METHOD FOR ACCELERATED MICROSCOPY OF LARGE SAMPLES

Abstract

A method for acquiring microscope-images of sample being larger than a field of view of a microscope, the method comprising creating a continuous relative movement between a sample and the microscope, wherein the optical axis of a microscope objective is substantially perpendicular to the vector of the relative movement, illuminating a part of the sample through the microscope objective, wherein the illuminated part of the sample is smaller than the field of view and forms an illumination slit, moving the illumination slit in a scanning direction across the field of view, and detecting light from the sample collected by the microscope objective, wherein the sample is moved in the same direction as the scanning direction or in a direction perpendicular to the scanning direction while the illumination slit is moved across the field of view.

Claims

1. A method for acquiring a microscope-image of a sample being larger than a field of view of a microscope, the method comprising: creating, by at least one control unit, a continuous relative movement between a sample and the microscope, wherein the optical axis of a microscope objective is substantially perpendicular to the vector of the relative movement; illuminating, by a light source controlled by the at least one control unit, a part of the sample through the microscope objective, wherein the illuminated part of the sample is smaller than the field of view and forms an illumination slit; moving, by a scanning unit controlled by the at least one control unit, the illumination slit in a scanning direction across the field of view; and detecting, by a detector controlled by the at least one control unit, light from the sample collected by the microscope objective; characterized in that the sample is moved in the same direction as the scanning direction or in a direction perpendicular to the scanning direction while the illumination slit is moved across the field of view by the scanning.

2. The method of claim 1, further comprising: correcting the acquired microscope-image to compensate for 1-dimensional image deformations or for 2-dimensional image deformations caused by the continuous movement of the sample.

3. The method according to claim 2, wherein in the case of the sample being moved in the same direction as the scanning direction, the correction comprises image contraction in scanning direction, the extent of contraction depending on the relative speed of the sample movement and the movement of the slit of the slit illumination.

4. The method according to claim 2, wherein in the case of the sample being moved in a direction perpendicular to the scanning direction, the correction comprises image shearing perpendicular to the scanning direction, the extent of shearing depending on the relative speed of the sample movement and the movement of the slit of the slit illumination.

5. The method according to claim 1, wherein the detector is a camera, controlled by the at least one control unit in such a manner that the image acquisition frame-rate is at least approximately synchronized with the time it takes the slit-illumination to move from a starting position at one end of the detector to an opposite end of the detector;

6. The method according to claim 5, wherein the detector is controlled by the at least one control unit in such a manner that at a time only a strip-like zone of the detector is activated to detect light, with the activated zone being moved across the detector in at least approximate synchronism with the movement of the image of the illumination slit on the detector so as to keep the image of the illuminated part of the sample in the centre of the activated zone of the detector and wherein the active zone is moved from a starting position at one end of the detector to an opposite end of the detector

7. The method according to claim 1, wherein the sample is illuminated using structured illumination.

8. The method according to claim 1, wherein the sample is moved in the same direction as the scanning direction at a speed which amounts to 5% to 50% of the speed of the movement of the illumination slit, preferably at a speed which amounts (i) 12.5%, (ii) 25%, (iii) 33% or (iv) 50% of the speed of the movement of the illumination slit.

9. The method according to claim 1, wherein the sample is moved in a direction perpendicular to the scanning direction at a speed which amounts to 5% to 100% of the speed of the movement of the illumination slit, preferably at a speed amounting to (i) 100%, (ii) 50%, or (iii) 33% of the speed of the movement of the illumination.

10. The method according to claim 1, wherein each position of the sample is imaged multiple times, preferably 7 times (i), 3 times (ii), or 2 times (iii) during a single sweep.

11. The method according to claim 10, wherein parameters of the scanning method are changed between repeated scanning events of the scanned position, wherein the changeable parameters include type of illumination or focus position.

12. The method according to claim 5, wherein the speed of the movement of the sample with respect to the speed of the movement of the illumination slit is adjusted in order to compensate for delays caused by the time it takes the illumination slit to return to its initial position.

13. The method to claim 1, wherein the scanning unit is controlled such that, when the activated zone on the detector reaches the end of the length of the detector, illumination of the sample by the light source is interrupted while the illumination slit is moved back to the starting position so as to complete one scanning cycle.

14. The method of claim 13, wherein the scanning cycle is repeated while the sample is moved until a desired area of the sample has been covered by the illumination slit, wherein a image of a portion of the sample is created in each scanning cycle.

15. The method of claim 14, wherein the images of portions of the sample partially overlap with images created from adjacent portions of the sample.

16. A microscope device comprising: a microscope objective; at least one control unit; a sample stage for holding a sample; a mechanism for creating a continuous relative movement between a sample and the microscope; a light source; an illumination slit-forming element; a scanning unit; and a detector; the device being configured to: create, by at least one control unit, a continuous relative movement between a sample and the microscope; wherein the optical axis of a microscope objective is substantially perpendicular to the vector of the relative movement; illuminate by a light source controlled by the at least one control unit, a part of the sample through the microscope objective, wherein the illuminated part of the sample is smaller than the field of view and forms an illumination slit; move, by a scanning unit controlled by the at least one control unit, the illumination slit in a scanning direction across the field of view; and detect, by a detector controlled by the at least one control unit, the illumination slit in a scanning direction across the field of view; and detect, by a detector controlled by the at least one control unit, light from the sample collected by the microscope objective; characterized in that the sample is moved in the same direction as the scanning direction or in a direction perpendicular to the scanning direction while illumination slit is moved across the field of view by the scanning.

17. A non-transitory computer readable medium comprising instructions thereon which when executed on a processor cause a microscope comprising: a microscopic objective; at least one control unit; a sample stage for holding a sample; a mechanism for creating a continuous relative movement between a sample and the microscope; a light source; an illumination slit-forming element; a scanning unit; and a detector, to create by at least one control unit, a continuous relative movement between a sample and the microscope, wherein the optical axis of a microscope objective is substantially perpendicular to the vector of the relative movement; illuminate, by a light source controlled by the at least one control unit, a part of the sample through the microscope objective, wherein the illuminated part of the sample is smaller than the field of view and forms an illumination slit; move, by a scanning unit controlled by the at least one control unit, the illumination slit in a scanning direction across the field of view; and detect, by a detector controlled by the at least one control unit the illumination collected by the microscope objective; characterized in that the sample is moved in the same direction as the scanning direction or in a direction perpendicular to the scanning direction while illumination slit is moved across the field of view by the scanning.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 shows how resolution is reduced with increasing stage/sample movement speed relative to the speed of the illumination slit when both move perpendicular or antiparallel.

[0025] FIG. 2 outlines image acquisition and correction procedure for perpendicular movement of sample and illumination.

[0026] FIG. 3 outlines image acquisition and correction procedure for movement of sample and illumination in parallel.

[0027] FIG. 4 shows the achieved resolution in scan-direction as a function of the speed of the illumination slit relative to the sample speed.

[0028] FIG. 5 schematically shows the use of a spatially modulated slit-illumination in embodiments of the present disclosure.

[0029] FIG. 6 shows a schematic overview of an microscope according to the disclosure.

[0030] The accompanying drawings illustrate exemplary embodiments of the disclosure and serve to explain, by way of example, the principles of the disclosure.

DETAILED DESCRIPTION

[0031] The present disclosure will now be described more fully hereinafter with reference to the accompanying figures, in which preferred embodiments are shown. The method, however, may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. It should be noted that these figures are intended to illustrate the general characteristics of the methods utilized in certain embodiments. However, the figures may not precisely reflect the precise structure or performance characteristic of any given embodiment. Moreover, in the figures like reference numerals designate corresponding parts throughout the different views or embodiments.

[0032] The present disclosure also aims at mapping large sample-areas with a continuously moving sample to acquire a fully resolved microscope-image with the final desired magnification without sacrificing resolution in scan-direction. To this end the direction of a continuously moving sample is made not antiparallel to the movement of a slit-shaped illumination pattern. In one embodiment of the present disclosure, the sample is continuously moved perpendicular to the scanning direction.

[0033] In another embodiment, the sample is moved in parallel to, i.e. in the same direction as the scanning direction.

[0034] FIG. 1 shows how resolution drops with increasing scan-speed relative to the speed of the illumination slit for the case when both move in antiparallel fashion as known from methods for acquiring images of continuously moving samples according to the prior art. As outlined above, in the event that slit-illumination and stage move at equal speed, image compression and motion blur together cause a more than twofold resolution drop in scan-direction, corresponding to a (unidirectional) virtual magnification of 50% of the nominal objective magnification.

[0035] However, as also shown in FIG. 1, if sample stage and slit-illumination move in accordance to an embodiment of the present disclosure with equal speed perpendicularly to each other, the unilateral resolution-drop is 25% only, instead of 45%, Thus one can turn a 40× objective into a virtual 30× with very little resolution loss, and if the stage moves even slower, the virtual magnification remains even closer to the nominal one and the resolution-loss shrinks even further. When the stage movement is slowed to ⅓ the speed of the slit-illumination, resolution in scan-direction is nearly unaffected, dropping less than 5%. Thus, given the speed of modern high-speed cameras, one can capture images from an illumination slit moving over the sample at a speed of 30 mm/s, and since one can record image stripes from a sample moving at ⅓ this speed, one can acquire high-resolution images in “lawn-mower fashion” without stop&go and without a significantly reduced resolution.

[0036] However, images acquired in accordance with embodiments of the present disclosure are distorted in 2-dimensions and need to be straightened mathematically as illustrated schematically in FIG. 2. The corresponding correction of the acquired microscopic image to compensate for 2-dimensional image deformations caused by the continuous movement of the sample can be performed directly after image acquisition or can be done subsequently.

[0037] Another embodiment of the disclosure is depicted schematically in FIG. 3. In this embodiment, the sample is moved in parallel to, i.e. in the same direction as the slit-illumination and the slit-illumination is set back to its starting position on the camera-chip each time it has swept over the field of view of the camera. In an example making use of the embodiment, the illumination slit moves twice as fast as the sample, thus each camera-exposure captures an image-field corresponding to 50% of the field of view of the camera. As a result of the movement of the sample with respect to the illumination slit, the acquired image is stretched out (in this example twofold) in scan-direction and hence needs to compressed (in the given example twofold) in order to establish the original magnification. The corresponding correction of the acquired microscopic image to compensate for 1-dimensional image deformations caused by the continuous movement of the sample can be performed directly after image acquisition or can be done subsequently.

[0038] It has to be noted that the numeric values given in the above embodiment is an idealization, as assumes a negligible fly-back time for the illumination-slit scanner. To accommodate a realistic fly-back time, one has to decrease the speed of the moving sample accordingly, otherwise one will miss fractions of the sample. Thus to scan 50% of the camera field of view takes the time to complete a camera-sweep plus the flyback-time. Respective hardware specific adaptations fall also under the scope of the present disclosure.

[0039] FIG. 4 plots the achieved resolution in scan-direction as a function of the speed of the illumination slit relative to the sample speed with respect to the embodiment of the present disclosure in which the sample is moved in parallel to, i.e. in the same direction as the slit-illumination. In FIG. 4 zero means a stationary sample, and 0.5 means that the sample moves at half the speed of the slit-illumination. Ratios>0.5 yield dis-continuous images with missing zones.

[0040] The ordinate plots the unilateral resolution in scan-direction, whereby 1 means diffraction limited resolution, whereas values>1 denote a resolution enhancement. As the achieved resolution depends on the time a given sample-element remains within the illumination slit during the scan, it not only depends on the relative speed of sample movement, but also on the width of the illumination-slit. The values shown in FIG. 3 show the maximally obtained resolution using a diffraction-limited slit-width.

[0041] It as to be noted that in this variant of the disclosure, in which the sample is moved in parallel to, i.e. in the same direction as the slit-illumination, the moving sample never degrades resolution, instead it even provides a (unilateral) resolution increase, which is maximal (2-fold) when the sample moves at the maximal allowable speed (½ of the speed of the slit illumination), the limit beyond which the resulting image becomes discontinuous.

[0042] As outlined above, the underlying principle of the disclosure is that a signal, especially a fluorescent signal from a given fluorophore in a continuously moving sample is excited only during the short time when the moving slit-illumination and the moving sample-element overlap, and emitted photons are captured by the detector only during this short time of spatial overlap. However, in cases where the slit-illumination undergoes scattering in focal-planes above and below the focal-plane of the objective, the camera will collect background signal from fluorophores, which are not directly illuminated, and this, in turn, will cause image-blurring. This detrimental effect is reduced by unidirectionallly restricting the active area of the camera to form an active window, which follows the slit illumination while it scans from one end of the field of view of the camera to the other end. The smaller this active window is made, the fewer emission photons originating from not directly illuminated regions can contribute to the image hence the better blurring is suppressed.

[0043] The merit of the disclosure over the current state of the art is outlined by the following experiment:

[0044] Goal is to scan 96 wells of a micro-plate at diffraction-limited (using a 40× objective with NA 1.25 and Nyquist-sampling at 525 nm) pixel resolution of 107 nm. Using, for example, a rolling shutter chip of 4.432×2.368 pixel with 4.6 μm pixel size and a frame-rate of 120 fps (as provided, for example by a Fairchild LTN 4323), one can move the sample at 15.2 mm/s and thus acquire an image stripe of 474 μm width and 110 mm length in 7.2 s. 16 such stripes are needed for scanning a single row of wells, and eight such rows make up a whole plate. Thus the disclosure enables to scan a whole micro-plate in 8×16×7.2 s, that is little more than 16 minutes. Covering the same area by stitching single image-tiles acquired with the conventional stop&go approach according to the prior art would require to accelerate and stop ca. 45.000 times. Even with the fastest stages one maximally visits 6 position/s, thus reading the whole micro-plate in conventional fashion would take at least two hours, that is 7.5× longer.

[0045] As shown in FIG. 4, image-resolution is enhanced in scan-direction beyond the nominal resolution of the orthogonal direction. It can increase up to 2-fold in the direction of the scan-axis, while it remains unchanged along the slit-axis. The concept of structured illumination microscopy (SIM), using a spatially modulated slit-illumination as in the prior art would allow to increase resolution along this axis, too, and would provide a further increased sectioning beyond slit-confocality.

[0046] This is outlined in FIG. 5, where a—in slit-direction intensity-modulated—illumination-stripe (fill-factor 50%) is scanned so as to record a continuous image-stripe. Three such image stripes, each side-shifted 113rd of a period relative to the previous one, need to be recorded from a given sample-region before one can calculate a sectioned image from these three “phase images”. To record these individual image stripes one can scan the sample continuously from one end of the desired region to the other, turn there and scan in reverse direction after phase-shifting the pattern 113rd of a period. By repeating this procedure at the origin of the scan, one obtains the third phase image. The three such obtained phase-images can undergo a suitable SIM-analysis and can be combined to yield image lanes, which not only feature resolution enhancement in the direction of the slit-illumination, too, but also further removal of out-of-focus information beyond that afforded by the slit-confocal scan. Thus, with respect to the above example, a continuous SIM approach allows to obtain a sectioned SIM-image from a complete micro-plate in little more than ½ hour, still 4× faster than a slit-confocal stop&go approach.

[0047] As outlined in FIG. 4, a stage-speed as fast as 50% of the speed of the illumination slit provides gap-less image stripes composed of subsequent exposures. If the stage-speed is lowered to ⅓ of the slit-speed, each emitting area will be revisited twice, if it is lowered to ¼ of the slit-speed, each emitting area will be revisited three times and so forth. Thus the above three exposures for a SIM image could also be achieved by lowering the scan-speed such that the emission-slit passes every area of the sample three times, with an orthogonal phase-shift in-between each of the three passages. Compared to the above “lawn-mower” approach, where long stripes are recorded between turning points, this affords a 1.S× faster scan, as the scan-speed needs to be reduced twice only in order to obtain three phase-images. However, the resulting resolution improvement drops from 1.4× to 1.25×. Following the same reasoning, one could also acquire SIM-images composed from 7 phase-images by reducing the stage-speed to 1/8 of the speed of the slit-illumination, thereby revisiting each sample area 7 times.

[0048] The above feature of the disclosure, namely that areas of the sample can be imaged several time in a single, uninterrupted scan, i.e. during a single sweep can be applied for applications other than SIM, too. For instance, (i) one can change the excitation wavelength of the illumination-stripe between imaging events of the same area or at the same position of the sample in order to obtain a multicolor image in a single sweep, (ii) one can change the focal-plane between imaging events of the same area or at the same position of the sample so as to acquire an image stack in a single sweep, or (iii) one can use an intentionally tilted sample plane, which automatically yields images taken at as many focal planes as there are imaging events of the same area or at the same position of the sample.

[0049] FIG. 6 shows a schematic overview of an microscope according to the disclosure suitable to perform the method according to the disclosure. The microscope comprises: A sample (1), which is placed and held in the focal-plane (2) of a microscope objective (3), can be moved continuously and with steady speed by a stage (4), which can move orthogonally to the chip-coordinates of an area detector (5), on which, after passing objective (3) and a tube-lens (6), an image of the sample (1) is recorded. The fluorescence image is elicited by a slit-shaped illumination (7), generated by a light-source (8) and a slit-forming optical element (9), for instance a Powell-lens, and it is scanned over the sample with the help of a deflecting element (10), which can move the illumination slit (7) strictly parallel to one of the moved axes of stage (4). Illumination and emission beam are combined by means of a suitable dichroic element (11). At least one control-unit (12) synchronizes the stage (4), camera (5), light-source (8) and scan-unit (10). The at least one control-unit (12) is further configured to perform the method as described above.

[0050] In particular embodiments, the at least one control unit is at least one computer system configured perform one or more steps of one or more methods described or illustrated herein. In particular embodiments, one or more computer systems provide functionality described or illustrated herein. In particular embodiments, software running on one or more computer systems performs one or more steps of one or more methods described or illustrated herein or provides functionality described or illustrated herein. The control unit may include a storage for data or instructions. For example and not by way of limitation, storage may include a hard disk drive (HOD), a floppy disk drive, flash memory, an optical disc, a magneto-optical disc, magnetic tape, or a Universal Serial Bus (USB) drive or a combination of two or more of these. Storage may include removable or non-removable (or fixed) media, where appropriate. Storage may be internal or external to computer system, where appropriate. In particular embodiments, storage is non-volatile, solid-state memory. In particular embodiments, storage includes read-only memory (ROM). Where appropriate, this ROM may be mask-programmed ROM, programmable ROM (PROM), erasable PROM (EPROM), electrically erasable PROM (EEPROM), electrically alterable ROM (EAROM), or flash memory or a combination of two or more of these. This disclosure contemplates mass storage 96 taking any suitable physical form. Storage may include one or more storage control units facilitating communication between processor and storage, where appropriate. Where appropriate, storage may include one or more storages.

[0051] The scope of this disclosure encompasses all changes, substitutions, variations, alterations, and modifications to the example embodiments described or illustrated herein that a person having ordinary skill in the art would comprehend. The scope of this disclosure is not limited to the example embodiments described or illustrated herein. Moreover, although this disclosure describes and illustrates respective embodiments herein as including particular components, elements, feature, functions, operations, or steps, any of these embodiments may include any combination or permutation of any of the components, elements, features, functions, operations, or steps described or illustrated anywhere herein that a person having ordinary skill in the art would comprehend. Furthermore, reference in the appended claims to an method or device or a component of an device or system being adapted to, arranged to, capable of, configured to, enabled to, operable to, or operative to perform a particular function encompasses that apparatus, system, component, whether or not it or that particular function is activated, turned on, or unlocked, as long as that apparatus, system, or component is so adapted, arranged, capable, configured, enabled, operable, or operative. Additionally, although this disclosure describes or illustrates particular embodiments as providing particular advantages, particular embodiments may provide none, some, or all of these advantages.