阅读说明：本技术 用于空间解析荧光相关光谱学的光垫显微镜 (Light pad microscope for spatially resolved fluorescence correlation spectroscopy ) 是由 麦克尔·克诺普 马尔特·瓦克斯穆特 热雷米·卡普拉德 于 2012-02-14 设计创作，主要内容包括：本公开内容教导了显微镜(1),其具有用于照明样品或物体(18)的照明光路(20)和用于观察所述样品的观察光路(40,50),所述显微镜包括在照明光路中的照明光路聚焦装置,所述照明光路聚焦装置限定沿着所述照明光路的照明方向和其垂直方向延伸的基本上二维的样品或物体照明区域(22)。所述显微镜进一步包括在照明光路中的用于有选择地照明所述基本上二维的物体照明区域的部分(10)的照明区域界定装置(27),其中所述基本上二维的物体照明区域的该部分在所述照明方向和其垂直方向中的至少一个方向上被界定。(The present disclosure teaches a microscope (1) having an illumination light path (20) for illuminating a sample or object (18) and an observation light path (40,50) for observing the sample, the microscope comprising an illumination light path focusing means in the illumination light path defining a substantially two-dimensional sample or object illumination area (22) extending along an illumination direction of the illumination light path and a direction perpendicular thereto. The microscope further comprises an illumination area defining means (27) in the illumination light path for selectively illuminating a portion (10) of the substantially two-dimensional object illumination area, wherein the portion of the substantially two-dimensional object illumination area is defined in at least one of the illumination direction and a direction perpendicular thereto.)

1. A microscope having an illumination light path (20) for illuminating a sample (8) and at least one observation light path (40,50) for observing the sample (8), the microscope comprising an illumination light path focusing device (23) in the illumination light path (20), the illumination light path focusing device (23) defining a substantially two-dimensional object illumination region (22), the object illumination region (22) extending along an illumination direction of the illumination light path (20) and a direction perpendicular thereto, and

illumination area defining means (27,29) in said illumination light path (20), said illumination area defining means (27,29) for selectively illuminating a portion (10) of said substantially two-dimensional object illumination area (22), wherein said portion (10) of said substantially two-dimensional object illumination area is defined in at least one of said illumination direction and said perpendicular direction.

2. The microscope as claimed in claim 1, wherein the illumination path focusing means (23) comprises at least one of a cylindrical lens (23), a distortion shaping lens, a one-dimensional array of spherical lenses or a one-dimensional array of aspherical lenses.

3. The microscope of claim 1 or 2, wherein the illumination path focusing means (23) comprises at least one distortion shaping mirror.

4. Microscope according to any one of the preceding claims, wherein the illumination area defining means (25) comprise at least a first aperture (29), the first aperture (29) being adapted to define the portion of the substantially two-dimensional object illumination area (22) in the illumination direction.

5. The microscope of any one of the preceding claims, wherein the illumination area defining means comprises at least a second aperture (27), the second aperture (27) being adapted to define the portion of the substantially two-dimensional object illumination area in a direction perpendicular to the illumination direction.

6. The microscope of any preceding claim, wherein the illumination region defining means (23,27) comprises a beam shaper.

7. The microscope of any one of the preceding claims, wherein the viewing direction of the at least one viewing optical path (40,50) is substantially perpendicular to the illumination direction and the substantially two-dimensional object illumination area (22).

8. The microscope of any preceding claim, further comprising an adjustable detection aperture (573) in the detection light path (50), the adjustable detection aperture (573) being capable of reducing the effective viewing area in one or two dimensions.

9. The microscope of claim 8, wherein the portion (10) of the substantially two-dimensional object illumination area (22) and the effective observation/observation area are congruent and/or coincide.

10. The microscope of claim 9, wherein the portion (10) of the substantially two-dimensional object illumination area (22) and the effective observation/observation area are movable through the sample (8) in unison and/or coincidentally.

11. The microscope of any one of the preceding claims, further comprising an additional objective (61) for illuminating and observing a sample (8) arranged in the sample illumination area.

12. The microscope as claimed in claim 11, wherein the additional objective (61) is a component of a fluorescence microscope (6).

13. The microscope of claim 11 or 12, wherein the additional objective (61) is part of an inverted microscope (6).

14. A method of detecting a sample, the method comprising:

illuminating a two-dimensional portion of the sample by focusing an illumination beam (20) onto a substantially two-dimensional object illumination area (22), the substantially two-dimensional object illumination area (22) extending in an illumination direction of the illumination beam (20) and a direction perpendicular thereto;

wherein illuminating the two-dimensional portion further comprises delimiting the substantially two-dimensional object illumination area (22) to selectively illuminate a portion (10) of the substantially two-dimensional object illumination area (22), wherein the portion of the substantially two-dimensional object illumination area is delimited at least in the illumination direction.

15. The method of claim 14, further comprising viewing the substantially two-dimensional object illumination area in a viewing direction, the viewing direction being substantially perpendicular to the illumination direction.

16. The method of claim 14 or 15, further comprising moving at least one of the substantially two-dimensional object illumination area (22) or the portion (10) of the substantially two-dimensional object illumination area across the sample (8).

17. The method of any one of claims 14-16, further comprising determining signal fluctuations in said portion (10) of said substantially two-dimensional object illumination area.

18. The method of any one of claims 14-17, further comprising determining a fluorescence intensity contrast for calibration.

Technical Field

The present invention relates to optical microscopes, in particular to optical microscopes with a defined focal volume (focal volume) and methods suitable for the determination of fluctuations in the concentration of a sample.

Background

Diffusion fundamentally contributes to the movement of soluble molecules, and thus to the spatiotemporal aspects of many biological processes, from regulation of cell division and signal transduction within cells, to hormonal regulation at tissue origin, and to morphogenic gradients during development. Quantifying the diffusive properties of biomolecules in complex cellular environments is very challenging. Devices and methods that enable direct analysis of diffusion processes are very rare and are all specifically tailored to individual problems.

The determination of the properties and behaviour of biomolecules, in particular proteins, and in particular in their natural environment, is a key step in elucidating and analyzing their function and the mechanisms behind cellular and developmental processes. Fluorescence Correlation Spectroscopy (FCS) [1,2] is a known method for analyzing the mobility of molecules, which provides information on the mobile and immobile parts of the labeled molecules, their diffusion properties and concentrations, and the co-diffusion of different labeled molecules interacting.

Confocal laser scanning microscopy (confocal microscope) is the instrument of choice for living cell imaging with high resolution [3] and for FCS, since confocal laser scanning microscopy in combination with hypersensitive photon counting using Avalanche Photodiodes (APDs) makes diffraction limited imaging possible. FCS measurements using confocal laser scanning microscopy have been applied to quantify the kinetics of protein complex formation involved in signalling (4 by EMBL and 5 by others) to study the maturation of mrnps with export-competence [6,7] or to characterize morphogenic gradients [8 ]. FCS measurements using confocal laser scanning microscopy are referred to as confocal FCS in this disclosure. However, confocal FCS experiments are still challenging due to the inherent limitations imposed by the serial practice of confocal FCS data acquisition from one point to another, along with the low overall fluorescence photon yield of photons input into the sample due to out-of-focus illumination [9 ]. In general, confocal laser scanning microscopes allow the determination of only one or a few points per cell at a specific selected position [4,10,11 ].

In other words, confocal FCS does not provide sufficient spatially resolved information to produce images of cells and other biological samples that would otherwise be able to visualize the dispersion process across the entire cell or organism and other protein parameters (e.g., protein interactions) from the FCS.

Another disadvantage of the known method and device is that diffuse spatially resolved imaging is limited or impossible.

Disclosure of Invention

The present disclosure teaches a microscope having an illumination light path for illuminating a sample or object and an observation light path for observing the sample. The microscope includes an illumination light path focusing arrangement in the illumination light path, the illumination light path focusing arrangement defining a substantially two-dimensional sample or object illumination region extending along an illumination direction of the illumination light path and a direction perpendicular thereto. The two-dimensional object or sample illumination area may be considered a light strip or light sheet (light-sheet). Since the illumination light is shaped into the two-dimensional sample illumination area, the illumination light path focusing means may be understood as a light path shaping means.

The microscope further comprises in the illumination beam path an illumination area defining means for selectively illuminating a part of the illumination area of the substantially two-dimensional object, wherein the part of the illumination area of the substantially two-dimensional object is defined at least in the illumination direction and/or in a direction perpendicular thereto. The light strip is thus delimited in at least one of the illumination direction and its perpendicular direction, substantially forming part of the substantially two-dimensional object illumination area, also referred to as "light pad". The thickness of the substantially two-dimensional object illumination area and the thickness of the light pad are much smaller than the illumination direction length and its perpendicular direction width. For example, the length in the illumination direction and the width in the perpendicular direction of the portion of the substantially two-dimensional object illumination area may be about 6 times or more than about 6 times the thickness of the portion of the substantially two-dimensional object illumination area.

The illumination path focusing (and shaping) device may include at least one of a cylindrical lens, an anamorphic shaping lens, a one-dimensional array of spherical or aspherical lenses. The illumination path focusing (and shaping) means may further comprise at least one distortion shaping mirror.

The illumination area defining means may comprise at least a first aperture for defining the portion of the substantially two-dimensional object illumination area in the illumination direction. The illumination area defining means may further comprise at least a second aperture for defining the portion of the substantially two-dimensional object illumination area in a direction perpendicular to the illumination direction. At least one of the first and second apertures may be adjustable and may be a circular aperture or a rectangular hole or slit.

The viewing direction of the viewing optical path may be substantially perpendicular to the illumination direction. The substantially two-dimensional object illumination area is then adjusted to lie within the focal plane of the detection objective. In this respect, the microscope may be based on a single plane illumination microscope having an illumination area which is defined at least in the illumination direction. Other viewing directions may also be used in conjunction with the present disclosure.

The detection or viewing optical path may include at least one spatial filter allowing to delimit the detection area to the portion of the substantially two-dimensional illumination area.

The observation and detection of the sample may be carried out using one or more arrays of detector pixels (such as at least one of a CCD camera or an EM-CCD camera) onto which the substantially two-dimensional object illumination and detection areas are projected/imaged using the observation optical path.

The present disclosure also teaches methods for viewing/detecting a sample. The method includes illuminating a two-dimensional portion of the sample by focusing an illumination beam onto a substantially two-dimensional object illumination area extending in an illumination direction of the illumination beam and a direction perpendicular thereto, wherein illuminating the two-dimensional portion further includes defining the substantially two-dimensional object illumination area to selectively illuminate a portion of the substantially two-dimensional object illumination area, wherein the portion of the substantially two-dimensional object illumination area is defined in at least one of the illumination direction and a direction perpendicular to the illumination direction. This portion of the substantially two-dimensional object illumination area may be referred to as a light pad.

The method may comprise moving at least one of the substantially two-dimensional object illumination area or the portion of the substantially two-dimensional object illumination area through the sample. This can be used to scan the entire sample. The movement of the illumination area of the substantially two-dimensional object and/or the part thereof in the sample may be performed in 3D by moving at least one illumination objective, by moving the illumination beam path focusing means or an element thereof, by scanning with a scanning unit, by moving the illumination area defining means and by changing the collimation of the illumination beam path, which may be done by manipulating the wavefront, for example with a Spatial Light Modulator (SLM) or with a mirror having a variable curvature.

In order to obtain an image of the distribution of e.g. fluorescent molecules over the illumination area and the detection area of the substantially two-dimensional object, the recordings of the light from the sample over each pixel of the detector pixel array over a certain time period may be combined.

Light from the sample may be recorded at each pixel of the detector pixel array at a series of short time intervals, and a spatio-temporal correlation analysis may be applied to the recordings to obtain FCS data for each pixel on the detector array.

The method may further comprise determining signal fluctuations in the portion of the substantially two-dimensional object illumination area. The temporal tracking of the fluorescence intensity over each pixel or over an area or portion of the substantially two-dimensional object illumination area may be analyzed for fluctuations. The fluctuation analysis may be at least one of a temporal autocorrelation analysis, a temporal cross-correlation analysis between signals from different pixels, a temporal cross-correlation analysis between signals from different spectral channels, a photon count histogram, a photon coincidence analysis between signals from different pixels, a photon coincidence analysis between signals from different spectral channels, and other methods known to those skilled in the art.

Drawings

Further aspects and details of the invention will become apparent upon reading the detailed description with reference to the drawings in which:

FIG. 1 shows FCS imaging based on light sheet with diffraction limited light pad;

FIG. 2 shows the microscope of FIG. 1a in more detail;

FIGS. 3a to 3c schematically illustrate an optical light pad;

FIG. 4 schematically illustrates the optical properties of a light pad microscope;

FIG. 5 shows the ID-FCS assay in solution;

FIG. 6 shows the effect of cell culture medium on a focused light sheet;

fig. 7 shows FCS imaging results of protein concentration and mobility in MDCK cells and in wing imago discs from drosophila larvae recorded with the light pad microscope;

FIG. 8 shows confocal FCS measurements in vivo for comparison;

FIG. 9 shows 1D-FCS assays for adult discs of wings and 3T3 cells from Drosophila larvae; and

fig. 10 shows a spatially resolved analysis of HP1 α mobility in 3T3 cells by FCS imaging.

Detailed Description

The invention will now be described with reference to the accompanying drawings. It should be understood that the examples, embodiments and aspects of the invention described herein are merely exemplary and do not limit the scope of the claims in any way. The invention is defined by the claims and their equivalents. It is to be understood that features of one embodiment or aspect of the invention may be combined with features of one or more other aspects and/or embodiments of the invention, and it is to be understood that not all of the example and embodiment features are necessary to practice the invention.

The present disclosure teaches a new microscope called light-pad microscope 1. The light-pad microscope 1 consists of three modules, as shown for example in fig. 1a and in more detail fig. 2: (i) an illumination device 2 that produces a diffraction-limited light sheet 22 from an illumination beam 20; the diffraction-limited light sheet 22 is bounded in at least one direction, thereby defining the light pad 10; (ii) a detection unit 4 capable of observing along at least one of the detection optical paths 40 and 50 of the light sheet 22 and/or the focal area of the light pad 10; and optionally (iii) an inverted microscope 6 that can conveniently position a particular region of a specimen or sample 8 within the focus of the optical pad 10.

The light pad microscope 1 of the present disclosure provides complete spatial control of fluorescence excitation when only a cross section of the specimen 8 is illuminated by the light sheet 22, thereby avoiding unwanted out-of-focus exposure. At the same time, all emitted photons originate at the focal plane of light sheet 22 and no filtering of photons based on their spatial origin is required.

The light pad microscope 1 is based on two orthogonally arranged objective lenses, namely an illumination objective lens 21 and a detection objective lens 41. The illumination objective 21 and the detection objective 41 may be long working distance objectives, such as, but not limited to, 40x/0.8NA objectives. It will be appreciated that other objectives with different magnifications and/or numerical apertures may be used. The illumination objective 21 and the detection objective 41 may be identical or may be different objectives with different magnifications and/or numerical apertures. The illumination objective 21 and the detection objective 41 may be immersed in a petri dish 82 containing the sample 8.

The light pad microscope 1 may be used with any wavelength or any combination of wavelengths that are considered to be helpful for imaging or FCS, depending on the sample 8 and the dye used to study the sample 8. For the detection of GFP fluorescence, for example, a488 nm line of an argon laser 220 (as shown in FIG. 2, e.g.InnovaSabre SBRC-R-DBW/20 from Coherent) with an output of, for example, 2W can be used. The power of the illumination beam 20 may be adjusted, for example, by passing the illumination beam through a half-wave plate 222 (e.g., WPMH05M-488 from Thorlabs) placed in front of a polarizer 224 (e.g., a Glan-Taylor polarizer such as GL5-A from Thorlabs) located behind an acousto-optic tunable filter 226 (e.g., AOTF; AA. AOTFnC-400.650-TN, AA Opto-Electronic). Single mode fiber 228 (e.g., kineFLEX-P-3-S-458-640-2.0-2.0-PL, Point Source) can be used to transmit light to the arrangement contained on test board 7. In another embodiment, the laser light may be transmitted directly to the arrangement by using an optical element, such as a mirror (not shown).

To shape the illumination beam 20 to produce the light sheet 22, the illumination beam 20 may first be collimated and distorted to cut an approximately constant cross-section from a Gaussian profile in one direction. In the other direction, the illumination beam 20 is focused with a cylindrical lens 23 (e.g. having an f of 75.6 mm). In order to accurately position the sheet of light 22 in the sample, the illumination beam 20 passes through a scanning module 26, the scanning module 26 being composed of, for example, a galvanometer-driven mirror 262(VM-500+, GSI) disposed in the back focal plane of an F-theta scanning lens 264(S4LFT0061, F60 mm, sil Optics). A tube lens 24 (e.g., having f-245.60 mm) and an underwater illumination objective 21 (e.g., Plan-apochromatat 40x/0.8NA, Leica) having a long working distance of, for example, 3.3mm may be used to produce the optical sheet 22 in sample 8. The width of the optical sheet 22 can be adjusted to, for example, between about 20 μm and 200 μm by adjusting the size of a width-defining slit or a width-defining aperture (iris)27 placed at the back focal plane of the barrel lens 24. This allows illumination of only a portion of the sample, thereby preventing photobleaching of adjacent areas when performing assays or image acquisition. The mirror 25 between the scan lens 264 and the tube lens 24 deflects the light beam by 45 deg. so that the illumination objective 21 is directed at 45 deg. to the horizontal plane (bottom of the petri dish) containing the specimen or sample 8.

A multi-slit arrangement in the illumination path 20 is used to define the light sheet 22 to the light pad 10 by an illumination area defining means. The illumination area defining means may comprise a width-defining slit or aperture 27 for defining the width w of the light sheet 22. The illumination region defining means may further include a length-defining slit or aperture 29 disposed between, for example, a tube lens 24 and the illumination objective lens 21 for defining the length l of the illumination region or light sheet 22 in the illumination direction of the illumination optical path 20. The light sheet 22 may thus be defined to a length l and a width w to form the light pad 10 (having a width w and a length l) as shown in fig. 1b and 3 a. The upper image of fig. 3a shows a top view of the light pad 10 seen through the detection objective 41 in the z-direction of fig. 1a, 1b and 2. The lower image of fig. 3a shows the light sheet 22 and the light pad 10 as seen along the x-direction of fig. 1a, 1b and 2, showing the thickness d of the light sheet 22 and the light pad 10. The adjustment of scanning module 26 and the positioning of length-defining slit 29 and width-defining slit 27 provides for convenient positioning of light pad 10 within selected areas of sample 8 (fig. 1a-1b and 3a-3 c).

In order to collect the emitted fluorescence, a detection objective 41, which is an observation objective, is arranged at an angle of 90 ° to the illumination objective 21 (fig. 2). To avoid reflected laser light, a dichroic mirror 44 (e.g. BS HC R488, AHF analytechnik) may be placed in front of a detection tube lens 43 located behind the observation objective 41 if an excitation wavelength of 488nm is used. A flip-mirror 45 may be used to flip the fluorescence between the two alternative detection paths, first detection path 40 and second detection path 50. Between the sample 8 and the flip mirror 45, the first detection optical path 40 and the second detection optical path 50 are superimposed and use the same elements. The present disclosure may be implemented using both the first detection optical path 40 and the second detection optical path 50 or using only the first detection optical path 40 or only the second detection optical path 50. The flip mirror 45 can be omitted if only one optical path is used.

The first detection path 40 may be used for intensity light sheet imaging and may include a band pass filter 47 (e.g., BrightLine HC525/45, AHF analytechnik) and a keplerian telescope 46 that may affect the overall magnification. The fluorescent signal is focused along the first detection optical path 40 onto an electron multiplying charge coupled device (EM-CCD) camera 48 (e.g., Quantum EM:512SC, Photometries, Inc.), which camera 48 is referred to as an imaging camera 48. The pixel size of this imaging camera 48 may be at 131x131nm²(actual size on chip: 16X16 μm²) In the sample plane, the field of view corresponds to 67x67 μm in the sample²Area. Smaller or larger chip pixel sizes and/or chips of different geometries may be used.

For light pad microscopes 1 using the imaging camera 48 and the first detection light path 40, the sample 8 can be 3D imaged using a motorized stage 84 holding the sample 8 as shown in fig. 1a and 2. In the case of roundish objects (e.g. microspheres or yeast cells), image stacks can be obtained at equidistant sample positions along the detection optical axis (z-axis). In the case where a laterally extending object is used as the sample 8, such as a drosophila larvae wing adult disc, the image stack can be acquired horizontally (in the y-z direction). A larger effective field of view can be obtained by tiled imaging (tiled imaging) of the adjacent areas.

The second detection beam path 50 can be used for FCS imaging (1D-/2D-FCS recording) and/or intensity imaging. The second detection optics 50 may include a bandpass filter 55 (e.g., BrightLine HC525/45, AHFAnalysentechnik) and a scan module 56. Spatial filtering may be achieved by a spatial filter 57, which spatial filter 57 may comprise two lenses, e.g. a first lens 571 and a second lens 572 which are achromatic doublets (e.g. f 60mm, Thorlabs); and an adjustable slit 573 (for example 07SLT701, Melles-Griot corporation) disposed at an imaging plane of the first doublet 571. The adjustable slits 573 can also provide a definition of the light pad 10 arranged perpendicular thereto. The scanning module 56 enables illumination zones for the sample 8The area of intra-domain FCS imaging is located and the spatial filter 57 allows to adjust the size of the image of this illuminated area. In other words, a section or portion of the light pad 10 may be selected for imaging or detection along the second detection light path 50. This defines the light pad 10 (fig. 1b and 2a) together with the lateral delimitation of the light sheet 22 in the illumination beam path 20 by means of a spatial filter 27 (aperture). Finally, a focusing lens 59, for example an aspherical lens (f ═ 40mm, Thorlabs corporation), focuses the fluorescence on a second EM-CCD camera (e.g., SamBa SE-34, Sensover corporation) called FCS camera 58. The total magnification of the second detection light path 50 may be, for example, 39x, which may result in a pixel size of 190x190 nm in sample space²(actual size on chip: 7.4X 7.4. mu.m)²) And producing a sample corresponding to 124x94 μm²The maximum field of view. Other magnifications and other pixel sizes and different detector arrays may also be used in the present disclosure and may also be configured for the microscope and the sample 8 to be investigated.

For the acquisition of one-dimensional (1D) -FCS data, the optical path 50 (fig. 1a and 2) to the FCS camera 58 can be used. To improve temporal resolution, the EM-CCD of FCS camera 58 may be operated in a line scan mode, if desired. Between subsequent exposure intervals, instead of transferring a full frame (complete frame) from the sensor area to the memory area of the chip, only a single line is shifted. A portion of the line or the entire line is then transferred to a frame grabber card (e.g., PCI-1422, National Instruments, inc.) via an a/D conversion and chip amplification chain. The readout process is a time-limited step of CCD image acquisition, which significantly improves the temporal resolution, such as 40 μ s (or 25,000 lines/second) for a portion of e.g. 120 pixels of a line. For an in vivo measurement, for example to expand the field of view, 340 pixels (half lines) with a temporal resolution of 70 μ s may be acquired. To increase the number of photoelectrons per data point, the spatial filter 57 in the second detection optical path 50 may be configured in such a way as to illuminate a plurality of lines.

The fluorescent signal can be collected, for example, for 30-60s per assay. To assess the overall background of the assay, the laser may be turned off before and/or at the time of the assay (e.g., 5 s).

Two-dimensional (2D) -FCS measurements can be performed according to the same procedure as in 1D-FCS. The spatial filter 57 may be adjusted in order to illuminate the 20 lines of the EM-CCD chip of the FCS camera 58. The corresponding 20 lines are then transferred between subsequent exposure intervals, for example to a storage area, and then converted, amplified and transferred to a frame capture card as a single frame. Here, a temporal resolution of, for example, 700 μ s can be achieved using the present technique when a frame size of 20 lines of 340 pixels is used. As for 1D-FCS, the total background of the signal can be determined at the beginning of each assay or at the time of performing each assay, for example, in 5 s. (time series or 2D fluctuation curve recordings of images are shown in FIG. 1D and FIG. 4 g).

Although the imaging optical path 40 with the imaging camera 48 and the separate FCS optical path 50 with the FCS camera 58 have been described in the above embodiments with reference to fig. 1 and 2, a composite imaging and FCS optical path with a single camera or imaging device for both FCS and imaging may also be used. The flip mirror 45 may be omitted.

The laser power for imaging the FCS can be determined at the focal plane using an illumination lens, for example a Nova II power meter (Ophir Optronics, jershire, israel) equipped with a PD300 detector. In a typical example of an in vivo experiment in 1D-/2D-FCS, the light sheet 22 may have a focal laser intensity of several kW^-2Within the range of (1). This corresponds to the lower limit of the focal intensity typically used in conventional confocal FCS devices. Thus, the intensity used to perform one FCS measurement in the confocal apparatus can be measured, for example, 20 times (along 20 pixels). Thus, in addition to avoiding out-of-focus illumination, the light pad microscope 1 also provides at least about 20 times higher efficiency in this embodiment compared to typical confocal FCS devices.

Furthermore, the axial scanning of the illumination objective 21 and the synchronous scanning of the detection area by the detection scanning module 56 or the synchronous scanning of the detection spatial filter/slit 573 enable scanning of the optical pad 10 within the field of view of the detection objective 41. Or the cylindrical lens 23 may be scanned to achieve the same effect. Alternatively, controlled collimation/non-collimation of the beam, for example by replacing the mirror 25 with a spatial light modulator, for example a mirror with adjustable curvature, may also be applied to achieve the same axial scanning of the optical pad 10.

Each pixel of the FCS camera 58 collects light emitted from a corresponding viewing volume element (fig. 1b) in the light pad 10. The temporal and spatial concentration (and fluorescence) fluctuations of individual fluorescent molecules, such as GFP-labeled proteins, are produced when the molecules undergo movement across individual volume elements in the light pad 10, for example driven by diffusion. These fluctuations can be recorded with high time resolution and single photon sensitivity using an EM-CCD chip as the FCS camera 58. For each individual of the volume elements, a spatio-temporal correlation analysis of the temporal tracking of the intensity of each pixel (fig. 1c) provides statistical information about the frequency and speed of the fluorescent molecule inflow and outflow motion (fig. 1 d-f). At calibration, this information is converted into a spatially resolved map of protein mobility, e.g., protein concentration and diffusion coefficient as in the case of soluble molecules.

The optical properties of the light pad microscope 1 are such that: the light pad microscope 1 produces a diffraction limited light pad, the length of which along the illumination axis is, for example, about 4 μm, providing an array of multiple nearly confocal single volume elements (see fig. 1b, 3a-3c and 4). The width of the light pad 10 is adjustable by means of a slit 27 in the illumination light path 20. The pixels of the FCS camera 58 may, for example, have a maximum available pad area of 3.8x65 μm²(fig. 4a) is subdivided into lines of 20 340 pixels, but other subdivisions are possible. The observation volume amounts to about 0.31fL and corresponds to an almost isotropic Point Spread Function (PSF) (fig. 4 e-h). The size of the observation volume is determined by the thickness of the light sheet 22, which may be about 0.7 μm (1/e along the z-axis) and the lateral resolution of the detection objective 41²) Or less than 0.7 μm. This produces an appearance that is, for example, about 1.6 times larger than a standard confocal microscopeThe volume was examined (FIGS. 4 i-l).

The light-pad microscope 1 can be constructed on a vertically standing test plate 7, as shown in fig. 1a and 2. The vertical configuration of such a device has the additional advantage that: the vertical configuration enables the use of a conventional petri dish 82 (without the need for 3D cell culture or embedding the sample in a gel, e.g. agarose) with a diameter of e.g. 60mm, filled with medium directly submerged in 2 objectives (fig. S1b, fig. 2). The vertical configuration also avoids optical aberrations due to changes in the refractive index of the air-glass, glass-media or media-agarose interface that may be encountered when using conventional light sheet microscope configurations with dry objective lenses for illumination. In order to allow free movement of the sample 8, the petri dish 82 may be mounted on a three-axis motorized platform 84 (e.g., stepper motors: LN-Mini23 manipulator block XY and LN-Mini Z vario, Cell biologyTracing/Luigs & Neumann, capable of positioning the sample 8 with an accuracy of 50 nm).

The light pad microscope 1 may be designed such that: the sample 8 can be viewed from the bottom (e.g. through the glass bottom of a petri dish, if a petri dish is used) with the aid of an optional conventional inverted microscope 6 (e.g. Olympus IX 70) equipped with an objective lens 61. For example, a long working distance dry objective lens, such as a 20x/0.4NA lens, may be used. The best lens can be selected by those skilled in the art for the needs of the application. Due to its larger field of view, inverted microscope 6 allows for easier and faster localization of larger samples of biological sample 8 in the pad or rapid selection of cultured cells suitable for FCS. In positioning the samples, transmitted light illumination may be used, for example, white light emitting diodes placed on the petri dish 82, in order to view them.

Alternatively, standard confocal fluorescence images, image stacks and FCS data can be acquired on the inverted microscope 6, for example if a confocal laser scanning microscope (e.g. Leica TCS SP5 AOBS smdfcs equipped with HCX PlanApo CS 63x/1.2NA water immersion objective) is used. For excitation, 488nm lines of Ar laser or another excitation wavelength may be used. Fluorescence can be detected using a photomultiplier tube for imaging and an avalanche photodiode (e.g., SPCM-AQR-14, Perkin-Elmer Optoelectronics, Inc.) for imaging and/or FCS. In this embodiment, the diameter of the detection aperture may be fixed at 1 Airy spot. The laser power in sample 8 is well below about 200 μ W for FCS and below 500 μ W for Confocal Laser Scanning Microscope (CLSM) acquisition as measured in front of the objective lens. To acquire FCS data, incident beam 60 is placed at a location of interest in a previously acquired image, and laser illumination and detector readout is initiated for 30-60 s.

Fig. 4a shows an illuminated slide sheet imaged with an imaging camera (z-axis is the view direction) and visualized by illumination with Alexa488 dissolved in water. The dashed area highlights the optical pad 10, and in this particular embodiment, the optical pad 10 is adjusted to 3.8x65 μm²The light sheet 22 is thin enough to provide a small enough single viewing volume element (fig. 1 b). To characterize the thickness of the light sheet, a horizontal mirror may be placed in the center of the petri dish 82 filled with water, and the petri dish 82 is positioned in the focal plane of both the illumination objective 21 and the detection objective 41 along with the horizontal mirror to reflect the light sheet 22 directly into the detection objective 41, as shown in fig. 4 b. After the dichroic mirror 44 and the emission filter 47 are removed from the first detection beam path 40, the focal section of the light sheet 22 is imaged with an imaging camera 48. The resulting image is shown in fig. 4 c. Fitting the mean horizontal intensity distribution with a Gaussian function yields a 1/e of 700 + -10 nm²Full width of time (fig. 2 d).

The total PSF was characterized by analyzing the image stack of individual fluorescent microspheres 20nm in diameter (see axis definition in fig. 4 e). Fitting of the image stack of microspheres according to a 3D Gaussian function (FIG. 2f), a lateral (x-y)1/e of 370 + -20 nm can be used²Radius and axial (z)1/e of 410 + -40 nm²Radius, which means that the PSF is almost isotropic, with a single observation volume of 0.31 fL. The transverse and axial intensity distributions extracted from the 3D stack were compared by yeast cells expressing the membrane protein Pma1 fused to GFP (fig. 4g-h), confirming the isotropic profile of the PSF.

Fig. 4i-l shows a direct comparison of the same microspheres characterized using a Leica SP5 confocal microscope equipped with a 1.2NA water immersion objective and used for confocal FCS. According to the same protocol, a transverse 1/e of 240. + -.10 nm is obtained²Radius and axial (z)1/e of 600 + -20 nm²The radius, i.e., the PSF, is significantly more anisotropic, having a volume of 0.19 fL. Overall, the PSF of the light pad microscope is isotropic and about 1.6 times greater than that of a standard confocal microscope, and thus small enough to enable FCS measurements. Furthermore, in contrast to conventional light sheet microscopes, the light pad microscope 1 enables isotropic 3D imaging of the sample 8 without the need to image the sample from different directions by rotating the sample or deconvolving the image.

FIG. 5 shows an example of in vitro 1D/2D-FCS recordings. In this application, the light pad microscope 1 is used for FCS of in vitro fluorescence samples. 1D-FCS data were recorded for FluoSphere fluorescent microspheres with a diameter of 20nm as sample 8. Fig. 5a shows 250 individual autocorrelation functions (ACFs) calculated from 1D-FCS recordings along the dashed line shown in the inset. The fluorescent microspheres tended to form heterogeneous aggregates, which were clearly visible in the raw data of the image (bright spots in the inset shown in fig. 5 a) and caused significant amplitude and attenuation heterogeneity of the ACFs. Thus, after removing the spikes (spikes) corresponding to the aggregates from the intensity time trace, the ACF shown in fig. 5a is obtained. When near-surface 1D-FCS recordings were made, pure Brownian motion of the fluorescent microspheres was observed at the bottom of the Petri dish 82, whereas in the center of the droplet, the resulting motion could be observed to be mainly convective. The quality of the aggregate-filtered FCS data allows us to distinguish between two modes of transport: the ACF (fig. 5b) can be fitted with a one-component anomalous diffusion model (using equation (2) defined below) to obtain different anomalous parameters 1.0 and 1.85, respectively, the former representing pure diffusion and the latter directional motion, as would be expected from the motion of large aggregates as can be seen in a movie.

To show the performance of the light pad microscope 1And to verify the specifications measured, FIG. 5c shows the 1D-FCS record for diffusion of Alexa488 solution in distilled water. The 60 individual ACFs are shown in fig. 5c, and the recordings from the pixel ranges marked in fig. 5d show only small variations in amplitude and time dependence. Fitting ACF with a one-component free diffusion model, previously determined axial focal radius of 410nm and independently determined 320 μm by cross-correlation analysis (see below and FIG. 5e)²s^-1The effective transverse focal radius of 490. + -.30 nm can be achieved (retrieval) (FIG. 5 d). For comparison, we calculated the theoretical expected value: since we applied 3-pixel binning to the data (FIG. 5f), we applied a 3x190 nm long rectangular detection profile and 1/e with 370nm as determined above²The gaussian function of the radius is convolved to obtain an effective detection profile that fits well to a 1/e with 500nm²The radius of the Gaussian function is consistent with the experimental result. The effective focal volume including the combining (binning) was 0.57 fL. The number of molecules obtained from the fit can be converted into a concentration profile (fig. 5d), which is almost constant along the line, so that the fluorophore concentration used can be well obtained. Thus, 1D-FCS of Alexa488 adequately confirmed imaging-based PSF assays in an independent manner.

In addition to the calculated ACF, one can calculate the spatial cross-correlation as a function of the inter-pixel distance as shown in FIGS. 5e-f and by fitting equation (2) globally to the CCF for pixel distances of 0-4, we obtain 320 + -10 μm as independent fitting parameters²s^-1The diffusion coefficient of (c).

Figure 6 shows the effect of the refractive index of the cell culture medium on the focusing of the light sheet 22. Objectives designed for water are expected to cause optical aberrations when operated in a medium having a refractive index different from that of water. In view of the long working distance of the objective lenses 21 and 41 that can be used with the light pad microscope 1, even small differences may show an effect that is not negligible. To evaluate the effect of this difference on the data obtained from 1D-/2D-FCS recordings with light-pad microscope 1, we observed the position of the beam waist of the slide 22 when using Alexa488 dissolved in water or in a buffer with a different refractive index (see fig. 6 a-b). When illuminated with a sufficiently high intensity, the beam waist of the illumination beam may be identified as a dark narrow region due to fluorophore saturation near the illumination focus. This effect can be used to accurately determine the position of the light pad 10. When Phosphate Buffered Saline (PBS) was used instead of water, a shift of 4.5 μm or 0.14% of the working distance of the light pad 10 towards the illumination objective 21 could be observed (see fig. 6 b). This result indicates that the refractive index of the growth medium has been taken into account and that the system needs to be adjusted accordingly by appropriate refocusing and repositioning of the detection objective.

FIG. 6c shows Alexa488 1D-FCS ACF at a concentration of 250nM dissolved in water, 1xPBS and 1xPBS with Ca2 +. Fitting ACF with a one-component free diffusion model, PBS and PBS/Ca when compared to water²⁺The resulting amplitude was significantly reduced by 48% and 64%, respectively. In contrast, for diffusion related times, PBS and PBS/Ca were observed when compared to water²⁺Only a small relative increase of 9% and 13%, respectively. The differences in diffusion related times may be attributed to the different viscosities of water and PBS (0.99 and 0.89mPa s at 25 ℃ respectively); [12,13]. This indicates that the different focus radii of the refractive indices of the media have no effect and that the reduced amplitude is due to an increased defocus signal. To illustrate the effect of spherical aberration on amplitude, we applied a correction factor of 0.52 measured in vivo in PBS and a correction factor of 0.52 measured in PBS/Ca to the concentration values obtained in the data fitting²⁺The in vivo measurement correction coefficient in (1) was 0.36.

Data processing analysis

The following provides embodiments for data processing and data analysis that may be used with the present disclosure. It will be appreciated by those skilled in the art that other methods of data processing and data analysis may be used depending on the parameters to be studied. Known data processing and data analysis methods for the diffusion of fluorescent molecules (or fluorescent particles) can be used in the present disclosure.

For theFor each pixel measured 1D-FCS and 2D-FCS, the intensity time trace F from pixel x in line y can be extracted from the image file after subtracting the data obtained for the first 5s or another part of the total acquisition time as background signal and after converting the pixel grey values to photoelectron numbers as described previously_x,y(t) of (d). From the obtained fluorescence intensity trace and confocal FCS measurement, an autocorrelation function (ACF) and a cross-correlation function (CCF) were calculated according to the following formulas.

The slow variations due to e.g. photo bleaching can be corrected by means of a moving average. The resulting ACF and CCF can be fitted by using, for example, Matlab software (The MathWorks corporation) using a non-linear least squares Levenberg-Marquardt algorithm with The following general model functions.

Where N is the number of (apparent) molecules whose lifetime in the non-fluorescent state is τ_blinkThe molecular fraction theta of (A) represents the molecular scintillation and takes into account the time of diffusion dependence tau_diff,i＝w₀ ²/(4D_i) Abnormal diffusion of the two components f₁,f₂＝1-f₁Transverse and axial focal radius w₀And z₀Component i ═ 1,2 apparent diffusion coefficient D_iAnd exception parameters α_i. In the case of CCF, the pass includes the pixel size δ and the pixel index x₁、y₁、x₂、y₂The second index term of (2) takes into account the pixel shift, and for ACF, the term is 1. Curve fitting was performed without any scintillation contribution, Θ being 0, for the 1D-FCS and 2D-FCS data for fluorescent microspheres and Alexa 488. For green fluorescent proteinConfocal and 1D-FCS data of classes, the non-fluorescence lifetime can be set to 100. mu.s. For 2D-FCS data, a temporal resolution of 0.7ms can ignore the flicker contribution, i.e., Θ equals 0. For a one-component fit, set f ₁1. By being adjusted to have 0<R_adj ²<1 degree of freedom R squared (R)_adj ²) Goodness of fit may be estimated. This can be used for R_adj ²An acceptable fit result of 0.8 or higher.

In this way, fitting parameters, goodness of fit R, can be established_adj ²And pixel intensity profiles (1D-FCS) and maps (2D-FCS). To exclude noisy data and/or unsatisfactory fit and to sort out those regions for which the resulting concentration, diffusion coefficient and component scores are plotted, the intensity and/or R may be determined_adj ²The atlas is thresholded to produce a binary mask.