METHOD AND CONFIGURATION FOR THE OPTICAL DETECTION OF AN ILLUMINATED SPECIMEN
A method and a configuration for the depth-resolved optical detection of a specimen, in which a specimen or a part of the specimen is scanned by means of preferably linear illumination. The illumination of the specimen is periodically structured in the focus in at least one spatial direction. Light coming from the specimen is detected and images of the specimen are generated. At least one optical sectional image and/or one image with enhanced resolution is calculated through the specimen. Images are repeatedly acquired and sectional images are repeatedly blended while changing the orientation of the linear illumination relative to the specimen and/or spatial intervals between lines exposed to detection light from the illuminated specimen region are generated for the line-by-line non-descanned detection on an area detector or a camera and/or, during a scan, light is further deflected upstream of the detector through the line in the direction of the scan of the specimen.
This application claims the benefit of U.S. provisional patent application 60/990,016 filed on Nov. 26, 2007, the contents of which are hereby incorporated by reference herein.
FIELD OF THE INVENTIONThe present invention relates generally to the field of microscopy, and more particularly to microscopy in which structured illumination is used for depth discrimination in the wide field and for enhancing resolution and contrast.
BACKGROUND OF THE INVENTIONIn microscopy, structured illumination is used for depth discrimination in the wide field [1]1 and for enhancing the resolution and the contrast [2]. Generally, a grating or another periodic structure is projected into the specimen [3] or an interference pattern is generated in the specimen by means of interference of coherent component beams [4]. By shifting the illumination structure, images are generated that differ from one another with different phase angles of the period structure. Subsequently, these images are suitably blended with one another so as to obtain an optical sectional image and/or an image with enhanced contrast and enhanced resolution. The disadvantage is that the signal from out-of-focus regions of the specimen is detected as well, which, because of the limited dynamic region of the detector, leads to a reduced signal-to-noise ratio. The strength of the out-of-focus signal limits the useful sample thickness. This is of considerable significance, especially in cases in which the frequency of the structure approaches the diffraction-limited threshold frequency of the optical system, and the contrast of the structure is therefore necessarily low. This invariably applies to cases in which the objective is enhancement of contrast and resolution. Bracketed references refer to the list of references at the end of the specification prior to the claims.
One solution to this problem aims at a partially confocal detection which is made possible by structuring a line of light and detecting the thereby excited fluorescent light by means of a slit detector [5]. However, this method has a number of disadvantages. Structuring occurs only along the line. As a result, the effects of contrast and resolution enhancement are limited to this direction. Thus, especially in cases of nonlinear [6], but also linear, structures [7], the discrepancy between the one direction with enhanced resolution and all other spatial directions is significant. It is necessary to scan the line in a random direction in the specimen plane and to set the phase angle of the period structure. In the prior art, this requires separate actuators for controlling the relative phase angle and the scanning procedure.
SUMMARY OF THE INVENTIONIt is accordingly an object of the present invention to overcome the drawbacks of the prior art.
According to the present invention, the advantages of structured illumination in the wide field (few optical components, high parallelization) are combined with the advantages of structured illumination along a line (partially confocal suppression of the background signal for maximum contrast, high intensities in the focus for nonlinear and linear specimen interactions). The proposed configuration makes it possible to rotate the scanning direction rapidly, variably, and precisely, and to adjust the relative phase angle of the imaged structured periodic structure by means of only two scanners. In addition, it enables a variably adjustable confocal detection while allowing only very low losses of light in the detection beam path. In this context, reference is also made to DE 10155002 A1, the disclosure of which is hereby incorporated by reference as if such disclosure were fully set forth herein.
A solution according to the present invention is preferably a line-scanning microscope with as few components in the detection beam path as in a wide-field system. This includes an objective lens which is corrected for an infinite beam path, a barrel lens, a main color divider, an emission filter and a camera. In the excitation beam path, a beam-shaping unit which shapes the light beam of a light source. The light beam has been intensity-modulated by a modulator into a line that is modulated along the line width. In an example illustrated in
According to another embodiment of the present invention (not shown in
Disposed further along the optical axis are a first scanner, a second scanner orthogonal to the first scanner, and a scanning lens. The axis of rotation or swivel axis of the scanner is disposed substantially orthogonal to the axis of rotation of the first scanner. The scanner is used to shift the line in the specimen in the x-direction, and the scanner is used to shift the line in the y-direction.
Both scanners and are disposed near the conjugate pupil plane.
Next, shifting the phase of the structured line and scanning the image field by means of the interaction of the two scanners (9) and (23) with the AOM (5) will be described.
Without loss of generality, an example will be described, wherein the line in the specimen is oriented along the x-direction and scanning of the image field takes place in the y-direction, perpendicular to the x-direction. This also requires an orientation of the beam-shaping unit (8) to generate an orientation of the line in the x-direction.
During this line orientation, the scanner (23) serves to change the phase angle of the structure between two and more acquired images, while the scanner (9) is responsible for the scanning procedure in the y-direction.
From the images acquired at different phase angles (“phase images”), a sectional image is calculated (reconstructed). In this context, reference is made to DE 10155002 A1.
If, during a linear scan by the scanner (9) over a time Δt=t3−t1, the camera synchronously acquires an image with an exposure time of at least Δt, the result obtained is equivalent to a wide-field image of the specimen. At the same time, the out-of-focus background is detected as well. According to the present invention, confocal filtering can be used if the modulator (5), synchronously with the scanning procedure, periodically switches the illumination on and off in the y-direction as each phase image that is needed to calculate a sectional image is acquired.
One advantage is that even during the switched-off intervals, the scanner, in addition to the continuous scanning motion with on and off switching, can be rapidly moved to the next position with switched-on illumination in which the illuminated scanning procedure is continued. The scanner could also move step-by-step, similar to a stepping motor.
The method according to the present invention leads to an exposure in the camera plane, which exposure is structured in the y-direction (see
This procedure of shifting the specimen illumination line by line is repeated until all sections of the specimen in the image field have been scanned, so that M images per phase angle are obtained as a result of this acquisition procedure.
As an alternative, to set the spatial interval between exposed lines, first, images could be acquired at several phase angles of the periodic structure, and subsequently the spatial interval could be changed for the acquisition of several more phase images.
In addition to the method already described above, each of these images can be created by repeatedly acquiring each image, preferably at the lowest possible intensity to spare the specimen, using the same scanner settings and then taking the mean. This method can reduce artifacts due to bleaching phenomena taking place in the specimen. By blending M images per phase angle, it is now possible to adjust the confocality.
Specifically, it is necessary to subtract the exposed background between the exposed receiver lines, which was detected by the receiver, for the individual images. This background can be readily identified on the receiver since the exposed lines in the specimen can be unambiguously linked to regions that are separated from one another on the camera.
If all M images of a phase angle are simply summed up, a result corresponding to the wide-field image is obtained. Summing up the images after selection of the lines that correspond to the relevant illuminated lines in the focus of the specimen leads to a confocal image. In this step, the image regions neighboring the selected lines are, as described, masked and not analyzed. This corresponds to the function of a virtual slit diaphragm, since the unused, masked image regions correspond to the detection sites of the out-of-focus scattered light. The confocality can be varied between 1 Airy unit (2 lines selected) and M Airy units (virtual slit diaphragm).
Compared to nonconfocal detection, the speed of image acquisition is decreased by the factor M. Based on an image acquisition of 50 images/s, at M=5, a complete image can be obtained in 100 ms (at a phase angle of the structure). However, it should be noted that for each structure orientation, N=3 to 5 images at different phase angles must be acquired. Thus, in the case of a linear structure with 3 structure orientations, typically, 9 images must be taken [7], which, at M=5, leads to an image acquisition time of approximately 1 s per plane.
A slightly more favorable situation results if the scanner (9) does not scan the image field uniformly (at speed vs) but moves at a maximum speed vmax during the times in which the laser is switched off. Although this makes higher demands on the control and synchronicity of the scanners, it increases the image acquisition time by the factor
i.e., approximately M-fold (if vmax>>vs) or up to the maximum image acquisition speed of the camera.
When linear structured illumination is used, it should be remembered that the length of the higher orders transmitted through the circular pupil is shortened as the structuring frequency increases (see
b=(2√{square root over (1−f2)})−1
results. For a typical structuring frequency of 90% of the threshold frequency (f=0.9), a widening of 15% results. At 95% of the threshold frequency, this widening increases to 60%. This widening does not have an influence on the resolution that is determined by the structuring frequency and the transfer function of the objective lens, but it does influence the suppression of the out-of-focus background and must be taken into consideration for confocal filtering.
A structured line is generated on the specimen by the interfering first diffraction orders. The spatial interval of the diffraction orders is s; a denotes the size of the pupil. The ratio between s and b is the structuring frequency f that has been standardized to the threshold frequency.
The lines parallel to the x-direction seen in
The phase angle (double arrow) of the projected structure is determined by the relative constant offset of the two scanners (9) and (23) during the scan, while the direction of scan preferably perpendicular thereto (arrow) is defined by the relative speed of the two scanners. However, the specimen can also be scanned with scanner (9) only. This simplifies system control. Depending on the scan mode, it must be ensured that the image field generally determined by the detector is illuminated as homogeneously as possible even when the line is rotated.
In the configuration described so far, shaping of the structure projected into the specimen is ensured by the beam-shaping unit (8). According to the invention, the unit (8) can be a combination of a line-shaping optics system (7) with a periodic structure (13). The line-shaping optics system can comprise a Powell lens. The period structure can be a phase structure, an amplitude structure or a combination of the two. In addition, the entire beam-shaping unit (8) can be replaced with a diffractive optical element (see also DE 10155002 A1). This element can generate one or more structured lines on the specimen at a minimum spatial interval M in order to reduce the number of shifts.
A potential problem of sequential scanning of the sample with M line patterns arises when the specimen moves during the time of image acquisition. This is a fundamental problem of the method for the structured illumination and should be minimized as much as possible by minimal image acquisition times. This is why the sensitive detection with minimum fluorescence losses between the specimen and the detector is so important. An alternative to sequential scanning with M line patterns that makes it possible to acquire a single image instead of M line images and yet allows confocal detection will be described below. To this end, one takes advantage of the fact that the line scanner scans the specimen sequentially line-by-line. This makes it possible to implement a discrete line-by-line deflection of the detection light by an additional element in the detection beam path so that a line pattern as shown in
On the area detector, these scan jumps generate spaced-apart signals of the illuminated specimen which approximately correspond to the spaced-apart regions of the detector as described in detail above especially in connection with
When using optics systems with higher numerical apertures, such as are normally used in microscopy, polarization must be taken into consideration if a structured illumination with the highest possible contrast of the structure in the specimen plane is to be obtained. Maximum contrast is possible only if the polarization of the illuminating light is perpendicular to the connecting line of the diffraction orders in the pupil plane (i.e., perpendicular to the position of the line in an image plane), as shown in
the beam path of
The invention is not limited to the embodiments described above.
Within the context of the actions and knowledge of those skilled in the art, modifications and changes can be covered by the inventive thoughts,
For example, the present invention can be applied analogously to other illumination distributions, such as multi-point configurations (U.S. Pat. No. 6,028,306) and other point configurations, including Nipkow disks, and to detection in wide field.
While the invention has been illustrated and described in connection with currently preferred embodiments shown and described in detail, it is not intended to be limited to the details shown since various modifications and structural changes may be made without departing in any way from the spirit of the present invention. The embodiments were chosen and described in order to best explain the principles of the invention and practical application to thereby enable a person skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.
REFERENCES
- [1] Neil M. A. A., Juskeitls R., Wilson T.: “Method of obtaining optical sectioning by using structured light in a conventional microscope”, Opt.Lett. 22 (24): 1905-1907, 1997
- [2] Lukosz W., Marchand M., “Optische Auflösung unter Überschreitung der beugungsbedingten Auflōsungsgrenze”, Optics Acta 16, 241-255, 1963
- [3] Heintzmann R., Cremer C., “Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating”, in Proc. of SPIE 3568; 185-196, 1998
- [4] Nell M. A. A., Juskaltls, A., Wilson, T., “Real time 3D fluorescence microscopy by two beam interference illumination”, Opt. Comm. 153: 1-4, 1996
- [5] U.S. Pat. No. 6,947,127 B2, 2005
- [6] Heintzmann R., Jovin T. M., Cremer C., “Saturated patterned excitation microscopy—a concept for optical resolution improvement” JOSA A, 19 (8):1599-1609, 2002
- [7] Gustafsson, M. G. L., Agard, D. A., Sedat, J. W., “Doubling the lateral resolution of wide-field fluorescence microscopy by structured illumination”, In Proc. of SPIE 3919: 141-150, 2000
- [8] Gustafsson M. G. L., “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution”, PNAS 102: 13081-13086, 2005
Claims
1. A method for the depth-resolved optical detection of a specimen, wherein a specimen or a part of the specimen is scanned by means of linear illumination,
- the illumination of the specimen being periodically structured in the focus in at least one spatial direction,
- light coming from the specimen being detected and images of the specimen being generated,
- and at least one optical sectional image and/or one image with enhanced resolution being calculated through the specimen determined,
- comprising the steps of
- repeatedly acquiring images, and repeatedly blending sectional images while changing the orientation of the linear illumination relative to said specimen.
2. The method of claim 1, wherein the line is rotated about the optical axis, and the images are generated and the sectional images are blended at different angles of rotation.
3. The method of claim 1, wherein, to generate lines, a beam-shaping unit is jointly rotated with means for structuring the illuminating light.
4. A method for the depth-resolved optical detection of a specimen, wherein a specimen or a part of the specimen is scanned by means of preferably linear illumination,
- the illumination of the specimen being periodically structured in the focus in at least one spatial direction,
- light coming from the specimen being detected and images of the specimen being generated,
- and at least one optical sectional image and/or one image with enhanced resolution being calculated through the specimen is determined, comprising the steps of
- generating spatial intervals between lines exposed to detection light from the illuminated specimen region for the line-by-line non-descanned detection on an area detector or a camera.
5. The method of claim 4, wherein during the preferably linear illumination and detection, the illumination is repeatedly switched on and off.
6. The method of claim 4, wherein during the scanning of the specimen, the light is repeatedly interrupted so that a spatial interval is formed between two illuminated specimen regions.
7. The method of claim 4 for the confocal generation of images, wherein an image is calculated by partially or completely masking the spatial intervals between camera regions associated with the exposed specimen regions and the images thus obtained are blended.
8. The method of claim 7, wherein the images are blended in such a manner that neighboring scanned regions of the specimen are properly scaled and adjacently aligned in the blended image.
9. A method for the depth-resolved optical detection of a specimen, wherein a specimen or a part of a specimen is scanned by means of preferably linear illumination,
- the illumination of the specimen being periodically structured in the focus in at least one spatial direction,
- light coming from the specimen being detected and images of the specimen being generated,
- and at least one optical sectional image and/or one image with enhanced resolution being calculated through the specimen is determined, wherein, during a scanning procedure, light is further deflected upstream of the detector through the line in the direction of the scan of the specimen.
10. The method of claim 9, wherein the speed of the light deflection is greater than the speed of the relative movement between the specimen and the illuminating light.
11. The method of claim 9, wherein the light is deflected step-by-step.
12. The method of claim 9, wherein the light is deflected continuously.
13. The method of claim 9, wherein, during a rotation of the illuminating line, the polarization of the illuminating light is synchronously rotated with the rotation.
14. The method of claim 9, wherein repeated scanning takes place and the position of the periodic structure on the specimen and/or the position of the illuminating light on the specimen is shifted.
15. The method of claim 9, wherein several images with different image phases are acquired and sectional images are calculated therefrom.
16. The method of claim 9, wherein the images are acquired with different image phases with a constant spatial interval between illuminated/detected sections.
17. The method of claim 9, wherein the position of the spatial interval is changed and several images with different image phases are acquired for each position and sectional images are calculated therefrom.
18. The method of claim 9, wherein first the position of the spatial interval for one position of the structure is changed and specimen images are acquired, and subsequently this procedure is repeated for the next position of the structure.
19. The method of claim 9, wherein the position of the spatial interval is changed in such a manner that substantially all specimen regions are sequentially illuminated line-by-line and the specimen light is detected.
20. The method of claim 9, wherein the light is interrupted by decreasing the intensity by means of an electro-optical and/or acousto-optical modulator.
21. The method of claim 9, wherein, for the purpose of the periodic structuring of the illumination, a light beam is divided into several component light beams, which light beams are interferometrically overlapped and shaped into a line.
22. The method of claim 9, wherein the light resulting from a nonlinear interaction of the illumination with the specimen or a part of the specimen... [word or words missing] and is detected.
23. The method of claim 9, wherein linear scanning is carried out simultaneously with several lines.
24. The method of claim 9, wherein the optical section thickness or optical resolution is varied as structures with different modulation frequencies are imaged.
25. The method of claim 9, wherein during illumination with several wavelengths, the section thickness is identically set by adjusting each modulation frequency.
26. A configuration for the depth-resolved optical detection of a specimen, comprising
- means for the preferably linear illumination of the specimen with at least one wavelength,
- means for spatially structuring the illuminating light in at least one plane,
- means for generating a relative movement between the specimen and the illuminating light,
- means for imaging the light influenced by the specimen on at least one detector,
- means for calculating at least one optical sectional image and/or one image with enhanced resolution from the spatial information of the light influenced by the specimen, and means for changing the orientation of the linear illumination relative to the specimen.
27. The configuration of claim 26, further comprising a jointly rotatable unit comprising a beam-shaping unit to generate lines and means for structuring the illuminating light in the beam path.
28. A configuration for the depth-resolved optical detection of a specimen, comprising
- means for the preferably linear illumination of the specimen with at least one wavelength,
- means for spatially structuring the illuminating light in at least one plane,
- means for generating a relative movement between the specimen and the illuminating light,
- means for imaging the light influenced by the specimen on at least one detector,
- means for calculating at least one optical sectional image and/or one image with enhanced resolution from the spatial information of the light influenced by the specimen,
- an area detector or a camera for the non-descanned detection of the specimen light, and
- means for interrupting the light during the scan so as to generate a spatial interval between illuminated specimen regions and/or to generate spatial intervals between lines exposed with detection line from the illuminated specimen region on the area detector.
29. The configuration of claim 28, wherein intensity control means are disposed in the illuminating beam path.
30. The configuration of claim 28 or 29, wherein an electro- or acousto-optical modulator for light interruption is provided.
31. A configuration for the depth-resolved optical detection of a specimen, comprising
- means for the preferably linear illumination of the specimen with at least one wavelength,
- means for spatially structuring the illuminating light in at least one plane,
- means for generating a relative movement between the specimen and the illuminating light,
- means for aging the light influenced by the specimen on at least one detector,
- means for calculating at least one optical sectional image and/or one image with enhanced resolution from the spatial information of the light influenced by the specimen, and
- a scanner disposed in the detection beam path in order to expand the specimen light discretely on the detector or continuously on the detector during the line-by-line scan.
32. The configuration of claim 31, characterized in that at least one scanner is provided as a means for generating the relative movement.
33. The configuration of claim 31, wherein the means for structuring the illumination is an optical element that is preferably rotatable about the optical axis and that is structured relative to its transparency.
34. The configuration of claim 31, wherein in order to set different image phases of the structure, the position of at least one scanner can be adjusted.
35. The configuration of claim 31, wherein in order to set different frequency structures, gratings of different periodicities that can be rotated into the beam path are provided.
36. A microscope, preferably a laser scanning microscope, having the configuration of claim 26, 28 or 31.
Type: Application
Filed: Jun 28, 2011
Publication Date: Jan 26, 2012
Inventors: Michael KEMPE (Jena), Ralf Wolleschensky (Jena), Michael Schwertner (Jena)
Application Number: 13/170,807
International Classification: H04N 7/18 (20060101);