Method of observation of the emission of light from a sample by dynamic optical microscopy

Abstract

Method for observing an emission of light from a sample in a medium of refractive index nL disposed against a surface of a transparent support of refractive index nS, greater than nL, the emission of light comprising luminous components oriented toward the support and forming an angle and including supercritical luminous components and critical or subcritical luminous components, implementing an observation device applying filters to the signal; and transforming the signal into an image zone of the sample Modulation of the signal is carried out, in which components of the emission of light are allowed to pass through so as to obtain image zones, the modulation pertaining to all or some of the collected signals; and at least one useful image zone of the sample is produced by combining image zones, the combination evidencing differences between the image zones related to the modulation.

Claims

1. A method for observing a light emission of a sample in a medium with a refractive index n.sub.L, the sample being arranged on a surface of a transparent support of refractive index n.sub.s, which is greater than n.sub.L, the light emission comprising luminous components of a given amplitude and phase, oriented toward the support and forming an angle with a direction perpendicular to the surface, said luminous components including supercritical luminous components, for which the angle is strictly greater than a critical angle .sub.c=arcsin(n.sub.L/n.sub.s), and also including critical or subcritical luminous components, for which the angle is less than or equal to the critical angle .sub.c, the method comprising: capturing, using an observation device, at least part of the light emission from a region of interest of the sample and obtaining a captured luminous signal comprising luminous components arising from the supercritical luminous components of the light emission; applying filters, using the observation device, to the captured luminous signal in order to selectively decrease the amplitude and/or change the phase of certain luminous components of the captured luminous signal to obtain a filtered luminous signal; and transforming, using the observation device, the filtered luminous signal into an image zone of the region of interest of the sample; wherein: the method realizes a modulation of the filtered luminous signal, in which luminous components arising from critical or subcritical luminous components of the light emission are allowed to pass through in order to obtain a plurality of image zones of one and the same region of interest of the sample, the modulation pertaining to all or some of the luminous components of the captured luminous signal arising from the supercritical luminous components of the light emission; and at least one useful image zone of the sample is produced by combining the plurality of image zones to produce at least one enhanced image zone for the region of interest, the combination evidencing differences between the image zones associated with the modulation.

2. The method according to claim 1, wherein the luminous components of the captured luminous signal being the subject of said modulation are emitted from the supercritical luminous components of the light emission for which the angle is within a predetermined range in accordance with a range of depths to be explored in the sample.

3. The method according to claim 1, wherein the image zones obtained by using the observation device and generating, by combining, the useful image zone of the sample, are successively obtained by successively applying filters to the captured luminous signal.

4. The method according to claim 1, the method further comprising: a) taking a plurality of image zones in a same region of interest of the sample using the observation device and a plurality of filters, each filter being used to take one image zone of said plurality of image zones, the plurality of filters being such that: a filter of said plurality of filters allows the passing through of, in the filtered luminous signal, luminous components arising from the supercritical luminous components of the light emission; the filters of said plurality of filters all allow the luminous components of the captured luminous signal arising from the critical or subcritical luminous components of the light emission to pass through, and act substantially identically between them on the luminous components of the captured luminous signal arising from the critical or subcritical luminous components of the light emission; and there are at least two filters of said plurality of filters that act substantially differently between them on the amplitude or the phase of at least some of the luminous components of the captured luminous signal arising from the supercritical luminous components of the light emission; and b) producing a useful image zone of the sample by a calculation combining the plurality of image zones taken in step a) to evidence differences between the image zones of the plurality of image zones of the sample.

5. The method according to claim 1, the method further comprising: a) taking at least two image zones of a same region of interest of the sample using the observation device and two filters, each filter being used to take one of the two image zones, the two filters being such that: one of the two filters allows the passing through, in the filtered luminous signal, of the luminous components from the supercritical luminous components of the light emission; and the other filter acts substantially identically to said one of the two filters on the luminous components of the captured luminous signal arising from the critical or subcritical luminous components of the light emission, and decreases substantially more than said one of the two filters the amplitude of at least some of the luminous components of the captured luminous signal arising from the supercritical luminous components of the light emission; and b) producing a useful image zone of the sample by a calculation combining the two image zones of the sample taken in step a), wherein the calculation comprises an algebraic difference between the two image zones of the sample.

6. The method according to claim 1, wherein the filters used also partially reduce the amplitude of all or some of the luminous components of the captured luminous signal arising from the critical and subcritical luminous components of the light emission.

7. The method according to claim 1, wherein luminous components of the captured luminous signal arising from luminous components of the light emission that form the same angle are processed substantially identically by a same filter for decreasing the amplitude or changing the phase.

8. The method according to claim 1, wherein the sample showing a phenomenon to be observed having a given characteristic time, the image zones are successively taken at time intervals of less than or equal to half of the characteristic time.

9. The method according to claim 1, wherein: one of the image zones of the sample is obtained using a neutral filter that allows to pass through, in the filtered luminous signal and without any decrease of amplitude, all the luminous components of the captured luminous signal arising from the supercritical luminous components of the light emission; and another image zone of the sample is obtained with a total filter, which cancels in the filtered luminous signal all the luminous components arising from the supercritical luminous components of the light emission.

10. The method according to claim 1, wherein the observation device comprises a full-field immersion lens and the filters are located in a rear focal plane of the immersion lens and/or in a conjugate plane of said rear focal plane.

11. The method according to claim 10, wherein the filters comprise a diaphragm that allows, in an open position, the passing through of luminous components of the captured luminous signal arising from the supercritical luminous components of the light emission and allows, depending on a degree of closure of the diaphragm, to obscure the luminous components of the captured luminous signal arising from the supercritical luminous components of the light emission having an angle greater than a limit value related to said degree of closure.

12. The method according to claim 1, wherein the sample to be observed is biological.

13. A method for observing a light emission of a sample in a medium having a refractive index n.sub.L, the sample being arranged on a surface of a transparent support having a refractive index n.sub.S, the refractive index n.sub.S being greater than the refractive index n.sub.L, the light emission being oriented toward the transparent support and forming an angle with a direction perpendicular to the surface, the light emission comprising supercritical luminous components for which the angle is greater than a critical angle .sub.c=sin.sup.1n.sub.L/n.sub.S, critical luminous components for which the angle is equal to the critical angle .sub.c, and subcritical luminous components for which the angle is less than the critical angle .sub.c, the method comprising: capturing at least a portion of the light emission from a region of interest of the sample to obtain a captured luminous signal, the captured luminous signal comprising the supercritical luminous components, the critical luminous components, and the subcritical luminous components; applying filters to the captured luminous signal to selectively decrease an amplitude and/or change a phase of certain luminous components of the captured luminous signal to obtain a filtered luminous signal; modulating the filtered luminous signal to obtain a first image zone of the region of interest of the sample by allowing luminous components arising from at least the supercritical luminous components and the subcritical luminous components to pass through an aperture of a diaphragm; modulating the filtered luminous signal to obtain a second image zone of the region of interest of the sample by obscuring luminous components arising from the supercritical luminous components with the diaphragm and allowing luminous components arising from at least the subcritical luminous components to pass through the aperture of the diaphragm; and combining at least the first image zone and the second image zone to obtain at least one useful image zone of the region of interest of the sample, the combination evidencing differences between the first image zone and the second image zone associated with the modulation.

14. The method of claim 13, modulating the filtered luminous signal to obtain the first image zone comprising allowing luminous components arising from the critical luminous components to pass through the aperture of the diaphragm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Other features and advantages of the present invention will become apparent from the following description of non-limiting exemplary embodiments, with reference to the accompanying drawings, wherein:

(2) FIG. 1 shows a schematic view of a microscopy device allowing to implement a method according to the invention;

(3) FIG. 2 shows a variant according to the invention of the apparatus shown in FIG. 1, suitable for confocal microscopy;

(4) FIG. 3 shows a variant according to the invention of the apparatus shown in FIG. 1, suitable for TIRF microscopy;

(5) FIG. 4 shows an example of a filter for the implementation of a method according to the invention;

(6) FIGS. 5a and b show the fluorescence emission components according to different configurations and have been commented on above;

(7) FIGS. 6a and 6b illustrate the image zones of a same cell respectively with and then without the luminous components of the captured luminous signal from the critical or subcritical luminous components of the light emission;

(8) FIG. 6c shows a useful image zone of the same cell obtained by combining the image zones of FIGS. 6a and 6b;

(9) FIG. 7 is a graphical representation of the light intensity of the spots obtained from a same test sample, firstly by the method described in the document FR-A-2943428 (obscuring of critical or subcritical components) and secondly by a method according to the invention (modulation applied to the supercritical components, and then demodulation).

(10) For reasons of clarity, the dimensions of the various elements shown in these figures are not necessarily in proportion to their actual dimensions. In the figures, identical references correspond to identical elements.

DETAILED DESCRIPTION

(11) FIG. 1 shows a schematic view of a fluorescence microscopy device 100. It comprises an immersion lens 110, of which the ON is greater than or equal to 1.33. A glass support 20 is arranged above the immersion lens 110. Oil is disposed between the immersion lens 110 and the glass support 20.

(12) A sample 10 to be observed is arranged on the glass support 20. This sample 10 comprises, for example, fluorescent elements dispersed in water.

(13) The rear focal plane of the lens is referenced with number 400. Excitation light is generated by a beam 200 from a light source, which passes through an excitation filter 210 and is reflected by a dichroic mirror 120 to illuminate the sample 10 after passing through the transparent support 20. An example of the path of the incident excitation light is indicated by the arrows pointing to the top of the figure. The incident excitation light may be partly reflected and is then filtered by an emission filter 130 so that the image formed on an image plane comprises only the fluorescence light emitted by the sample 10.

(14) The fluorescent light emitted by the sample 10 passes through the transparent support 20, the dichroic mirror 120, and the emission filter 130.

(15) According to the embodiment shown in FIG. 1, this light is reflected off a mirror 140 and the rest of the device works with the reflected light.

(16) A lens 150, called tube lens, allows the focusing of the light on an intermediate image plane 410.

(17) Then the light is parallelized by a lens 160 and focused by a lens 180 onto the image plane 430, where the image is acquired by a suitable device, notably by a camera 300. The planes 430 and 410 are conjugate image planes of the observation plane.

(18) The lenses 160 and 180 are arranged so that a conjugate plane 420 of the rear focal plane of the immersion lens 110 is located between the lenses 160 and 180.

(19) A variable-aperture diaphragm 170 is arranged in the rear focal plane of the immersion lens. This diaphragm acts as a filter for the luminous components of the captured luminous signal. It can be in open position and allow the passing through of all the luminous components of the captured luminous signal arising from the luminous components of the light emission. It can be in a partially closed position and obscure part of the luminous components of the captured luminous signal.

(20) More specifically, the light rays that are emitted following a certain angle by the fluorescent emitters of the sample 10 located in the observation plane intercept the rear focal plane 400 of the lens (or any conjugate plane 420 of the plane 400) at a certain distance r() from the center (defined by the optical axis) of this plane. r() is an increasing function of . For aplanatic lenses, for example, r() is approximately proportional to sin(). Thus, all the rays emitted at the angle (conically) describe a circle of a radius of r() in the rear focal plane.

(21) When the diaphragm 170 is arranged in the rear focal plane 400 of the lens 110, the relationship between r() and sin() is:

(22) r()=n.sub.if.sub.osin(), where f.sub.o is the focal distance of the immersion lens 110 (usually of the order of a few millimeters) and n.sub.i is the index of the immersion medium used for the lens (usually oil).

(23) According to an embodiment, there is an immersion lens with a magnification G=100 and the focal distance of the tube lens 150 is f.sub.t=200 mm.

(24) We then have f.sub.o=f.sub.t/G=2 mm. In this configuration we get: r (.sub.c)=2.66 mm.

(25) If the diaphragm 170 is arranged in the conjugate plane of the rear focal plane, one should take into account the magnification factor related to the optical system. For example, in the plane 420, it is necessary to introduce a multiplication factor G=f160/f150 where f150 is the focal distance of the tube lens 150 and f160 is the focal distance of the lens 160.

(26) The luminous components of the captured luminous signal arising from the critical or subcritical luminous components of the light emission intercept the rear focal plane following a closed centered disk of a radius of r(.sub.c). The luminous components of the captured luminous signal arising from the supercritical luminous components of the light emission, where .sub.c<<.sub.max, form an open ring in the rear focal plane r(.sub.c)<r()<r(.sub.max). The implementation of a diaphragm 170 centered on the optical axis and having an opening r(.sub.c) thus allows to obscure all supercritical luminous components.

(27) The selection is thus made at the emission. As a result, the lighting system does not need to be changed from that of a standard epifluorescence observation device. It is thus possible to illuminate with a source of non-coherent light, such as a standard white light, obtained in particular using a mercury lamp. This results in several advantages, such as the absence of significant additional cost (compared to TIRFM microscopy technique, where a laser light is required) as well as the possibility to obtain a homogeneous field (possibly allowing quantitative measurements).

(28) According to one embodiment, actuation means of the diaphragm operate at the video frame rate (typically in the magnitude of a few tens of Hertz) in order to pass alternatively from the open position to the closed position with the speed of image acquisition. It is thus possible to have information on the volume and the surface simultaneously.

(29) This imaging method is particularly suitable for imaging biological samples, in particular for the study of biological processes in living cells, such as cell adhesion phenomena, endocytosis/exocytosis, . . . .

(30) FIG. 2 shows a schematic view of a variant according to the invention of the device of FIG. 1 where the elements present before the image plane 430 are identical in both embodiments. In the device of FIG. 2, a pinhole-type mask 190 that comprises a hole 195 is arranged in the image plane. A mono-detector 350 allows a point-by-point acquisition of the light passing through the hole 195. It is thus possible to obtain a configuration that allows performing the confocal microscopy.

(31) FIG. 3 shows a schematic view of a variant according to the invention of the device of FIG. 1 where the elements of the microscopy device are similar, but where the light source differs.

(32) In the device of FIG. 3, the light 250 is coming from a laser and the illumination of the sample is produced by total internal reflection. It is thus possible to obtain an improved TIRF type device.

(33) Note that the rear focal plane of the commercial lenses is usually located inside the lens and is therefore difficult to access. It is thus often recommended to realize a system for imaging the rear focal plane to be able to insert the filter system 170 between the sensor and the lens.

(34) According to one embodiment, an inverted fluorescence microscope of the Ti type of Nikon is used that comprises a module (ref. TI-T-BHP, MEB55810) that enables imaging the rear focal plane and positioning an annular mask to enable the (external) phase contrast with large numerical aperture lenses. It is possible to put into this type of module a diaphragm 170 of the device according to the invention. The system of centering and adjusting the position of the plane is quite suitable for a diaphragm filtering supercritical angles. The system comprises a plurality of positions for different lenses.

(35) FIG. 4 shows a schematic view of a diaphragm 170 to be arranged in the rear focal plane 400 of the immersion lens 110 or in a conjugate plane 420 of said focal plane. The diaphragm 170 comprises a peripheral area 176 apt at obscuring light. This area 176 is either actually mobile (as in a camera), or the diaphragm is replaced by another, for example, by turning a motor-driven rotary filter wheel.

(36) The diaphragm 170 may be an iris diaphragm, such as that sold by Thorlabs. Its aperture is adjusted by moving mechanical moving parts (not shown).

(37) Another possibility is to use a wheel with openings or semi-transparent materials distributed on sectors of the wheel and rotate it. In this case, the diaphragm can, for example, be achieved by a circular hole of a suitable diameter in an opaque material. This allows the obtaining of very short transmission/shutter cycles, which, for example, keep pace with the acquisition pace of images with a camera.

(38) According to one example of an embodiment, a Nikon Ti-U type inverted fluorescence microscope is used with a base of the binocular tube of the phase TI-T-BPH, an oil-immersion 100 lens with a numerical aperture of 1.49. A fluorescence filter cube, which contains a transmission filter, a dichroic plate, and an excitation filter, is used. The light source used is a fiber source of the commercial reference Nikon Itensilight with a 130 W Hg lamp and a generator C-HGFI. The camera used is an EMCCD Andor Ixon+ camera, cooled to approximately 75. The diaphragm 170 used is the iris diaphragm produced by Thorlabs.

(39) The diaphragm is then positioned in the Nikon MEB55810 module (TI-T-BPH) instead of the phase ring. The position of the diaphragm is adjusted by means of the Bertrand lens of the microscope and by displacement of the module by means of screws for centering and axial position. The procedure followed is the same as the adjustment of the phase ring supplied by the manufacturer with the module.

(40) Observations have been conducted on embryonic human kidney cells marked by Choleratoxin (which binds to glycolipids on the membrane and the constituents of lipid rafts) coupled to Alexa 488 and excited by the Nikon Intensilight Hg 130 W lamp (conventional lamp). The filter cube used consists of an excitation filter with a bandwidth from 450 to 490 nm, a dichroic mirror of 500 nm, and an emission filter with a bandwidth from 510 to 550 nm.

(41) FIGS. 6a and 6b show two images obtained with an open diaphragm (6a) and with a closed diaphragm to hide all the supercritical luminous components (6b). The pause time (T=300 ms) and the gain (G=0) are identical for images 6a and 6b. The image 6c is obtained as the absolute value of the difference between the images 6a and 6b (that is to say, between the intensity of the signals associated with the images).

(42) It is noted that the two images 6a and 6b appear to be identical. However, image 6c is well contrasted. One advantageously observes intensity variations that are associated with membrane phenomena that are difficult to distinguish in the other two images, because they are embedded among other information coming from the inside of the cell.

(43) It should be noted that these observations have been advantageously made with a classic lamp and that it was not necessary to implement a laser to obtain them.

(44) Measurements of the lateral resolution have been performed with fluorescent beads Fluosphere (marketed by Invitrogen) of the excitation/emission: 580/605 nm deposited by spin (spin-coating) on a standard glass slide (thickness 0.13-0.16 mm), and then immersed in distilled water.

(45) FIG. 7 illustrates the profile of the fluorescence intensity of these beads (normalized signal intensity on the ordinate as a function of lateral displacement on the abscissa, expressed in microns). It should be noted that the C2 profile, which corresponds to the useful image zone obtained by the method according to the invention is narrower than the C1 profile, which corresponds to the image obtained by the method described in document FR-A-2943428. The corresponding improvement in resolution is 20-25%.

(46) The invention is not limited to these types of embodiments and should be interpreted in a non-limitative way and encompassing all equivalent embodiments.