or a plasma, and reacts with a slide carrying reactive elements, for example proteins, cells, sub-cellular fractions, bacteria , virus, etc., previously deposited on the slide. After this first reaction, the slide is brought into contact with a developing reagent.
• reactive elements for example proteins, cells, sub-cellular fractions, bacteria , virus, etc.
• This signal may be a radioactivity, a colored reaction resulting from an enzymatic amplification, or a fluorescence signal. It is in the latter case that the resolution required by the increasing density of the deposits can be reached.
• the present application relates to a fluorescence reading device for serology slides or molecular biology hybridization. It also relates to any device comprising such a device, as well as the use of these devices and / or devices in analysis or diagnostic methods.
• illumination and “lighting” are synonymous. They refer to the excitatory light of fluorescence.
• the object of the present invention is in particular to provide a reading device for serology slides or molecular biology hybridization which avoids the disadvantages mentioned above, by ensuring the recording and processing of fluorescence signals, reliably and automatic.
• a particular feature of the devices of the invention resides in particular in the use of light-emitting diodes to provide a channelized excitation light, and in the arrangement of the excitation source and the recovery optics, giving great reliability to the reading and analysis.
• a particular object of the invention thus resides in a device for reading and / or analyzing slides comprising a reactive zone carrying micro deposits of reactive elements, said device comprising means for placing a blade, means for illuminating the reactive zone and recovery optics, characterized in that:
• the illumination means of the reactive zone comprise light-emitting diodes (LEDs) arranged in channels to allow illumination obliquely with respect to the optical axis, that is to say the axis in which the fluorescent light emitted by micro deposits is captured by the recovery optics;
• LEDs light-emitting diodes
• the device comprises at least two diode channels each emitting a clean excitation light; and -
• the recovery optics includes an objective forming the image of micro deposits on a sensor.
• the axis of the diode channels is oblique with respect to the optical axis with an angle greater than or equal to 15 °; and preferably greater than or equal to 20 ° and / or;
• the device comprises at least two diodes, each diode emitting a clean illumination light having a wavelength in the near UV or in the visible, the wavelengths being sufficiently spaced to allow the selective excitation of the fluorescent molecules ; preferably, the excitation wavelengths are spaced at intervals equal to or greater than 100 nm; and or
• each of the diodes follows a distinct direction
• the device comprises elements homogenizing the illumination of the area of deposits on the blade; and or
• each channel comprises successively at least one diode, a collimation optics, a filter intended to restrict the spectrum of the exciting light emitted by this diode and, optionally, an optical device intended to standardize the spatial distribution of the light and / or a condenser directing light towards the reactive zone of the blade; and or
• the recovery optics comprises a first objective having a focus coincides with the reactive zone of the blade, a filter holder, preferably a filter carousel, and a second objective forming the image; and or
• the device further comprises a solid base and / or a console, ensuring the maintenance of the means for placing the blade, the illumination means of the reactive zone and the recovery optics.
• three diodes are grouped in the same channel, in the vicinity of the optical axis.
• the device is controlled or operated by a dedicated software, typically which corrects the signal for all the causes of disturbances: randomness of deposit, irregularities of illumination and variations in the quality of the fluorescent reagents.
• the software is able to perform comparisons of fluorescence levels of the same deposit at different wavelengths and different deposits at the same wavelength.
• the software uses pre-recorded images of uniform, fluorescent or simply scattering surfaces to calculate a fine correction of the fluorescence of the deposits at different wavelengths; and or
• the device comprises three excitation light channels whose wavelengths are sufficiently wide apart to allow the selective excitation of different dyes; preferably, it comprises three excitation light channels, one centered around 365 nm, the second around 470 nm, the third around 594 nm. Other combinations of wavelengths are possible, from the near UV to the infrared; and or
• the exciter light uniformizing device is an optical guide of diameter adapted to allow multiple reflections of the light, or else a Kohler-type device.
• the device that homogenizes the illumination is of Kohler type.
• the homogenization can be improved by adding a diffuser, for example of the holographic type; and or
• the support is a microscope slide, or any other parallel-faced support
• the exciter light reaches the sample through the slide
• the objective of the sensor - side pick - up optic has a focal length equal to or less than that of the object - side lens, achieving a magnification of less than or greater than 1, depending on the sensor used; and or
• the filter carousel is motorized and coupled to the change of the excitation wavelength
• the recovery optics form the image of the deposits on a matrix sensor, for example of the CDD (Charge Coupled Device) type; and or
• the device comprises a blade identifier reader; and or
• the device comprises an automatic blade feeding device.
• Another subject of the invention relates to a serological analysis method comprising the incubation of a serology slide comprising a reactive zone comprising a series of deposits of biological agents, for example infectious agents, pathogens, allergens or autoantigens, with a serum sample of a patient, or a dilution thereof, and then revealing the antibodies (eg IgG and / or IgM) of the sample attached to the deposits by means of labeled reagents, characterized in that the reading and the analysis of the labeling (eg, fluorescence) are carried out by means of a device as defined above.
• biological agents for example infectious agents, pathogens, allergens or autoantigens
• the analysis method comprises three analysis wavelengths, selectively exciting three dyes: the first associated with the deposits, prior to the serological reaction, the second associated with the G-type immunoglobulin developer, and the third associated with the
• the dye associated with the deposition is excitable around 365 nm
• the dye associated with the G-type immunoglobulin developer is excitable around 470 nm
• the dye associated with the immunoglobulin developer of type M is excitable around 594 nm.
• Another object of the invention are the functionalities of the software which carries out the analysis and which preferably comprises:
• the three digital shots at the three wavelengths corresponding respectively to the control fluorescence 1 of the quantity deposited, the fluorescence 2 measuring the immunoglobulins of type G and the fluorescence 3 measuring the immunoglobulins of the M type and / or
• the invention therefore relates to a medium comprising software for implementing a device according to the present invention, implementing the formulas of Example 3 or other similar formulas.
• the invention also relates to the use of a device as defined above for serological analysis or in molecular biology.
• the analysis in molecular biology comprises the analysis of ribonucleic or deoxyribonucleic acids of a biological sample.
• the glass slide carries, for example, single-stranded DNA deposits intended to capture the fluorescent cDNAs originating from the sample.
• excitation wavelengths may be chosen in the vicinity of 543 nm for endogenous labeling with cyanine 3, in the vicinity of 635 nm for labeling with cyanine 5, in the vicinity of 488 nm for Hybridized part labeling with Sybr Green ® (Molecular Probes, Eugene, Oregon). The list is not exhaustive.
• kits including biological analysis, comprising the use of a device as defined above.
• the invention is applicable in many fields, in particular for histological or serological analysis in a medical, veterinary, environmental, agri-food, etc. context.
• the invention relates to a device adapted to the analysis of slides.
• the main components are shown in Figure 1.
• the device advantageously comprises a solid base (1) carrying a plate (2) for the purpose of maintaining the positioning of the various elements relative to each other.
• Each light emitting diode (3) is enclosed in a case (4) containing the homogenizing device, the assembly constituting a lighting channel fitted into a support (5) on which is fixed the blade holder chamber (6). Beyond this is screwed the first objective (7) which is itself fitted into a sleeve integral with the case of the filter carousel (8). Symmetrically, beyond the carousel, there is the second objective (9) screwed on the CCD camera (10).
• the lighting of the diodes by the housing (11), the positioning of the filter holder and the starting of the camera are controlled by a microcomputer, which also contains the interpretation software according to the invention.
• the optical axis of the recovery optics is horizontal.
• the light-emitting diodes preferably have an electric power of between 500 and 5000 mW.
• a set of convergent lenses is placed in order to give the light beam an approximate parallelism. A divergence of less than 10 degrees is preferred.
• a preferred value of the window is an interval equal to or less than 40 nm.
• the exciter wavelengths are spaced from each other, one in the near UV, one in the blue, one in the yellow, orange or red.
• a preferred value of the difference between the excitatory wavelengths is an interval equal to or greater than 100 nm.
• the homogenizing device consists of a diffusing surface at the inlet of a mixing optical guide consisting of a tube of transparent material with a high refractive index of between 20 and 40 centimeters in length. mm, and preferably between 6 and 10 mm in diameter.
• a mixing optical guide consisting of a tube of transparent material with a high refractive index of between 20 and 40 centimeters in length. mm, and preferably between 6 and 10 mm in diameter.
• an input lens concentrates the beam at a suitable angle, ( Figure 2)
• the homogenizing device is constituted by a diffusing surface placed at the focus of a collimating lens whose image is formed on the object (so-called Kohler assembly) (FIG. 3) .
• the diffuser is of holographic type, with very high efficiency.
• the blade to be measured is embedded in a removable support which is itself slid into a slot.
• the support is provided with an opening to the right of the active zone of the blade.
• a locking device such as an abutment ball immobilizes said support in the position where its window is in the axis of the optical recovery.
• the blade to be measured is directly inserted into the device through a slot and positioned by supports on slides, and springs on the opposite face.
• the blade is provided with a polarizer, for example an indentation on an angle, which prevents an erroneous introduction.
• the diode holder and the housing of the blade advantageously constitute a rigid assembly that can be made from various materials, possibly mixed. It may in particular be composed of (or based on) plastic material, metal and / or any rigid material at temperatures of 37 ° C or higher.
• the block is composed of black (delrin, rilsan) black or anodized aluminum alloy or painted to avoid reflections.
• the housing of the blade, or the removable blade holder are made of metal to prevent any distortion detrimental to maintaining the focus.
• the lenses have a focal length of between 20 and 40 mm.
• the camera-side lens has a lower focal length than the objective lens.
• the photoelectric matrix preferably has a number of pixels greater than 10,000.
• the CCD sensor can be that of a digital camera such as
• Nicon's "CoolPix” or preferably on a camera such as Hamamatsu 5885, Qicam Qicam, Sony SVS, or any other brand. It is not necessary to cool the sensor, the oblique orientation of the exciter light provides an excellent signal-to-noise ratio.
• the optical axis is vertical. In a preferred embodiment, it is horizontal.
• the blade protrudes from the measuring slot and allows the reading of a bar code or an electronic tag.
• the device according to the invention therefore comprises a barcode reader or a communication antenna with the electronic tag.
• a particular object of the present invention relates to blades provided with electronic tags on which the characteristics of the reading and the results of the reading can be recorded.
• a particular object of the present invention relates to the reading algorithm specific to serology slides.
• Three images are recorded, with three different fluorescences.
• the first image which relates to a fluorescence marker systematically attached to the deposit at the time of manufacture of the blades, is analyzed in terms of "clusters", that is to say related elements, which are the deposits.
• clusters that is to say related elements, which are the deposits.
• the advantage of doing so is that there is no position to position a grid on the image of the deposits, which would be anyway imprecise due to the inevitable distortions of the optics. It is also freed from the game in the slides of the housing of the blade and an automatic analysis becomes possible.
• the other two images corresponding to two other fluorescent markers, are respectively associated with the immunoglobulin G and immunoglobulin M responses of the patient.
• micro deposits being tainted with great uncertainty as to their quantity, it is an element of the invention to use one of the fluorescent labels as a control of the amount of material deposited and to use the corresponding signal to correct the signals representative of the serological reaction. It is another aspect of the invention to use images of a slide carrying a diffusing or fluorescent element to measure local illumination differences and then to use these results to correct the fluorescence of the deposits for the irregularities of illumination. It is another element of the invention to report the fluorescence associated with the antibodies of a subject having fixed on an antigen deposit, to that of a reference deposit of pure imunoglobulins, respectively of type G or type M, made fluorescent by the same fluorescent anti IgG and anti IgM developer respectively. This will be better understood in Example 3.
• the device of the invention further comprises a means for ensuring a displacement of the blade perpendicularly to the optical axis so as to be able to make images of different areas of the blade and also to be able to measure an increased number deposits.
• a means for ensuring a displacement of the blade perpendicularly to the optical axis so as to be able to make images of different areas of the blade and also to be able to measure an increased number deposits. According to this mode, up to 40 000 deposits would be readable, this number being an order of magnitude, and can be exceeded according to the techniques of deposits and the number of pixels of the sensor.
• an automatic blade feeder having a blade magazine, an identifier reading device and a transfer mechanism through the reading device is used.
• the devices according to the invention are suitable for any type of microscope slide.
• the term "blade” means any rigid object-holder element that can be used to immobilize a biological deposit, thereby delimiting a reactive zone. It may be for example a solid lamella, a membrane, a filter made transparent, etc.
• the blade can be made of (or based on) any known and conventional material, such as plastic, glass, nylon, biological polymers, silica, etc.
• Preferred slides are glass microscope slides. Their dimensions are usually standard, about 26mm x 76mm.
• the blades are provided with a polarizer, for example in the form of a notch in a corner.
• the blade (or microarrays) used for the diagnosis does not comprise more than 400 deposits, for example, which makes it possible to form the fluorescence image thereof. only one shot.
• the installation of the blade can be carried out either by a support, or by sliding the blade in a slot.
• This slot made of a black material is provided with countersinks avoiding deterioration of the deposits during an introduction, even erroneous.
• Figure 1 is a general diagram of a device according to the invention.
• Figure 2 is a schematic diagram of a mixer guide type diode lighting channel.
• Figure 3 is a schematic diagram of a diode lighting channel with Kohler type illumination.
• Figure 4 shows a control screen of the acquisition sequence.
• Figure 5 shows the result sheet corresponding to Figure 4.
• the fluorescences of the IgG and IgM controls are arbitrarily standardized to 10,000.
• the invention can be implemented for the analysis of serology slides.
• the blade In the serological mode, the blade carries a series of biological deposits ("spots"), for example infectious agents, pathogens, autoantigens or allergens. Deposits are carefully marked, this marking constituting an identification code.
• the liquid sample to be tested is a patient's serum, usually diluted in a suitable buffer.
• the treatment of the blade can be carried out by manual means, consisting of dipping in successive baths, or by automatic means as described in the patent application FR0403365.
• the invention can also be implemented for molecular biology analysis, either in fluorescence mode, as previously described, or in optical density mode.
• the blades carry a nylon support on which the spots are deposited, the lighting is diffused by the nylon and the spots absorb the light. They are detected and quantified as dark spots on a light background.
• Example 1 Description of a device according to the invention This embodiment is described in relation to FIG.
• the light-emitting diodes are:
• Nichia-NCCUOOl or NCCU0033 for UV excitation associated with Semrock filter FF 409-Ex02; Lumileds LXHL -MBlD for excitation at 470 nm, associated with the filter
• the homogenizer is of the light guide type.
• the blade holder is made of black DeMn.
• Recovery optics include:
• the image is projected on the sensor of an SVS Vistek SV084 "S” camera with 658x494 pixels.
• the assembly is controlled by a microcomputer carrying control, reading and analysis software according to the invention.
• a serology slide carries 12 deposits, arranged in 3 rows and 4 columns at a pitch of 0.5 mm.
• the first two deposits of the first line are immunoglobulin IgG and IgM controls respectively.
• Other deposits are infectious agents.
• the slide is first incubated for thirty minutes with the patient's serum. Then, after rinsing, the slide is incubated for ten minutes with the secondary antibody reagents which are fluorescein-labeled goat anti-human immunoglobulin G antibody, and a human anti-human immunoglobulin goat anti-human Ig antibody. Texas red, mixed. Deposits having fixed IgGs have excitable green fluorescence at 470 nm and IgM-containing deposits have excitable red fluorescence at 594. Only the image excited at 365 nm is shown ( Figure 4).
• the algorithm aims to correct fluorescence spots: hazards of deposits, irregularities of illumination, and reagents.
• Zi 2 and Zi 3 are called relative fluorescences. They are used to quantify the IgG and IgM antibody sample concentrations that are specific to the i antigen.
• Relative fluorescences for example Zj 2 , have the expected properties. Indeed :
• spot size, illumination, fluorescent reagents, relative fluorescence z; 2 is proportional to the specific signal Fj 2 which represents the intensity of the serological reaction, or the hybridization reaction.
• Fj 2 represents the intensity of the serological reaction, or the hybridization reaction.

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• Health & Medical Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Physics & Mathematics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

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• 2004
  • 2004-08-02 FR FR0408520A patent/FR2873815B1/fr not_active Expired - Fee Related
• 2005
  • 2005-08-01 WO PCT/FR2005/002009 patent/WO2006024772A1/fr not_active Ceased
  • 2005-08-01 AU AU2005279081A patent/AU2005279081A1/en not_active Abandoned
  • 2005-08-01 JP JP2007524369A patent/JP5060292B2/ja not_active Expired - Fee Related
  • 2005-08-01 CA CA002576779A patent/CA2576779A1/fr not_active Abandoned
  • 2005-08-01 US US11/572,694 patent/US7812952B2/en not_active Expired - Fee Related
  • 2005-08-01 EP EP05793765A patent/EP1774295A1/fr not_active Withdrawn

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