CN101351735B - Device and method for capturing image of a sample originating from organism - Google Patents

Device and method for capturing image of a sample originating from organism Download PDF

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Publication number
CN101351735B
CN101351735B CN200680049546XA CN200680049546A CN101351735B CN 101351735 B CN101351735 B CN 101351735B CN 200680049546X A CN200680049546X A CN 200680049546XA CN 200680049546 A CN200680049546 A CN 200680049546A CN 101351735 B CN101351735 B CN 101351735B
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image
sample
cell
light
luminescent
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CN101351735A (en
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秋吉龙太郎
铃木浩文
福冈庄尚
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Olympus Corp
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Olympus Corp
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Abstract

A novel apparatus and a novel method of imaging a luminescence phenomenon of a sample of biological origin using an optical image formation device are provided. In the inventive method and apparatus, an illumination image acquired by illuminating the sample and a luminescence image acquired with the light emitted by a luminescence phenomenon of a cell in the sample without illuminating it are superposed to generate a superposition image. In the superposition image, an analysis region is determined. Thereby, even when the light detected in a luminescence observation is feeble, a luminescent cell and/or a luminescent site in a cell in a sample can be specified.

Description

Obtain the apparatus and method of the image of sample originating from organism
Technical field
The present invention relates to a kind of method and apparatus of the low-light level imaging that the sample originating from organism (or sample) such as cell, bacterium and other biological sample is sent, in particular, the present invention relates to by the low light level imaging that biological luminescence is produced this sample is observed method and apparatus with various measurements.
Background technology
In recent years, the increasing technology of using biogenic imaging samples in the research of life science, this sample originating from organism such as cell have for example GFP of fluorescence protein, and/or photoprotein, for example luciferase, photoprotein are expressed in arbitrary cell by transgenosis.Can express fluorescence or photoprotein, simultaneously with cell in addition arbitrarily certain protein (protein labeling) mix, and, if this fluorescence or photoprotein will only be expressed based on the activation of specific hereditary control band in the downstream that fluorescence or photoprotein encoding gene are inserted or replace specific hereditary control band.Therefore, up to now, in cell and/or outside in the various biological phenomenons that take place, want to make that the fluorescence or the photoprotein of expressing with the activation that protein is represented or heredity is regional are observed, and using optical microscope to detect and/or surveying observed protein or when and how hereditary zone is represented, and/or the protein that obtains in cell and outside will how to show, just use this fluorescence or photoprotein as reporter molecules or probe molecule.
Fluorescence protein, relatively stable in cell such as GFP, and the fluorescence that therefrom sends is bright, therefore be extensive use of these protein stablize with the imaging of carrying out the cell inner structure easily (for example, referring to Mason (1999) Fluorescent and luminescent probes for biological activity, second edition).On the other hand, such as the photoprotein of luciferase by chemiluminescence and/or the luminous exciting light (exciting light is harmful for cell or other biological sample usually) that does not have the activating fluorescent dyestuff of biological luminescence.Therefore, for viewing duration under optical microscope, the expectation photoprotein does not form injury to it as a kind of probe to the biological specimen imaging, and has also reported the example that some use the imaging technique of this photoprotein.For example, people such as Rutter are by using the gene expression imaging to luciferase in single living cell of photon counting video camera, observe and in the signal that insulin sends, comprise MAP kinase (Rutter, White, Tavare (nineteen ninety-five) " Involvement of MAPkinase in insulin signalling revealed by non-invasive imaging of luciferase geneexpression in single living cells " Current Biology Vol.5890-899.).People such as Sternberg have also reported use photon counting video camera and cooling CCD video camera detection bioluminescence (Sternberg, Eberl, Kongsbak, Molin (1997), " Detection of bioluminescencefrom individual bacterial cells:a comparison of two different low-light imagingsystem " J.Bioluminescence and Chemiluminescence Vol.12:7-13.).In addition, people such as Takasuka also report by using the photon counting video camera gene expression imaging of luciferase to be observed the dynamic change (Takasuka of prolactin promoter activity in single living cell, White, Wood, Robertson, Davis (1998), " Dynamic changes in prolactin promoteractivation in individual living lactotrophic cells " Endocrinology Vol.139:1361-1368.).
As mentioned above, in to above-mentioned cell when the fluorescence of gene expression and/or photoprotein imaging, the fluorescence of this fluorescence protein is brighter relatively, therefore can obtain the relatively clearly MIcrosope image of their fluorescence by the conventional optical microscope that is equipped with in the imaging system.Yet, light intensity from the photoprotein of cell or other biological sample (after this being called " cell etc. ") is very weak or trickle usually, therefore be difficult to be used for the video camera and the luminous MIcrosope image (can not observe the luminous of photoprotein by human eye in fact, usually) of imaging system acquisition of normal MIcrosope image.Therefore, in conventional system to the photoprotein imaging, be exclusively used in video camera or the image capture device of surveying low-light, be installed on the optical microscope (still such as hypersensitivity video camera or photon counting video camera, even have the hypersensitivity video camera, still need be on the light receiving surface of video camera to from the light integration of sample to minority minute or tens of minutes to produce piece image).
In this way, in the fluorescence or luminous observation of optical microscope, only be the light that protein sends on the light principle in the MIcrosope image.Therefore, not having the conditioned disjunction form in the zone that protein exists in sample is can not be observed.Observing under the situation of fluorescence protein, yet, can observe the form of cell and condition etc. because they have brighter fluorescence relatively, and owing to used exciting light from the autofluorescence emission of material outside the fluorescence protein in the cell etc.On the other hand, under the situation of the luminous observation of photoprotein, light from sample, very faint such as light from photoprotein, to such an extent as to can not produce image, unless its integration is some to tens of minutes, and, do not having outside the photoprotein under the radiative situation of material, just be difficult to obtain in sample position such as cell, form and these change (can not know whether that in the zone that does not have light cell exists).Yet at viewing duration, when observing living cells sample etc. especially for a long time, light intensity can change, and/or cell can move or be out of shape, and therefore is difficult to determine which part of which cell or cell is luminous.For example, the nervous process in luminous change phenomenon and some neurocyte in the very difficult analysis of cells matter.
In some prior aries,, carry out viewed in transmitted light with the luminous observation of photoprotein for the position and the form of observation of cell etc.In the case, however the light quantity in viewed in transmitted light and the luminous observation on the camera light receiving plane is very different.Therefore,, use different cameras (using different object lens), and different thus video cameras produces luminescent image (from the image of the light of photoprotein) and illumination image (image of transmitted light) respectively for viewed in transmitted light and luminous observation.Therefore, MIcrosope image illustrates the form of cell etc., and on different images, create the MIcrosope image of the distribution of showing photoprotein, the actual distribution of photoprotein or position are (usually in this just feasible very difficult accurately definite sample, video camera can be offset for the size and/or the position of the visual field of viewed in transmitted light and luminous observation, and actual distribution and the position of determining in this case, photoprotein in sample just become more difficult).In addition, when once observing a lot of cells, be not easy to find corresponding luminescent cell in corresponding with cell in the illumination image, the luminescent image.
In " luminescence imaging " of above-mentioned use photoprotein, if can survey or limit the distribution of photoprotein in the sample, obtain the form of cell in the same sample and/or condition etc. simultaneously, just can widen the range of application of " luminescence imaging ", and become useful in the analysis of the reaction that will in for various bioprocess, relate to etc.As mentioned above, yet, in the prior art, because the light of photoprotein is very faint and do not comprise that in sample the zone of photoprotein does not have light, just be difficult to about in some cell where and/or in the sample which cell show the information of photoprotein, although can survey the existence that photoprotein is represented in the sample or do not exist.In fact,, can carry out weak light detection, but in the case, the image that can obtain only is a mosaic image, and therefore is difficult to carry out labor, limit the zone that wherein will measure luminous quantity simultaneously by hypersensitivity image detection element.
Summary of the invention
Therefore, one object of the present invention is to provide a kind of apparatus and method, be used for above-mentioned biological luminescence is produced the cell and/or the other biological imaging samples of the low light level, wherein obtain the luminescent image of cell etc. by this way, wherein can determine the luminous position in luminescent cell and/or the cell.
Another object of the present invention provides a kind of like this method and apparatus, and it can obtain data from the specific region, and is used to analyze various phenomenons in cell.
According to an aspect of the present invention, the device that obtains biogenic sample image by optical imaging device is characterised in that this device comprises: illumination section is used to shine sample; Illumination image obtains part, is used to obtain the illumination image of sample; Luminescent image obtains part, and it obtains the luminescent image of cell in the sample; And image processing section, be used to superpose illumination image and luminescent image, with the generation superimposed image, and institute's analyzed area in definite or the appointment superimposed image.In this structure, optical imaging apparatus can be an optical microscope usually.In the case, illumination image is to use from the illumination light of illumination section to pass through to carry out the transmitted light MIcrosope image that viewed in transmitted light obtains under optical microscope, and luminescent image is the illuminating microscope image that obtains by luminescence phenomenon in the observation by light microscope cell.This illumination image obtains part and luminescent image and obtains and partly preferably include shared MIcrosope image collecting device, by this equipment acquisition of transmission light microscope image and illuminating microscope image.
According to said structure, superimposed by the luminescent image that the luminescence phenomenon of observing in the sample cell obtains with the illumination image that obtains by the illumination sample, therefore wherein the form of cell and/or condition etc. can be observed, and therefore, can be more in the past than the luminous position of in sample and/or cell, locating cell in the luminescent image more accurately.Especially, when obtaining illumination image (transmitted light MIcrosope image) and luminescent image (illuminating microscope image) by all purpose microscope image capture device, it is very easy that superimposed image just becomes.Even in viewing area, have in a lot of cells of similar form, wherein by watching with luminescent image and illumination image stack image that obtain, that cellular morphology in the sample is shown, can determine luminous cell immediately, and therefore, the zone that will watch in image is promptly analyzed (analyzed areas) such as zones that will monitor in the luminescence phenomenon and just is easy to found.In addition, can in luminescent image, obtain the condition of sample, therefore, even when cell moves and/or is out of shape, can easily judge which cell, wherein or how to move and/or be out of shape, and corresponding, can easily find luminous how the variation.In addition, according to structure of the present invention, although only by in luminescent image, observing, in the cell density of sample, be difficult to the overlapping of a plurality of cells that difference takes place at random, but can specify the zone with suitable cell density and judge whether to exist superpose cell subsequently, making thus can more accurate analysis luminous quantity.
In device of the present invention, as mentioned above, transmitted light MIcrosope image that superpose each other and illuminating microscope image are preferably by the collection of shared MIcrosope image collecting device.At this moment, research according to the present application people, know, be designed to make that at the lens subassembly of object lens that form sample image on the light receiving surface of MIcrosope image collecting device and optical microscope the square value of ratio (NA/ β) is 0.01 or bigger between numerical aperture (NA) and the magnification (β) that is incident upon the sample image on the light receiving surface of object lens, the light that the image capture device of use such as ccd video camera only sends from individual cells can produce image (referring to Japanese patent application No.2005-267531).In addition, surprising, this image-forming condition can be used for all imagings that come from biological specimen, and it is difficult to imaging in the past, even and known that under optical microscope eyes are difficult to the faint luminous component of directly seeing, such as bioluminescence, the image of cell etc. can obtain (for example 20 minutes) in the short period section.In addition, optical condition according to inventor's research, known the optical condition that the square value of the magnification (β) of numerical aperture (the NA)/projected image of image capture device object lens is represented be 0.071 or higher in, can in 1 to 5 minute short time, carry out imaging, make to obtain even the image of available cell etc. in graphical analysis.Therefore, in an embodiment of apparatus of the present invention, by light receiving surface and object lens and the lens subassembly that the MIcrosope image collecting device is set, square value with its NA/ β is set to 0.01 or higher, be more preferably 0.071 or higher, just can create the better superimposed image of transmitted light MIcrosope image and illuminating microscope image.
In addition, in device of the present invention, can also provide the fluoroscopic image that obtains the cell fluorescence image to obtain part.When optical imaging apparatus was optical microscope, fluoroscopic image was to carry out the fluorescence microscopy images that the sample Fluirescence observation obtains with optical microscope.Equally in the case, fluorescence microscopy images and illuminating microscope image can obtain by shared MIcrosope image collecting device.Well known to a person skilled in the art, in Fluirescence observation,, can survey various intercellular reactions by using the various fluorescent dyes responsive to environmental baseline such as pH, Ca concentration or film potential etc.Therefore, should be appreciated that fluoroscopic image that this Fluirescence observation is obtained and luminescent image are in conjunction with disclosing in the cell relation between phenomenon in the luminous position and various cell.Need explanation, the spatial relationship in the sample between cellular morphology etc. and luminous position can easily obtain by stack luminescent image and illumination image, and, according to this feature, can also further increase use in conjunction with luminescent image and fluoroscopic image.
In an embodiment of apparatus of the present invention, can provide the display part that shows superimposed image, easily on the display part, to watch the luminous position in the sample or in the cell to the user.In addition, in device of the present invention, can provide recording portion to assign to write down superimposed image, be used for the analysis and the demonstration of image.
In addition, as mentioned above, in observation of cell is luminous, especially in the process to transgene expression such as the luminescence imaging of the photoprotein of luciferase, photoprotein, the luminescent protein plasmagene is introduced cell, make and express corresponding photoprotein, mix simultaneously with protein (object of observation) arbitrarily, perhaps, this gene can be replaced or insert the downstream in genetic regulation district, and the photoprotein of the expression that therefore will obtain is used for surveying the arbitrary target protein of when and where expressing measurement as reporter molecules.Like this, when cell receives one of various excitations, in cell, often express the protein that to report by photoprotein.Therefore, in an embodiment of apparatus of the present invention, can be provided for providing the cell excitation of arbitrary excitation to supply with part to cell.For example, this cell excitation is supplied with part and can comprise the part that at least one is selected from had the group with the lower part: reagent supply part provides reagent to cell; The adjustment part, the temperature that is used to regulate sample; And the gas supply part branch, be used for providing gas to cell.
In addition, in one embodiment, said apparatus of the present invention can be the device that obtains the image of biogenic sample, comprises optical microscope; The MIcrosope image collecting device, the MIcrosope image that is used to gather observation by light microscope; And image processing equipment, be used to obtain MIcrosope image as view data and carries out image processing, it is characterized in that: this image processing equipment comprises the image overlay part, be used for producing superimposed image by stack transmitted light images and luminescent image, wherein under optical microscope, obtain transmitted light images, and gather the acquisition luminescent image by the light that luminescence phenomenon in the cell in the sample is sent with the MIcrosope image collecting device with MIcrosope image collecting device capturing sample image in the viewed in transmitted light of sample; Also comprise the image-region specified portions, be used to specify the analyzed area in the superimposed image.In this device, preferably, this MIcrosope image collecting device has light receiving surface, and this optical microscope comprises object lens and lens subassembly, it forms the image of sample on the light receiving surface of MIcrosope image collecting device, wherein the optical system of optical microscope is configured to and makes that the numerical aperture of object lens and the square value of the ratio between the magnification of projection sample image on the light receiving surface are 0.01 or higher.
According to an aspect of the present invention, provide a kind of device that uses optical imaging apparatus to obtain the sample originating from organism image, comprising: illumination section, this sample is used to throw light on; Illumination image obtains part, is used to obtain the illumination image of this sample; Luminescent image obtains part, is used for obtaining the luminescent image of this sample cell; Image processing section, be used to superpose this illumination image and this luminescent image are with the generation superimposed image, and the analyzed area in definite this superimposed image; Wherein, this optical imaging apparatus is an optical microscope; This illumination image is a transmitted light MIcrosope image of utilizing the illumination light of this illumination section to obtain in viewed in transmitted light under this optical microscope, and this luminescent image is to observe the light that luminescence phenomenon sends in this cell and the illuminating microscope image that obtains under this optical microscope; Wherein this illumination image obtains partly and comprises shared MIcrosope image collecting device with this luminescent image acquisition unit branch, and it gathers this transmitted light MIcrosope image and this illuminating microscope image.
According to a further aspect in the invention, provide a kind of method of utilizing optical imaging apparatus to obtain the sample originating from organism image, it is characterized in that this method comprises following step: this sample and obtain its illumination image throws light on; Do not throw light on sample and obtain the luminescent image of the light that the luminescence phenomenon of the cell in this sample sends; Superpose this illumination image and this luminescent image to produce superimposed image; And the analyzed area in definite this superimposed image, wherein this optical imaging apparatus is an optical microscope; This illumination image is the transmitted light MIcrosope image that obtains in the viewed in transmitted light under this optical microscope, and this luminescent image is to observe the light that luminescence phenomenon sends in this cell and the illuminating microscope image that obtains under this optical microscope; Wherein this transmitted light MIcrosope image and this illuminating microscope image are gathered by shared MIcrosope image collecting device.
According to another aspect of the present invention, provide a kind of method of utilizing optical imaging apparatus to obtain the image of biogenic sample, comprise following step: the irradiation sample also obtains its illumination image; The sample that do not throw light on obtains the luminescent image of the light that luminescence phenomenon sends in the sample cell; Stack illumination image and luminescent image are to produce superimposed image; Determine the analyzed area in the superimposed image.The method according to this invention is as above installed describedly, can produce the superimposed image of illumination image and luminescent image, and carry out the analysis of luminescence phenomenon thus, obtains to find luminous zone in sample that wherein occurs in illumination image or the cell.Like this, can carry out method of the present invention with said apparatus, but also can with other arbitrarily equipment carry out.In addition, in the method for the invention, can carry out the step of in the sample cell, obtaining fluoroscopic image and luminescent image and illumination image, and therefore luminescence phenomenon and the associating information that obtains from fluorescence can be got up, obtain in cell or sample, where to take place luminescence phenomenon simultaneously.
In said method, and in said apparatus, optical imaging apparatus can be an optical microscope.At this moment, illumination image is to use optical microscope to carry out the transmitted light MIcrosope image that viewed in transmitted light obtains, and luminescent image is the illuminating microscope image that obtains with luminescence phenomenon in the observation by light microscope sample cell, wherein preferably, by all purpose microscope image capture device acquisition of transmission light microscope image and illuminating microscope image.In addition, in optical microscope, preferably will be designed at the object lens and the lens subassembly that form sample image on the light receiving surface of MIcrosope image collecting device: making the numerical aperture of object lens and the square value of the ratio between the magnification of projection sample image on the light receiving surface is 0.01 or higher.
In one embodiment, for example for a long time to the luminescence phenomenon imaging time, irradiation sample and the step of obtaining illumination image are carried out repeatedly, therefore can check moving of the change of forms such as cell in the sample and/or position etc. arbitrarily.In addition, the cell that will observe in the gene in its cell combines to cell and certain specific incentives such as reagent excitation, electric excitation, gas excitation or thermal excitation is provided and during the luminescent protein plasmagene of expressing, selectively provides the step of above-mentioned at least a excitation to carry out before the step that obtains illumination image and luminescent image (maybe can also obtain fluoroscopic image) to cell.At this moment, for inspection condition before excitation is provided, the step that preferably will obtain cell illumination image and luminescent image was carried out before the cell excitation is provided.
In addition, obtain after the step of a series of above-mentioned various images and stack illumination image and luminescent image, can carry out the step that shows and/or write down the superimposed image of illumination image and luminescent image, to allow the customer inspection image and/or to carry out arbitrarily subsequently and handle.
In addition, optionally, after the designated analysis zone, condition for detailed inspection or observation analysis zone, can carry out the illumination image zone of demonstration, corresponding to the luminescent image of analyzed area zone and corresponding to the step of at least two (when also needing fluoroscopic image) in the fluoroscopic image zone of analyzed area corresponding to analyzed area.In the analysis of luminescence phenomenon, can carry out the step of the light intensity of measuring zone corresponding in the luminescent image and show the step that this light intensity changes with analyzed area.
Can know from top description, in luminescent image, usually there is not the area condition of photoprotein can not observe in essence in the sample, and the light of observing in image capture device or the video camera is very faint, therefore, even can survey the luminescence phenomenon of photoprotein, also be difficult to correct the acquisition and luminescence phenomenon where occur in the sample.According to apparatus and method of the present invention, produce the image that obtains by stack illumination image and luminescent image, therefore can analyze luminescence phenomenon and obtain to occur in the sample luminous position simultaneously.According to feature of the present invention, because luminescent cell and non-luminescent cell can determine respectively immediately, just can carry out any statistical study to the quantity of observing luminous cell quantity and not observing luminous cell from the superimposed image of illumination image and luminescent image.In addition, compared with prior art, can easily carry out the observation in time of the luminous intensity that only monitors luminescent cell.For example, when the sample inner cell moves in measuring process, just can the tracking illumination image and the superimposed image of luminescent image in luminescent cell.Past when increasing the magnification of micro objective, owing to increased the magnification of object lens, reduces from the signal of luminous position relatively, therefore may miss luminous position.Yet, according to the present invention,, just reduced failure owing to can on the superimposed image of luminescent image and illumination image, determine the luminous position of sample, for example miss luminous position.
We can say that above-mentioned the present invention has increased the use of photoprotein in cell and/or other biological imaging samples.According to the present invention, the past is only used various tests, measurement and the analysis of the expression of range protein in that photoprotein is carried out with spectrum, the pair cell to reporter molecules, can be undertaken by imaging, has wherein identified each cell.
Other purposes of the present invention and advantage will become apparent by following explanation.
Description of drawings
Figure 1A is a synoptic diagram, the overview of the structure of low-light-level measurement device first embodiment is shown, it comprises the optical microscope according to counter-rotating of the present invention, and Figure 1B is near the detailed synoptic diagram of the structure the sample container of Figure 1A, comprises the equipment that excitation is provided to the cell in the sample;
Fig. 2 A illustrates the structure (image processing equipment) of the computing machine 80 of low-light-level measurement device with the form of functional block diagram, and Fig. 2 B illustrates the synoptic diagram of the control panel that is connected with computing machine 80;
Fig. 3 illustrates the step that the present invention obtains the method for sample originating from organism image in a flowchart, and it is undertaken by low-light-level measurement device of the present invention;
Fig. 4 schematically illustrates the cyclical variation of the luminous intensity of observing in the NIH3T3 cell, wherein have clock gene combination expression vector wherein by luciferase gene is connected to, and shows plastid.In 24 hours cycle, ought luminously become very faint in (a), just can not obtain luminous image;
Fig. 5 illustrates the synoptic diagram of structure (optical system) of second embodiment of low-light-level measurement device of the present invention, and it carries out fluorescence simultaneously and bioluminescence is measured;
Fig. 6 is the synoptic diagram of structure (optical system) of the 3rd embodiment of low-light-level measurement device of the present invention, and it carries out fluorescence simultaneously and bioluminescence is measured, and the image spreading of carrying out digital zoom and optical zoom respectively;
Fig. 7 is that explanation will be introduced figure as the molecule condition of estimating in the plastid in the sample Hela cell of example 1, in the plastid luciferase gene is connected to the have tetracycline operator expression vector of (TetO2).Shown in Fig. 7 A, when the TetR homodimer is combined with the TetO2 zone, do not express the luciferase gene that is connected to TetO2.On the other hand, when giving the cell tetracycline, TetR homodimer structural change and separate with the TetO2 zone shown in Fig. 7 B, causes expressing luciferase;
Fig. 8 illustrates the time of adding tetracycline and illumination for exposure image and luminescent image in the example 1;
Each of Fig. 9 A, 9B and 9C illustrates in the example 1 image (expanded images in the frame of Fig. 9 A) that luminescent image obtains in the illumination image and Fig. 9 B among illumination image, luminescent image and the stacking diagram 9A that obtains.In Fig. 9 C, the square frame that is expressed as ROI-1 and ROI-2 is appointed as analyzed area;
Figure 10 illustrates the time dependent figure of (Hela cell) luminous intensity in the analyzed area of appointment among Fig. 9 C;
Each of Figure 11 A, 11B and 11C illustrates in the example 2 image that luminescent image obtains in the illumination image and Figure 11 B among illumination image, luminescent image and the stacking diagram 11A that obtains.Luminous position shows by pseudo-colours in the device of reality, still, shows with white among Figure 11 C;
Figure 12 illustrates among Figure 11 the time dependent figure of the luminous intensity of photobacteria in the PP-1 zone;
Each of Figure 13 A, 13B and 13C illustrates in the example 3 image that luminescent image obtains in the illumination image and Figure 13 B among illumination image, luminescent image and the stacking diagram 13A that obtains;
Figure 14 illustrates in the example 3 superimposed image (left side) and the illumination image (right side) that the photobacteria of progress is in time moved;
Figure 15 is the time dependent figure of luminous intensity of photobacteria in the example 3 (V.t.).
Preferred forms
Below, to some preferred embodiments detailed description the present invention of reference accompanying drawing, wherein same reference numbers is represented identical parts.
First embodiment of apparatus of the present invention
The structure of device
Figure 1A is the synoptic diagram of first embodiment of the invention preferred embodiment, and it obtains the image of sample originating from organism, with the low-light of measurement biological luminescence generation, and the imaging of execution cell and/or other biological sample.(after this being called " low-light-level measurement device ").
With reference to accompanying drawing, low-light-level measurement device of the present invention comprises the microscope of counter-rotating, comprise light source 2, lamp optical system 1, the light that makes light source 2 send becomes collimated beam and guides the bundle that obtains to sample 4, viewing optical system 5, be used to form the image and the eyepiece 6 of sample 4, the image that is used to expand sample 4 carries out eyes to be observed; Ccd video camera 8 (image acquisition section or MIcrosope image collecting device) has imageing sensor 7, is used to gather the MIcrosope image of sample 4; And computing machine 80 (image processing section or image processing equipment), having TV Monitor, ccd video camera 8 is connected to computing machine by signal wire 81.
The structure of optical microscope
The structure of above-mentioned optical microscope can be the microscope that reverses normally, wherein begins with described order from light source 2, and lamp optical system 1 is built with convergent lens 10, deflecting mirror 12, is used for the optical axis 11 and the convergent lens 14 of deflection illumination light.Light source 2 can be a coherent source, has the wavelength in the visible range, such as Halogen lamp LED, led light source, tungsten lamp, mercury vapor lamp etc.In addition, such as laser, and convert incoherent light to, can also be used as light source 2 by diffusion disk etc. from the light of coherent source.Although the wavelength of light source in visible-range, also can use infrared light usually.
Viewing optical system 5 is configured to has object lens 15, be used to form image, first relay lens 16, the deflecting mirror 17 of sample 4, be used for light and second relay lens 18 of deflection, combine the image (image of sample 4) that is used for from object lens 15 with first relay lens 16 and be formed into imaging surface from object lens 15.Between second relay lens 18 and imaging surface 19, conversion (change over) mirror 20 can be installed as and make the image that the changes sample 4 arbitrarily observation path between eyes are observed by eyepiece 6 and observed by ccd video camera 8.At this moment,, needn't use the type of mechanical switch, can use half-reflecting half mirror that light path is divided into two for conversion mirror 20.
In the sample viewed in transmitted light of said structure, with Koehler light source irradiation sample 4, as the situation of normal observation by light microscope transmitted light.Therefore, at first become parallel beam from the light of light source 2, and cause shining sample 4, form the image of light source 2 simultaneously in the pupil location of collector lens 14 by convergent lens 10.Subsequently, the light of irradiation sample 4 enters object lens 15 forming sample images by first relay lens 16 and second relay lens 18 on imaging surface 19 by sample 4, and the image of the sample 4 that obtains on imaging surface 19 is observed by eyepiece 6 observed persons.In addition, when ccd video camera 8 was gathered the image of samples 4, the light by second relay lens 18 was converted mirror 20 reflections, and the image of sample 4 is formed on the imageing sensor 7 of ccd video camera 8 thus.At this moment, the magnification of object lens can for example be 20 times.
As shown in the figure, sample 4 is arranged on the sample stage 3, wherein preferably as Figure 1B in detail shown in, sample 4 can be placed in the sample container 21 together with nutrient culture media, this container is such as the test board with lid 26.The bottom of sample container 21 is by having and the same characteristic of optical clear cover glass that is used for microscopical 0.17mm thickness, therefore the sample of the bottom of sample container 21 can use normal object lens to observe (at this moment, sample container 21 is not limited to this testing laboratory dish, and can use slide glass, microplate etc.).In addition, in order to keep the humidity in the sample container 21, sample container 21 can place water-bath 22, wherein provides pure water by ozzle 23, and keeps by the closing containers 30 that lid is arranged with water-bath 22.Subsequently, as Figure 1B in detail shown in, by air supply pipe 24 and from the Co of the cylinder outside the measuring equipment 25 2Ozzle 84 is with Co 2Gas is supplied with the upper surface of water-bath 22.Gas in the cylinder 25 is 5%Co for example 2And 95%O 2Mixed gas.Supply Co 2Gas is about 50mL/min to the flow velocity of the closing containers 30 that covers.In addition, heating plate 27 can be located at below the sample container 21.This heating plate 27 is adjusted temperature in the sample container with the temperature controller (not shown) by 0.5 ℃ stride usually.
In addition, as described later,, automatic dispensing device 100 can be set, as device to the cell excitation in order to carry out the test (make and express photoprotein) that produces luminescence phenomenon by the excitation cell.In automatic dispensing device 100, pump 102 obtains reagent solution and uses ozzle 103 that the reagent solution of scheduled volume is discharged into the sample container 21 from reagent container 101.As shown in the figure, when sample container 21 being put in the closing containers 30 that covers, preferably, before ozzle 103 release reagent solution, the lid 31 of closing containers 30 and the lid 26 of sample container are opened by the motor (not shown), with automatic dispensing device 100 coordinated manipulations, after discharging solution, lid 131 and specimen capsule cap 26 are closed by motor.At this moment, under following computer control, automatic dispensing device 100 can obtain MIcrosope image front and back and/or the operation of random time betwixt.
In addition, preferred on sample stage 3, in order to move horizontally sample stage 3 (directions X and Y direction) arbitrarily, two step motor (not shown) are installed as each other separately 90 ° in direction respectively.Two step motor can drive by sample stage controller (not shown) based on the instruction of computing machine 90, the location of the feasible sample stage 3 of control automatically.
In addition, preferably, around the object lens under the sample stage 3 15, object lens well heater 28 can be installed to be contacted with object lens 15, therefore can under the control of temperature control equipment (not shown), pass through 0.5 ℃ of temperature of stepped intervals, so object lens 15 outsides will remain on arbitrary temp with object lens well heater 28 adjustment object lens 15.Equally, around object lens well heater 28, be furnished with " Z axle object lens driving mechanism 9 ", be used for automatically along Z axle (optical axis direction) motion object lens 15.Z axle object lens driving mechanism 9 moves up and down object lens 15 by the pinion and rack (not shown).Carry out the rotary manipulation of pinion and rack knob by computer-controlled step motor (not shown).Perhaps, Z axle object lens driving mechanism 9 can be a friction roller mechanism.
The structure of MIcrosope image collecting device
CCD (charge-coupled image sensor) video camera 8 is used to receive the image of sample 4, and imageing sensor 7 wherein can have for example 1360 * 1024 pixels.In equipment of the present invention, as follows, ccd video camera 8 is gathered the transmitted light MIcrosope image of the viewed in transmitted light acquisition of passing through sample 4 and is passed through the illuminating microscope image of the luminous observation acquisition of sample 4, promptly observes the low-light of launching owing to luminescence of cell phenomenon in the sample 4.Therefore, ccd video camera 8 is gathered low-light in the luminous observation as photo-detector, preferably selects to have highly sensitive as far as possible detector.Therefore cooling system 29 is equipped with,, with Peltier device in its bottom for the preferred refrigeration CCD that use of ccd video camera 8, it can be reduced to the temperature of video camera+5 ℃ to-70 ℃ scope in, with the dark current of inhibition from ccd video camera 8.In addition, infrared section wave filter 13 preferably is installed, to end the infrared radiation of light as a setting on the light receiving surface of ccd video camera 8.The image that ccd video camera 8 is gathered is sent to computing machine 80 by signal wire 81 and shows on connected TV Monitor 37.In addition, ccd video camera 8 can be 3 template colour TV cameras, to gather the color lighting image.For the image capture device of MIcrosope image, for example, can use cmos image sensor, SIT video camera etc.
The structure of image processing equipment
Computing machine 80 can be constructed as the computing machine with CPU, and can carry out the various Flame Image Process of well known to a person skilled in the art of any type.The operation of the various piece of computing machine 80 controls low-light-level measurement device of the present invention, to obtain MIcrosope image as view data, and carry out from the illumination image of viewed in transmitted light acquisition and the luminescent image generation superimposed image that obtains from luminous observation, and the various processing of using this view data, such as using superimposed image to determine analyzed area.
The computing machine 80 of Fig. 2 A presentation function piece form.With reference to accompanying drawing, computing machine 80 comprises CPU 35, storer 41, be used to store the control program of control CPU 35 operations, signal processing circuit 33 and digital zoom circuit 34, being used to change the image that ccd video camera 8 gathers is view data, display control circuit 36 and TV Monitor 37, be used to show the image of ccd video camera 8 collections, the driver of record and reproducing control circuit 38 and recording medium 39, be used to write down and/or on TV Monitor 37, show image that ccd video camera 8 is gathered and/or the image that obtains by Flame Image Process, and control panel 40, be used to import user's order.On control panel 40, shown in Fig. 2 B, various types of buttons are provided, dwindle button 143, tracking cross button 45 and mode switch 48 such as reproduction button 43, record button 47, shutter release button 44, zoom large buttons 42, zoom, and the control panel 40 by being connected with CPU 35 thus, with user's order input computing machine 80.
In said structure, the MIcrosope image that is formed on the sample 4 on the imageing sensor 7 of ccd video camera 8 converts analog electrical signal to by imageing sensor, i.e. analog picture signal.The signal that obtains is sent to signal processing circuit 33 subsequently under the control of CPU 35, wherein the edge of any Flame Image Process carry out processing and amplifying, Filtering Processing and/or strengthen as to(for) picture signal.Subsequently, the picture signal of processing sends to digital zoom circuit 34 (digital signal processing part).34 pairs of analog picture signals from signal processing circuit 33 of digital zoom circuit are carried out the A/D conversion to produce data image signal, and after any digital processing, carry out as required and proofread and correct such as gamma, the data image signal that obtains is sent to display control circuit 36, be used for display image on TV Monitor 37, and be sent to the record and reproducing control circuit 38, be used for the dismountable recording medium 39 (for example, storage card, hard disk, disk, magneto-optic disk etc.) of recording image data to the holding tank (not shown).At this moment, the digital zoom circuit 34 of apparatus of the present invention is designed to shear the interior any zone of collection pixel region of whole ccd video camera 8, and the image-region that transmits shearing amplifies it with any magnification to display control circuit 36 or record and reproducing control circuit 38 simultaneously.Therefore, can on TV Monitor 37, only show the MIcrosope image that part that ccd video camera 8 is gathered is exaggerated, and/or only write down this parts of images.
In addition, in said structure, record and reproducing control circuit 38 can be designed as in response to the reproducing control signal from CPU 35 and read the data image signal of current record in the recording medium 39, and therefore this reading number picture signal can show the document image of sample 4 by in the display control circuit 36 input TV Monitors 37 on the screen of TV Monitor 37.
When operating above-mentioned image processing equipment, when the user opens record button 47 on the control panel, device executive logging pattern of the present invention, wherein CPU 35 provides the record controls signal to record and reproducing control circuit 38.The picture signal that provides from digital zoom circuit 34 in response to the record controls signal record of record and reproducing control circuit 38 is to recording medium subsequently.On the other hand, open when reproducing button 43, device of the present invention begins reproduction mode, and wherein CPU 35 provides the reproducing control signal to record and playback control unit 38, and it shows the view data of above-mentioned record on TV Monitor 37.In addition, when mode switch 48 is set to image acquisition modality, just output to digital zoom circuit 34 from the control signal of CPU 35, and just stand digital signal processing from a two field picture of ccd video camera 8 this moment and be attached in the storer 41 in response to promoting shutter release button 44.Subsequently, the view data that is attached in the storer 41 is compressed by any compressor circuit, and is recorded on the storage card by card reader/write device.
In the whole pixel region that the ccd video camera of carrying out on digital zoom circuit 34 8 obtains the shearing setting of arbitrary region by use zoom large buttons 42, zoom dwindles button 143 and tracking cross button 45 is carried out.In the shearing of image-region is provided with, " zoom center " and the frame of image-region at the center of the image-region that indicator gauge registration word zoom circuit 34 will be sheared on TV Monitor 37.Allow the user to operate tracking cross button 45 and the zoom center is set at an arbitrary position, watch TV Monitor 37 simultaneously, and allow to dwindle the size of the button 143 definite image-regions that will shear by zoom large buttons 42 and zoom.The magnification of the image that obtains from shear history can be in M=1.00 (the whole zone that ccd video camera is gathered) setting arbitrarily in the scope in (the whole zone that ccd video camera is gathered 1/4) to 4.00.
At this moment, the amplification of above-mentioned image can be carried out (optical zoom) by adjusting microscopical optical system, and does not use digital zoom circuit 34.For example, by the motor driven of step motor, carry out this optical zoom along the focal length of 18 of light shaft offset ccd video camera 8 and second relay lenss.In addition, in the lens arrangement of carrying out optical zoom (zoom lens), use the lens combination of three variable powers, the offset lens group of execution aberration compensation etc. and carry out the convergent lens (not shown) of focusing and adjusting, the focal length of lens can divide the manual or variation automatically of 10 steps.Can carry out the driving zoom lens, make the zoom motor (ultrasonic motor) be installed in around the zoom lens operate, to move zoom lens along optical axis based on the control signal of CPU 35 outputs.
In addition, in order to control the operation of (comprising light source 2) of automatic dispensing device 100 and irradiation apparatus 82, obtain picture signal that ccd video cameras 8 gather and can carry out synchronously with the execution of batch operation by automatic dispensing device 100 thereby computing machine 80 can be connected to these equipment.
Device by first embodiment is to the observation and the measurement of sample
As mentioned above, in the luminescence phenomenon of photoprotein was measured, its luminous intensity was very weak, therefore was difficult to survey light signal with ccd video camera, and can not observes the inner structure of cell usually.Therefore, in cannot observing as microscope is normal, adjust the focus of object lens and check the cell of observing in the sample simultaneously.Therefore, the focal position of object lens is wherein used from the light such as the light source of Halogen lamp LED and is shone sample by lamp optical system based on determining with the sample illumination image that obtains in the microscope viewed in transmitted light in the luminous observation.For example, by the focal position that object lens are set be: when increasing bioluminescent protein matter luminous intensity, object lens obtain the position and the approximate centre position on the optical axis between the position of the clearly luminescent image that obtains on the ccd video camera to converge of high-contrast image in viewed in transmitted light.After this, explanation is used the luminous formation method that carry out imaging of the device of the invention described above to photoprotein.
Fig. 3 illustrates the processing procedure in the method for luminescence imaging of the present invention with process flow diagram.With reference to accompanying drawing, at first, sample 4 (cell etc.) and culture solution are put into test board (sample container 21), and it is arranged in the device (step 1), and open illumination light (step 2) subsequently.Subsequently, on the light receiving surface of the imageing sensor 7 of ccd video camera 8, form MIcrosope image, and use the illumination image (step 3) that obtains sample from the signal of imageing sensor 7 by the sample 4 of object lens 15.Below, on the illumination image that obtains, specify desired zone, promptly have the zone (step 4) of suitable cell density.After this, platform moves with skew and has the center (step 5) of the appointed area of suitable cell density to the visual field.Subsequently, the data storage of illumination image (step 6), and close illumination (step 7), and finish obtaining of first illumination image subsequently in memory device.
Below, for cell inner expression photoprotein, encourage cell with the device that encourages cell at sample.The reagent of excitation cell can provide (step 8) by automatic dispensing device.At this moment, on platform, after step 8, finish irradiating step, and the illumination image data that obtain can be saved in (step 9) in the memory device.Subsequently, by irradiation, (step 10) also is saved in the memory device (step 11), and finish obtaining of first luminescent image thus to obtain luminous signal.Subsequently, for obtaining luminescent image repeating step 10 and step 11 up to the schedule time, predetermined luminous intensity or finished the execution of pre-determined number said process.At this moment, can carry out these processing by this way, wherein illumination image obtains with luminescent image and obtains in pairs, and perhaps, the luminescent image of every execution pre-determined number obtains, and carries out an illumination image and obtains.Carry out these Image Acquisition by rights for condition of cell activity, measurement etc.
Finish after illumination image and luminescent image obtain, the superimposed image that produces illumination image and luminescent image is demonstration (step 12) in image processing equipment (computing machine 80) also.Luminescent cell can be determined by superimposed image, and therefore, some cell can be easily specified even during measuring, moved also with reference to the demonstration of the illumination image that obtains and luminescent image.
Below, judge in superimposed image, whether can differentiate luminous position in the cell, and if can differentiate luminous position (step 13: be), specified measurement zone (step 15).Measured zone can be specified on monitor by any device, and such as mouse, cursor or pointer etc., it can carry out the Flame Image Process of various changes and limit zone on the screen by manual operation or by carrying out threshold value wherein.For example, a cell can be appointed as a measured zone, and the part of cell also can be appointed as a measured zone.
On the other hand, (step 13: not), just (therefore step 14) will luminously be plotted as easy as seen (step 15) in the specified measurement zone after the output that shows ccd video camera with pseudo-colours in be difficult to differentiating cell in the luminous position.At this moment, for example, a cell can be appointed as a measured zone, and intracellular part can also refer to be decided to be a measured zone.At this moment, not only can the excitation as the analytic target of cell before the appointed area, can also after carry out, and, based on the luminescent image or the illumination image that obtain after the excitation, the zone that measure can be displaced to the zone of the object that existence can suitably measure.If a zone is very faint or very not little luminous when selecting excitation, the zone that measure just can change to wherein can detect suitable luminous zone.Perhaps, have faint or do not have in luminous institute's analysis of cells in excitation, the cell that increases luminous quantity in response to excitation can be designated as the object that will analyze, and subsequently, the object that can optionally follow the tracks of detection is used for data and obtains and analyze.
In addition, time dependent luminous intensity can show with animated, or digitizing and show (step 16) with diagrammatic form.Execution analysis (step 17) like this, at last.Detailed says, uses the data that obtain, and carrying out relatively, luminous intensity, the light-emitting mode of determining cell, the comparison light-emitting mode of interior each position of identical cell of luminous intensity, comparison of each cell wait over time.From these results, can analysis of cells for the reaction of reagent, electricity, gas and thermal excitation.In addition, can also be when not carrying out excitation the activity of analysis of cells.
According to use of the present invention, for example, as shown in Figure 4, when observing the periodically variable sample of luminous intensity, even luminous disappearance also can observation post needs the variation of luminescence of cell intensity, and do not miss the cell of before luminous disappearance, being concerned about with the back, therefore, can specify some cell and follow the trail of its luminous intensity continuously.
Second embodiment of apparatus of the present invention
Second embodiment of the schematically illustrated apparatus of the present invention of Fig. 5 wherein further adds the structure of carrying out Fluirescence observation in the low-light-level measurement device shown in Figure 1 of first embodiment of the invention.
With reference to accompanying drawing, in the device of second embodiment of the invention, wherein similar with the device of first embodiment, digital zoom and/or optical zoom are carried out simultaneously, the optical system 49 that is used for exciting light is added to wherein, and viewing optical system 5 is revised as the fluorescence that makes from sample 4 is directed in the ccd video camera.
The optical system 49 that is used to excite is built with excitation source 50, collimation lens 51, deflecting mirror 52 and changeable dichroic mirror 53.Excitation source 50 is argon laser normally, has the output power of 10mW and the wavelength of 488nm.At this moment, excitation source 50 can be the gas laser in the visible range, such as helium-neon laser.Laser converts circular collimated beam to by collimation lens 51, has specific beamwidth, reflection on deflecting mirror 52, and enter the changeable dichroic mirror 58 that is installed in the viewing optical system 5.
Changeable dichroic mirror 58 have reflection excitation source 50 optical maser wavelength light and see through the fluorescence of sample 4 and the spectrum signature of the spectrum of luminous signal.Therefore, the laser that enters viewing optical system 5 reflects on changeable dichroic mirror 53, and enters object lens 15 from its bottom subsequently, and the sample 4 in sample container 21 converges and excite the fluorescence molecule in the sample 4.At this moment, changeable dichroic mirror 58 is placed in (not shown) in the retainer, and installs convertibly according to the wavelength fluctuation of excitation laser.In addition, if do not need to change the wavelength of excitation source 50, conventional dichroic mirror can be installed replace the switchable dichroic mirror 58 of use.
For object lens 15, can use to have high NA (numerical aperture) for example 0.9 or 1.0 or higher immersion liquid type object lens.When the fluorescence signal that sends and enter object lens 15 from sample 4 and luminous signal pass the viewing optical system 5 of device, these signals pass changeable dichroic mirror 58, advance to first relay lens 16 and second relay lens 18, and switching reflection on the mirror 20, and finally on the light receiving surface of the imageing sensor 7 of ccd video camera 8, form each image.Subsequently, in the device of first embodiment, be sent to computing machine 80 from the light signal of ccd video camera 8, wherein show and analyze this luminescent image, carry out luminous intensity measurement and signal analysis etc. in time, and the analysis result that obtains shows on the screen of the TV Monitor 37 of computing machine 80.
Can carry out Fluirescence observation with the mode of well known to a person skilled in the art.For example the fluorescent dye that uses in the Fluirescence observation can be Luo Daming green (RhG); TMR (tetramethyl isothiocyanic acid Luo Daming) and 5-Tamra (5-carboxyl tetramethyl isothiocyanic acid Luo Daming).In order to excite TMR and 5-Tamra etc., the helium-neon laser of the argon laser of wavelength 514.5nm and wavelength 543.5nm etc. can be used for distinguishing the excitation laser source.In addition, FITC (fluorescein isothiocyanate), TOTO 1, and acridine orange, Texas are red etc. can be used as fluorescent dye.
The 3rd embodiment of apparatus of the present invention
Fig. 6 illustrates the synoptic diagram of another embodiment of low-light-level measurement device of the present invention, and it can carry out digital zoom or optical zoom, and measures fluorescence and bioluminescence as Fig. 5 device simultaneously.
With reference to accompanying drawing, in an embodiment, measuring equipment itself is that optical microscope is enclosed in light box 54, and wherein breaking the barriers every the bottom of light box 54 56 is fixed on the chassis 55, and this box is designed to prevent that exterior light from entering viewing optical system 5 thus.At upper surface every light box 54, the resistance light lid 57 that install to separate, an and end of resistance light lid 57 is connected on the main body of light box 54 by hinge 58, make can rotary switch should resistance light lid 57.
In order to obtain illumination image, the light that is used to throw light on that for example comes the light source 2 of Halogen lamp LED, metal halide lamp etc. freely is introduced into upper surface with sample container 21 on the illumination sample stage 3 by optical fiber 60, and shines whole sample 4.In addition, in order to obtain fluoroscopic image, laser (exciting) light source 50 is installed on the outer light source box 62 of apparatus main body, and enters this device main body by the laser incident opening 64 on the outside framework from the laser that LASER Light Source 50 inspires.Laser for example can be the argon laser with 10mW output and 488nm wavelength.Laser by laser incident opening 64 access to plant bodies at first enters viewing optical system, and reflection on changeable dichroic mirror 53 enters object lens 15 from its bottom, and converges with its illumination to sample 4.Changeable dichroic mirror 53 is installed on the retainer and installs convertibly according to the oscillation wavelength of excitation laser.
With holding pin sample container 21 is fixed on the XY sample stage 3.XY sample stage 3 is designed to make it can move arbitrarily on the XY plane by gear-rack mechanism in the position on the XY plane.The object lens 15 that are installed under the sample container 21 are equipped with the mechanism 9 that drives object lens on the Z axle, so that it can move along optical axis (Z axle).By lead-in wire, the focus detection part 77 of installing outside Z axle object lens driving mechanism 9 and the low-light-level measurement device is communicated by letter and controlled by it.
In viewing optical system 5, the flashlight that object lens 15 are collected passes changeable dichroic mirror 53, and be not subjected to deflection subsequently, shown in above-mentioned first and second embodiment, flashlight by being installed in mirror bucket 66 bottoms lens 67 and converge on the light receiving surface of ccd video camera 8.Lens 67 are positioned at the bottom that is installed in the mirror bucket 66 on the pedestal 75 on chassis 55 with holding pin, and on pedestal 75, and ccd video camera 8 is arranged so that the focal position of Z axle of lens 67 is consistent with the approximate center of light receiving surface.Carry the main body base 76 that sample stage 3 and object lens 15 are installed on it and install, move up and down with respect to the top and the bottom 72 and 66 of mirror bucket by the post 73 that is fixed on the chassis 55.At this moment, optionally, can provide object focal point probe portion 70, it is connected to position sensing part 71, and therefore the output signal from position sensing part 71 can output to TV Monitor 37.
For ccd video camera 8, because faint from the light of sample, the preferred use has highly sensitive as far as possible video camera, as among first and second embodiment.The pixel quantity of ccd video camera 8 can be for example 1360 * 1024.In order to suppress dark current from ccd video camera 8, the bottom that the refrigerating system 29 that comprises the Peltier device is provided in ccd video camera with the temperature that reduces and keep ccd video camera 8 near 0 ℃.On the light receiving surface of ccd video camera 8, infrared section wave filter 13 being set, can be the infrared light of bias light to end.Yet as flashlight the time, infrared section wave filter 13 removed in the bottom from mirror bucket 66 before measuring with infrared light.The output of ccd video camera 8 is connected to signal wire 69, and will be sent to computing machine 80 by signal processing 74 from the sample image of ccd video camera thus, and shows on TV Monitor 37.3 type colour TV cameras can be used as ccd video camera 8, therefore can obtain colored illumination image.
In the operation, at first based on the illumination image of the sample 4 of light source 2 irradiation, the focal adjustments of object lens is to sample.For this reason, by Z axle object lens driving mechanism object lens 15 are moved q.s along optical axis, and each moves in the step at it, the optical output signal of analyzing from ccd video camera 8 by signal processing 74, be used to survey the position of object lens 15 on optical axis, wherein obtain the image of high-contrast.Subsequently, find the upside of object lens 15 on optical axis and two positions of downside that high-contrast image is provided, and pass through the calculating of signal processing 74, the approximate centre position between upper-lower position is defined as the focal position of luminous observation.After this, by operation Z axle object lens driving mechanism 9, mobile object lens 15 also are fixed to the focal position that obtains, and after this, fluorescence signal that sample 4 penetrates and luminous signal receive with ccd video camera 8.
In Z axle object lens driving mechanism 9, a plurality of object lens 15 are installed convertibly.When the object lens 15 with big magnification are used for observation of cell etc., the nutrient culture media in the mobile sample 4, because the less visual field, in some cases just can be so that the sample of cell etc. 4 moves on to outside the visual field.Therefore, preferred, sample 4 is at first observed with the object lens 15 with low magnification, such as * 10 and * 20, and after confirming and utilizing the desired location of mouse and keyboard appointment sample 4, by the enlarging function expanded view picture in apparatus of the present invention.At this moment, computing machine can be used as signal processing 74.
In order to study purposes of the present invention, carry out following test.At this moment, should be appreciated that following Example is used to illustrate use of the present invention and should not limits the scope of the invention.
Example 1
In example 1, use the low-light-level measurement device, wherein this measurement mechanism among Fig. 1 provides automatic dispensing device 100, in some cell in a plurality of Hela cells of introducing luciferase gene, observes luminous that the reagent excitation causes temporarily.
For sample, use the Hela cell of carrier wherein " pcDNA6/TR (obtaining) " continuous expression tetracycline repressor (TetR), and comprise and the plastid that has the luciferase gene that is linked to each other by the expression vector of genetically modified tetracycline operator (TetO2) " pcDNA4/TO (from In vitro gene co. acquisition) " from In vitro gene co..In the cell of introducing these two kinds of genes, shown in Fig. 7 A signal, TetRs at first is expressed as TetR with carrier (pcDNA6/TR), and TetRs forms homodimer, it combines with the TetO2 gene regions, suppresses transcribing of the luciferase gene be connected with the TetO2 district thus.Yet, shown in Fig. 7 B, when giving tetracycline with cell (reagent excitation), cause the structural change of TetR homodimer, cause the TetR homodimer to distinguish from TetO2, therefore will cause the transcript and expression luciferase protein matter of luciferase gene.Therefore, in this test example, import in the cell of said gene, observe the tetracycline excitation and make expressing luciferase and intracellular luminescence phenomenon.
In the device of Fig. 1, for object lens 15, use Olympus UApo/340 (20 *, N.A.0.75) and microscopical commercial object lens " Oil, 20 *, N.A.0.8 " or " 5 *, N.A.0.13 ".For ccd video camera 8, use the microscope image pick-up machine " DP30BW (Olympus) " of 5 ℃ of refrigeration.In this ccd video camera, CCD element (corresponding to imageing sensor 7) is 2/3 inch a type, and its pixel quantity is 1360 * 1024, and each pixel is of a size of 6 squares of μ m.Pass imaging len (relay lens) and the overall amplification of the sample image that forms at the CCD element is set to 4 times from object lens.In observation with during to the Hela cell imaging, whole device is covered by camera bellows.
Carry out above-mentioned test operation in the process below.
(1) preparation Hela cell sample, wherein co expression is expressed the carrier and the plastid that comprises the luciferase gene that links to each other with the expression vector of TetO2 of TetR all the time.For nutrient culture media, can use to comprise 10mM HEPES, have the D-MEM medium of 1mM luciferase.
(2) below, the sample in (1) is as sample, and collection illumination image and luminescent image.Illumination image and luminescent image are gathered in pairing always.The time shutter of an Image Acquisition (integral time of signal on ccd video camera) is 10msec to the illumination image setting, and luminescent image is set to 5min.
(3) subsequently tetracycline is added sample and transcribe, and after adding tetracycline, shown in process (2), gather the illumination image and the luminescent image of sample immediately in pairs to cause luciferase gene wherein.
(4) subsequently, as shown in Figure 8, the images acquired of process (2) repeats about 10 hours with the interval of 10min.The process of images acquired automatically performs by Computer Processing, and wherein in device of the present invention, the light signal that ccd video camera obtains converts numerical data automatically to.
(5) after finishing Image Acquisition, illumination image that obtains and luminescent image are applied to determine the Hela cell of expressing luciferase.Subsequently, the time dependent luminous intensity of Hela cell of expressing luciferase is digitized and analyzes.When on superimposed image, being difficult to determine the luminous position of Hela cell, with the output valve of pseudo-colours demonstration ccd video camera 8, in order easily to discern luminous position by human eye.
Fig. 9 A, 9B and 9C illustrate illumination image, luminescent image and superimposed image respectively.Figure 10 illustrates in ROI-1 and the ROI-2 zone in two cells luminous intensity over time, and each zone all is looped around in the square frame of the superimposed image that obtains in the above described manner of Fig. 9 C.Therefore, illustrate,, can determine which luminescence of cell and measure that it is over time luminous according to the present invention.
Example 2
In example 2, use with example 1 in the same device, carry out the luminous observation of photobacteria (P. photogen), and the luminous intensity that reagent encourages in the supervision cell is over time.Photobacteria is movable, luminous organism extensively exists in the nature with following form, (i) the independent live organism in the seawater, (ii) with the luminous organ symbiosis of fish or cephalopod, propagation in (iii) dead fish, the spoil, (iv) in the saltwater fish alimentary canal or cuttlefish supracutaneous habitual parasitic, and this bacterium demonstrates strong luminously continuously, is enough to observe by eyes under the oxygen condition.
In the observation, with Olympus UApo/340 (40 *, N.A.1.35) or Oil Iris " Oil, 40 *, N.A.1.35 " as object lens 15.The same in the condition of other images acquired and the example 1.
Test operation is carried out with following processes.
(1) photobacteria is present in as the symbiont body in the luminous organ of firefly squid (firefly squid), and it is opened and cultivate in comprising the LB nutrient culture media of 3%NaCl by isolated.Adding in this nutrient culture media glycerine to obtain 25% glycerite (v/v) is ultimate density.
(2) sample in (1) is used as sample, and gathers illumination image (observation image) and luminescent image.Illumination image and luminescent image are gathered all the time in pairs.The time shutter of an Image Acquisition (integral time of signal on ccd video camera) is 10msec to the illumination image setting, and luminescent image is set to 1min.
(3) use automatic dispensing device 100 subsequently, ampicillin is added in the sample, forbid that the cell membrane in the photobacteria is synthetic.Thus, suppress the propagation of photobacteria, make bacterium become extinct.Carry out like this and add ampicillin, wherein the operation of automatic dispensing device 100 and image acquisition are synchronous, wherein carry out images acquired immediately after adding ampicillin, and wherein illumination image and luminescent image are gathered in pairs.
(4) subsequently, the image acquisition of said process (2) description repeats after adding ampicillin through 1 hour with the interval of 5min.The process of images acquired automatically performs by Computer Processing, and wherein in device of the present invention, the light signal that ccd video camera obtains converts numerical data automatically to.
(5) after finishing Image Acquisition, illumination image that obtains and luminescent image are applied therefrom to determine strong photobacteria.Subsequently, arrange superimposed image with the formation animation with the time sequencing of gathering each image, and the change of following the trail of the luminous position that is accompanied by the photobacteria motion, and digitizing and the luminous change in time of analyzing photobacteria.At this moment, automatically perform the tracking of photobacteria with Computer Processing.
Figure 11 A, 11B and 11C illustrate illumination image, luminescent image and superimposed image (being expressed as pseudo-colours) respectively.Figure 12 illustrates among Figure 11 C in the PP-1 zone luminous intensity of photobacteria over time.According to example 2, illustrate, even motion during measuring such as the microbial body of bacterium can be determined also which microbial body is luminous and measure that it is over time luminous.
Example 3
In example 3, carry out the luminous observation of photobacteria (V.tischeri), wherein follow the tracks of the position of photobacteria and luminous intensity over time.The same in the condition of images acquired and the example 2.
Test operation carries out with the following step.
(1) photobacteria is cultivated in the agar LB that comprises 3%NaCl cultivates basal disc, and here that luminous intensity is high group classification also is suspended in the liquid LB nutrient culture media that comprises 3%NaCl.
(2) sample in (1) is used as sample, and gathers illumination image (observation image) and luminescent image.Illumination image and luminescent image are gathered all the time in pairs.The time shutter of an Image Acquisition (integral time of signal on ccd video camera) is 5msec to the illumination image setting, and luminescent image is set to 3min.
(3) subsequently, repeat the image acquisition that said process (2) is described.The process of images acquired automatically performs by Computer Processing, and wherein in device of the present invention, the light signal that obtains from ccd video camera converts numerical data automatically to.
(4) after finishing image acquisition, the illumination image and the luminescent image that obtain are applied.Because photobacteria is varying motion in time, just arrange the image of stack to form animation with the time sequencing of gathering each image, and follow the trail of the change of the luminous position that is accompanied by photobacteria (V.t.) motion, and the luminous change in time of digitizing and analysis photobacteria.At this moment, can automatically perform the tracking of photobacteria with Computer Processing.
Figure 13 A, 13B and 13C illustrate illumination image, luminescent image and superimposed image respectively, and Figure 14 illustrates photobacteria and changes mobile in time.Figure 15 illustrates photobacteria (V.t.) luminous intensity to be changed in time.Shown in example 3, even motion during measuring such as the microbial body of bacterium, it is time dependent luminous that the present invention also can measure the luminous component of appointment.As shown, only by the dynamic main body of tracking, and have nothing to do with having or not excitation, for example behavior and/or cytological analysis (propagation activity etc.) all is feasible.In addition, owing to can follow the tracks of the dynamic condition of bacterium, the motion that just can measure light-emitting zone in the sample, and have nothing to do with having or not excitation.
Although describe the present invention in detail, to those skilled in the art, also be conspicuous at other various feasible embodiment of scope of the present invention for specific embodiment.

Claims (21)

1. device that uses optical imaging apparatus to obtain the sample originating from organism image comprises: illumination section, and this sample is used to throw light on; Illumination image obtains part, is used to obtain the illumination image of this sample; Luminescent image obtains part, is used for obtaining the luminescent image of this sample cell; Image processing section, be used to superpose this illumination image and this luminescent image are with the generation superimposed image, and the analyzed area in definite this superimposed image;
Wherein, this optical imaging apparatus is an optical microscope; This illumination image is a transmitted light MIcrosope image of utilizing the illumination light of this illumination section to obtain in viewed in transmitted light under this optical microscope, and this luminescent image is to observe the light that luminescence phenomenon sends in this cell and the illuminating microscope image that obtains under this optical microscope; Wherein this illumination image obtains partly and comprises shared MIcrosope image collecting device with this luminescent image acquisition unit branch, and it gathers this transmitted light MIcrosope image and this illuminating microscope image.
2. according to the device of claim 1, it is characterized in that, this MIcrosope image collecting device has light receiving surface, and this optical microscope is included in object lens and the lens subassembly that forms the image of this sample on the light receiving surface of this MIcrosope image collecting device, and wherein the numerical aperture of these object lens and the square value that is incident upon ratio between the magnification of this sample image on this light receiving surface are 0.01 or bigger.
3. according to the device of claim 1, it is characterized in that this device comprises that fluoroscopic image obtains part, is used to obtain the fluoroscopic image of this cell.
4. according to the device of claim 3, it is characterized in that this optical imaging apparatus is an optical microscope; This fluoroscopic image is the fluorescence microscopy images that obtains in the sample Fluirescence observation under this optical microscope, and this luminescent image is to observe the light that luminescence phenomenon sends in this cell and the illuminating microscope image that obtains under this optical microscope; Wherein this fluoroscopic image obtains partly and comprises shared MIcrosope image collecting device with this luminescent image acquisition unit branch, and it gathers this fluorescence microscopy images and this illuminating microscope image.
5. according to the device of claim 1, it is characterized in that this device comprises the display part, be used to show this superimposed image.
6. according to the device of claim 1, it is characterized in that this device comprises recording section, be used to write down this superimposed image.
7. according to the device of claim 1, it is characterized in that this device comprises cell excitation supply part, is used to encourage cell.
8. according to the device of claim 7, it is characterized in that it is at least one that select that part is supplied with in this cell excitation: reagent is supplied with part, is used for providing reagent to this cell from comprise the group with the lower part; The adjustment part, the temperature that is used to regulate this sample; And the gas supply part branch, be used for providing gas to this cell.
9. method of utilizing optical imaging apparatus to obtain the sample originating from organism image is characterized in that this method comprises following step:
This sample and obtain its illumination image throws light on;
Do not throw light on sample and obtain the luminescent image of the light that the luminescence phenomenon of the cell in this sample sends;
Superpose this illumination image and this luminescent image to produce superimposed image; And
Determine the analyzed area in this superimposed image,
Wherein this optical imaging apparatus is an optical microscope; This illumination image is the transmitted light MIcrosope image that obtains in the viewed in transmitted light under this optical microscope, and this luminescent image is to observe the light that luminescence phenomenon sends in this cell and the illuminating microscope image that obtains under this optical microscope; Wherein this transmitted light MIcrosope image and this illuminating microscope image are gathered by shared MIcrosope image collecting device.
10. according to the method for claim 9, it is characterized in that this method comprises the step that shows this superimposed image.
11. the method according to claim 9 is characterized in that, this method comprises the step that writes down this superimposed image.
12. the method according to claim 9 is characterized in that, carries out twice or more times throw light on this sample and step of obtaining this illumination image.
13. the method according to claim 9 is characterized in that, this method comprises: the step of giving this cell excitation before the step of obtaining this illumination image and this luminescent image.
14. the method according to claim 13 is characterized in that, this method comprises: the step of obtaining this illumination image and this luminescent image before the step of giving this cell excitation.
15. the method according to claim 13 is characterized in that, giving the excitation of this cell in the step of giving this cell excitation is at least one excitation of selecting from the group that comprises following excitation: the reagent excitation; Electric excitation, the excitation by gas and the excitation by heat.
16. the method according to claim 9 is characterized in that, also comprises step: measure the light intensity in the zone corresponding in this luminescent image, and show this intensity variations with this analyzed area.
17. the method according to claim 9 is characterized in that, this method comprises: the step of obtaining the fluoroscopic image of cell in this sample.
18. method according to claim 17, it is characterized in that this method comprises such step: show in zone corresponding in this illumination image, this luminescent image with this analyzed area with the corresponding zone of this analyzed area and this fluoroscopic image in the corresponding zone of this analyzed area at least two zones.
19. method according to claim 9, it is characterized in that, this MIcrosope image collecting device has light receiving surface, and this optical microscope is included in object lens and the lens subassembly that forms the image of this sample on the light receiving surface of this MIcrosope image collecting device, and wherein the numerical aperture of these object lens and the square value that is incident upon ratio between the magnification of this sample image on this light receiving surface are 0.01 or bigger.
20. a device that obtains the sample originating from organism image, this device comprises optical microscope; The MIcrosope image collecting device is used to gather the MIcrosope image of this sample of this observation by light microscope; And image processing equipment, be used to obtain this MIcrosope image as view data and carries out image processing, wherein, this image processing equipment comprises the image overlay part, be used for producing superimposed image by stack transmitted light images and luminescent image, wherein obtain this transmitted light images, and the light that with this MIcrosope image collecting device luminescence phenomenon in the cell in this sample is sent is gathered and is obtained this luminescent image under this optical microscope gathering the image of this sample in the viewed in transmitted light of this MIcrosope image collecting device under this optical microscope at this sample; Also comprise the image-region specified portions, be used to specify the analyzed area in this superimposed image.
21. device according to claim 20, it is characterized in that, this MIcrosope image collecting device has light receiving surface, and this optical microscope comprises object lens and lens subassembly, be used on the light receiving surface of this MIcrosope image collecting device forming the image of this sample, wherein the numerical aperture of these object lens and between the magnification of this sample image of projection on this light receiving surface the square value of ratio be 0.01 or higher.
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