Method and apparatus for imaging the surface of a sample

Abstract

To provide a method for imaging a surface of a sample having topography by using confocal Raman and / or fluorescent microscopy as confocal microscopy: the method for preparing a first laser light source as an apparatus to be used to generate excitation radiation for Raman scattered light and / or fluorescence, and a second laser light source as a second apparatus or superluminescent diode (SLED) as a second apparatus having a focus position controllable independently of the first light source.SOLUTION: A first light source emits light of a first wavelength range, a second light source emits light of a second wavelength range, and the first and second wavelength ranges of the first and second light sources do not overlap. A focal plane of the second light source is moved into / onto the surface of a sample by a separately controllable focal position so as to establish topography of the sample by using a signal of the focal position of the second light source, and / or the sample is moved into the focal plane of the first light source because of the signal.SELECTED DRAWING: Figure 1b

Description

[Technical Field]

[0001] The present invention relates to a method and apparatus for imaging a surface, particularly a sample having topography, by scanning multiple regions of the surface using confocal microscopy. In confocal microscopy, a confocal image of a region of the surface is generated by a detector positioned within the imaging plane. Specifically, the present invention relates to, but is not limited to, so-called confocal Raman and / or fluorescence microscopy, or apparatus for confocal fluorescence and / or Raman microscopy.

[0002] In addition to methods for imaging the surface, specifically the surface of a sample having topography, instruments for imaging a sample using confocal microscopy or confocal Raman and / or fluorescence microscopy are further described. Raman or fluorescence measurements can be used to excite the sample with a light source and image chemically different materials based on the light emitted by the sample. [Background technology]

[0003] In confocal microscopy, light from a light source is guided through an objective lens to the sample and focused onto a basically point-like region (airy disk) on the sample surface. Simultaneously, the objective lens can be used to capture light emitted by the sample, specifically the emitted Raman light or fluorescence, and send it to the detector. Therefore, using this objective lens, it becomes possible to confocally image a region, i.e., a basically point-like region of the sample that is essentially perpendicular to the direction of illumination and / or beam detection paths. If the sample, objective lens, or illumination is moved, scanning in the x and y directions, and thus scanning the entire sample, is possible.

[0004] In confocal imaging, a light source, preferably a point-like light source, preferably a laser light source, is imaged onto a focal point resulting from the wave nature of light, on a point-like region, ideally on a single point of the sample (Abbe condition). This pixel is then focused onto a pinhole in front of a detector, preferably by the same optical system, i.e., the same objective lens. Instead of placing a separate pinhole in front of the detector, the detector itself can also represent the pinhole. When confocal imaging is used for microscopy, a significant increase in image contrast is achieved because only light from the focal plane of the objective lens contributes to the imaging.

[0005] Regarding confocal optical microscopy, Patent Document 1, which describes a microscope having a confocal objective lens in detail, is referenced.

[0006] Confocal measurements offer advantages in many applications, such as Raman and / or fluorescence measurements, because the existing scattered light background is very strongly suppressed. Since only light from the focal plane is measured, confocal microscopy also allows for the measurement of sample areas below the actual sample surface in the case of transparent samples.

[0007] Confocal Raman and / or fluorescence microscopy is known from published patent document 2.

[0008] However, the problem with confocal measurement or confocal microscopy is that, due to drift, topography, sample heterogeneity, roughness, and even the tilt of the sample, the imaged surface or region, especially the surface, often does not remain within the focal plane as the sample is scanned.

[0009] Therefore, for satisfactory results, many applications require extended methods and equipment for focus stability or focus tracking based on the surface being measured.

[0010] AFM microscopes or STM microscopes that can be combined with confocal microscopes are known from Patent Document 3. Using an AFM or STM tip, the sample can also be scanned, particularly in the z direction.

[0011] According to Patent Document 3, depth information can be obtained using an AFM chip. During AFM measurement, specifically AFM topography measurement, optical signals are also recorded so that the topography data obtained from the AFM topography measurement can be correlated with optical data. According to Patent Document 3, confocal measurement is always performed simultaneously with topography measurement. A disadvantage of Patent Document 3 is its limited scanning range, which is in the range of 100 μm to a maximum of 300 μm in the xy plane. Furthermore, the AFM chip can only provide depth information in the range of 5 to 10 μm in the z direction. Therefore, Patent Document 3 does not enable measurement of sample areas >300 μm and roughness >10 μm.

[0012] In confocal microscopy, particularly confocal Raman microscopy and / or fluorescence microscopy, on surfaces, especially large sample areas, especially >300 μm, and on craft surfaces, a problem arises in obtaining images because sufficiently flat sample topography is often not available. During scanning within a given plane, so-called XY scanning, the sample surface repeatedly moves away from the focal plane of the microscope, making it impossible to easily and completely image the sample surface or the sample itself.

[0013] Patent Document 4 describes a method and apparatus that first determines the surface topography of a sample for confocal microscopy, and then allows the surface to be imaged to move to the confocal plane using the surface topography values. Patent Document 4 proposes an apparatus having a surface topography sensor for this purpose. The surface topography sensor can preferably be a confocal sensor having an optical system with reflective and / or diffracting components. Alternatively, Patent Document 4 specifies contact sensors such as surface roughness gauges, AFMs, white light interferometers, triangulation sensors, or laser scanning systems. A disadvantage of the system in Patent Document 4 is that the surface topography of the entire sample is determined first, and then the actual fluorescence or Raman measurement is performed taking the determined surface topography into account. This results in a relatively large time lag between the topography and the fluorescence or Raman measurement. Instrument or sample drift or time-varying sample (e.g., due to evaporation or drying of the sample) can pose significant difficulties to this method.

[0014] As an alternative to the sequential measurements described above, i.e., Raman or fluorescence measurements after topographic determination, Patent Document 4 further describes a single-path method. In this case, first, distance measurements are performed at each raster point using a surface topography sensor, specifically a color sensor, and a surface topography-specific distance signal is then determined. This distance signal is then used directly with respect to this one raster point to track the focal plane or confocal plane of a confocal microscope, specifically a confocal Raman microscope, and this cycle is then repeated at the next raster point. This means that topography is measured first at each raster point, and that confocal measurements, specifically confocal Raman or fluorescence measurements, are initiated only after the completion of this measurement and the completion of topographic tracking. The confocal Raman or fluorescence signal is not detected over a long time range of the cycle, particularly during the overall topographic measurement and the movement of the sample from one raster point to the next.

[0015] In summary, the single-path method described in Patent Document 4 is also characterized by a complex back-and-forth switching between various measurement modes and measurement cycles. This involves large time intervals in between during which confocal signals, specifically Raman or fluorescence signals, cannot be detected. Furthermore, this method is extremely slow and complex.

[0016] Another disadvantage of the method described in Patent Document 4 is that, for example, the confocal color sensor used requires a very strong white light source that is as broadband as possible at the same time to achieve sufficient topographic resolution in a short measurement time, due to its measurement principle. In contrast to monochromatic light sources such as lasers typically used for Raman or fluorescence measurements, a strong broadband light source is not usually a point source but clearly has a macroscopic spread. However, only light from a point source can be projected onto the smallest diffraction-limited focus. This, in practice, limits the lateral resolution that can be achieved by topographic measurements with a confocal color sensor to about 10 μm, in contrast to diffraction-limited Raman or fluorescence measurements. The apparent difference of about an order of magnitude in the lateral resolution of topographic measurements compared to the lateral resolution of diffraction-limited Raman and / or fluorescence measurements leads to the fact that the focal plane for Raman or fluorescence measurements cannot be accurately tracked by topography of small samples in the lateral direction, especially in the case of high-resolution measurements of fine structures or rough porous surfaces.

[0017] As suggested in Patent Document 4, another disadvantage is that both Raman and fluorescence measurements, as well as confocal color topography measurements, use the widest possible spectral frequency range, ideally the practically full visible spectral range, especially when Raman or fluorescence measurements and topography measurements are performed with the same lens. Thus, it is difficult to avoid spectral overlap. If the wavelength ranges of topography measurements and Raman and fluorescence measurements are separated into two non-overlapping spectral ranges, as described in Patent Document 4, this places an extremely high demand on the coating of the common optical system, as the lens must be optimized for the overall resulting summed spectral range.

[0018] Accordingly, Patent Document 4 further introduces a simpler single-path method called the extended focusing method. No other light sources are required for surface detection other than the first excitation light source. Instead, the sample is tuned in the z direction, and a characteristic signal is recorded in the reflected Raleigh light. When the sample is in focus, the maximum Raleigh light is detected. During tuning, the characteristic signal in the Raleigh light is then used to track the average z position of the sample, which is then tuned around it using sample topography. Thus, at least the central z position remains in focus. Raman or fluorescence measurements are performed throughout the entire tuning period.

[0019] This indicates that the sample may remain outside the actual focus for the Raman or fluorescence signal for a relatively long period of time, and therefore, no Raman or fluorescence signal may be detected for extended periods, or only undesirable signals may be detected, resulting in a weak or blurred Raman or fluorescence signal. At the same time, this adjustment impairs the confocality of the measurement, as regions above and below the surface of the sample of interest also contribute to the measured Raman or fluorescence signal.

[0020] Therefore, the proven advantages of confocal optical microscopy, such as higher spatial resolution and greater depth of field, are lost in this method. [Prior art documents] [Patent Documents]

[0021] [Patent Document 1] German Patent Application Publication No. 199 02 234(A1) [Patent Document 2] German Patent Application Publication No. 10 2009 015 945(A1) [Patent Document 3] U.S. Patent No. 5,581,082 (B1) [Patent Document 4] International Publication No. 2011 / 131311(A1)

Summary of the Invention

Problems to be Solved by the Invention

[0022] Therefore, an object of the present invention is to provide a method and an apparatus capable of avoiding the disadvantages of the prior art, particularly the disadvantages of the prior art presented in International Publication No. 2011 / 131311 (A1). Specifically, a method and an apparatus will be provided that enable simple and rapid Raman and / or fluorescence measurements even for a non-uniform sample surface. Further, the present invention enables confocal imaging of a plane or a surface, specifically the surface of a sample, i.e., using confocal microscopy. This requires that the sample always be kept in focus during the entire measurement, even if there is an insufficiently flat sample topography of, for example, a curved sample.

Means for Solving the Problems

[0023] According to the present invention, this objective is solved in a method for imaging a plane or surface, specifically a surface having a topography, using confocal microscopy, specifically confocal Raman and / or fluorescence microscopy, wherein the instrument for performing this method comprises a first light source that generates excitation radiation for Raman and / or fluorescence microscopy, and a second simultaneously active light source for generating radiation in a narrow wavelength range that does not coincide with the wavelength range of the first light source, or, for example, the detected wavelength range such as Raman light / fluorescence. In this application, “simultaneously” means that the first and second light sources emit light onto the sample simultaneously, i.e., together, and that a topographic measurement of the sample for focus tracking can be performed on the one hand, and a Raman or fluorescence measurement on the other hand, simultaneously. Furthermore, the focal plane of the second light source is moved within / on the surface of the sample by a separate controllable focal position, and the sample topography thus determined is used to control / adjust the focal plane of the first light source from a control signal of the controllable focal position. The method according to the present invention ensures that Raman and / or fluorescence measurements remain active and within the focal point, unaffected by simultaneous topographic measurements of the sample for focus tracking. Thus, the method is characterized by the simultaneous performance of Raman and / or fluorescence measurements, as well as topographic measurements. Both the first and second light sources can be laser light sources, without limitation. Superluminescent diodes (SLEDs) may also be used as light sources. Similar to laser diodes, SLEDs are based on a pn junction and operated in the through-direction. The average wavelength emitted can be influenced by the selection of different semiconductor materials, such as indium arsenide (InAs), indium gallium arsenide (InGaAs), or indium phosphide (InP), based on the band gap. In contrast to laser diodes, SLEDs do not have a resonator exhibiting optical feedback via mirrors.

[0024] In the method according to the present invention, two transverse foci of approximately the same size are used, i.e., the focal sizes of the first and second foci are approximately the same, and therefore the same sample area can be evaluated. The first focal point is the focal point of the first light source, i.e., a laser light source for Raman or fluorescence excitation. The second focal point is the focal point of the second light source and is useful for determining the topography. The focal plane of the second light source is moved on the surface of the sample by a separately controllable focal position. The separately controllable focal position can be adjusted, for example, using an electrically focusing lens. Periodic excitation of the lens is particularly preferred, which causes its focal position to change periodically in the z direction. Whenever the focal point of the second light source is located on the surface of the sample, a signal is detected. This is due to the fact that, as a result of the confocal principle, light is back-reflected onto the detector with sufficient intensity to detect a signal only when the focal point of the second light source is located on the surface. The sample topography, determined from a controllable focal position control signal, is used to move the sample within the focal plane of confocal Raman and / or fluorescence microscopy. Thus, the present invention makes it possible to continuously hold the sample within the confocal plane of Raman and / or fluorescence measurement by continuously evaluating the focal plane of a second light source. Simultaneously, the Raman and / or fluorescence measurement can remain continuously active without being interrupted by the determination and adjustment of the topography. This significantly amplifies the Raman and / or fluorescence signal compared to that achieved by the control system in International Publication 2011 / 131311(A1), which only presents a tracking system. Therefore, the method according to the present invention makes it possible to compensate for thermal and / or mechanical drift, resulting in longer measurement times compared to the method known from International Publication 2011 / 131311(A1), due to the compensation of thermal and mechanical drift. High-resolution measurements, or measurements of porous or rough samples, are also greatly improved by the equivalent lateral resolution of topography and Raman and / or fluorescence measurements.

[0025] For example, Raman and / or fluorescence measurements of samples that dry or evaporate during measurement are also possible because the adjustments according to the present invention result in continuous tracking of the surface.

[0026] According to the present invention, specific adjustments / controls are used to hold the sample within the focal plane for Raman and / or fluorescence measurements.

[0027] The step of imaging a plane or surface, specifically the surface of a sample, by confocal microscopy, specifically Raman and / or fluorescence microscopy, is achieved by scanning multiple regions of the plane or surface, particularly the surface, using equipment for confocal imaging of the plane or surface, particularly the surface.

[0028] In this application, the terms surface topography or sample topography will be explained, as an example but not definitively, in relation to a confocal Raman microscope. In such a configuration, sample topography means sample non-uniformity greater than 1 nm, particularly greater than 10 nm, and preferably greater than 100 nm. Sample irregularity is the deviation of the sample surface in the z-direction, which is also called roughness.

[0029] The plane being scanned is the x / y plane. The resolution in the x / y plane of the Raman microscope or fluorescence microscope is at least 0.1 μm, preferably at least 1 μm, and preferably 1 μm to 50 μm.

[0030] According to the present invention, the position of the focal plane of the second light source is constantly determined. For this purpose, the focal position of the second light source can be changed, for example, using an electrically focusing lens, where the lens is preferably periodically excited at a frequency of, for example, 200 Hz to 800 Hz. In addition to lenses whose focus can be changed electrically, it is also possible to mechanically move the lens periodically. An alternative to the frequency range specified as 200 Hz to 800 Hz is a frequency range of 50 kHz to 1 MHz. When determining the position of the focal plane by changing the focal position of the second light source, it is necessary to adjust the modulation degree of the focus, or the roughness of the sample or the focal position of the topography.

[0031] The sample is moved to the confocal plane of confocal Raman or fluorescence microscopy, based on a second light source and the focal plane of the second light source determined, for example, using an electrically focusing lens. This movement into the confocal plane of Raman / fluorescence microscopy is therefore performed simultaneously with or at the same time as the measurement of the focal position of the second light source.

[0032] By using a second light source, specifically a second laser light source, and passing the focal point of the second light source through an electrically focusing lens, it becomes possible to continuously hold the Raman microscope at its focal plane or confocal plane, and thus operate confocal Raman microscopy even in the case of significant, i.e., non-planar sample topography.

[0033] In a particularly preferred embodiment of the present invention, readjustment of the focal plane of the first light source for Raman and / or fluorescence microscopy is defined to be performed, for example, by changing the distance between the microscope objective lens and the sample. This can be done by moving the sample table and / or the microscope objective lens.

[0034] Another preferred embodiment of the present invention is that light from the first and second light sources is guided to pass through the same microscope objective lens.

[0035] A particularly simple optical apparatus can be realized when the focal position of the second light source is controlled so that the focal point of the second light source moves along the optical axis of the microscope.

[0036] A simple determination of the sample topography is possible when the focal position of a second light source is periodically changed, and the sample topography is determined by the temporal progression of the maximum intensity of the reflected or scattered light from the second light source.

[0037] It is advantageous when the focal position of the second light source is controlled by a focusing lens within the beam path that affects only the beam path of the second light source. Electrically focusing lenses, whose focal length is determined by the application of voltage or current, are particularly simple in design and can be used to change the focal position.

[0038] Alternatively, the focal position of the second light source can be controlled using a sliding lens or several lenses.

[0039] It is particularly preferable that the lateral focal size of the second light source essentially corresponds to the lateral focal size of the first light source. This has the advantage that topographic determination on the one hand and Raman or fluorescence measurement on the other evaluate the same sample range and the same sample volume. On the other hand, if the lateral focal sizes of the two light sources differ significantly, in particular if the focal size of the first light source is significantly larger than that of the second light source, all topographic details such as pores or particles of the sample that are smaller than the focal size of the first light source will be missed from the topographic determination due to their small size, and therefore, Raman or fluorescence measurements at these sample locations will not be held within the focal point. With essentially identical lateral focal sizes for the first and second light sources, ideal topographic compensation is achieved because exactly the same topographic details are corrected or balanced, which can also be resolved by Raman or fluorescence measurement.

[0040] The required change in the distance between the microscope objective lens and the sample, determined by control / adjustment, can be achieved by moving the sample in the direction of the microscope's optical axis. Alternatively, the microscope can be moved in the direction of its optical axis, or the microscope objective lens can be moved in the direction of the microscope's optical axis.

[0041] It is particularly preferable that the wavelength range of the second light source does not overlap with the wavelength range of the detected Raman scattered light and / or fluorescence.

[0042] In addition to the method, the present invention further provides an instrument for imaging the surface of a sample by scanning a plurality of regions of the surface in the x / y plane, the instrument comprising means for confocal imaging of regions of the surface in the focal plane onto a detector, and having first and second light sources.

[0043] The light from the confocal Raman and / or fluorescence microscope, i.e., the light from the first light source, appears in a first wavelength range according to the present invention, and the light from the second light source is in a second wavelength range, where the first and second wavelength ranges do not overlap. The first wavelength range is usually selected such that it is determined by the range of the emission spectrum and / or Raman spectrum emitted by the sample being investigated, and the second wavelength range is above or below the first wavelength range that does not overlap with the first wavelength range. For example, the first wavelength range of the emission spectrum and / or Raman spectrum emitted by the sample being investigated can be 350 nm to 1000 nm, preferably 500 nm to 1000 nm, and particularly 532 nm to 650 nm. The second wavelength range can be 1000 nm to 2000 nm, preferably 1000 nm to 1500 nm.

[0044] As described above, in confocal microscopy, monochromatic light from a first light source for Raman and / or fluorescence measurements is guided to pass through an objective lens on its way to the sample and is therefore focused to essentially a single point on the sample surface. In particular, if the instrument is a confocal Raman microscope, the spectrometer can be configured to spectrally decompose the light emitted by the sample, i.e., Raman light or fluorescence. Such spectral decomposition can be performed, for example, within a spectrometer having a diffraction grating or prism. If the light thus decomposed is recorded by a CCD camera, it becomes possible to record the complete spectrum of the Raman light or fluorescence scattered by the sample. An advantage of spectral decomposition of Raman light within a Raman microscope is that, for example, by rotating the diffraction grating within the spectrometer, any spectral range can be selected for the detector for measurement.

[0045] The instrument, specifically a confocal microscope, preferably a confocal Raman and / or confocal fluorescence microscope, may have a sample table that can be moved in the x / y direction, which allows imaging of the sample surface by scanning the sample, for example. Alternatively or additionally, the objective lens or the microscope itself can be moved to obtain an image of the sample. It is also possible to record a spatial map of the spectral characteristics of the sample. Very high depth resolution is achieved, in particular, by confocal imaging.

[0046] Using a second light source, particularly in combination with an electrically focusing lens, the focal plane of the second light source can be moved in / on the surface of the sample by a separately controllable focal position according to the present invention. From the controllable focal position control signal, the sample topography is determined to control the focal plane of the first light source for Raman microscopy. This control / adjustment makes it possible to keep the sample surface constantly within the confocal plane for confocal Raman microscopy during scanning. For this purpose, the xy scanning of the sample is extended to xyz scanning, where z scanning helps to compensate for the sample topography. For this purpose, the sample table and / or lens can be moved in the z direction in addition to the x / y directions.

[0047] In contrast to International Publication No. 2011 / 131311(A1), the method according to the present invention does not require first recording surface topography for control / adjustment, and then subsequently tracking the sample surface into focus. Instead, continuous determination of the focal position using a second light source allows the sample to be simultaneously moved into the focal plane of the objective lens for confocal Raman and / or fluorescence microscopy. This has the advantage of obtaining a much stronger Raman and / or fluorescence signal than by mere tracking as in International Publication No. 2011 / 131311(A1).

[0048] The present invention will be described in detail below with reference to examples of embodiments. [Brief explanation of the drawing]

[0049] [Figure 1a] The main structure of a Raman microscope is shown, which has a first and a second light source, and the beam paths of the first and second light sources are depicted. [Figure 1b] This shows the main structure of a Raman microscope, which has first and second light sources, and in which the excitation focus of the first light source for Raman measurements is induced through the same objective lens as the focus of the second light source. [Figure 2a] This shows the adjustment of the electrically focusing lens for the second light source and the detected signal. [Figure 2b] This shows the adjustment of the electrically focusing lens for the second light source and the detected signal. [Figure 2c] The focal points of the first and second light sources and the main features of the non-uniform sample surface are shown. [Figure 3] The topography of the sample being investigated is shown. [Figure 4] Figure 3 shows an image of the sample, obtained by moving the sample into the confocal area for Raman and / or fluorescence spectroscopy without adjustment. [Figure 5] Figure 3 shows an image of the sample, which has been moved into the confocal area for Raman and / or fluorescence spectroscopy through adjustment. [Figure 6] The intensity profiles of the Raman signals with and without adjustment, as well as the surface topography of the sample, are shown. [Modes for carrying out the invention]

[0050] The present invention will be described below with regard to, but will not be limited to, examples of embodiments of an instrument for imaging a sample surface using scattered Raman light, a so-called confocal Raman microscope. Rather, the present invention includes all confocal microscopes, specifically confocal optical microscopes or fluorescence microscopes. A second light source having an electrically focusing lens can further be used for such a confocal microscope to move the sample into the confocal plane of the Raman microscope, depending on the surface topography.

[0051] Figure 1a shows the main structure of a first embodiment of a confocal Raman microscope for recording a sample surface. Confocal Raman microscopy can be used to analyze the chemical properties and phases of liquid and solid components to a diffraction-limited resolution range of approximately 200 nanometers. It does not require marking the sample with a fluorescent substance, as is done in fluorescence microscopy. The confocal structure provides depth resolution, allowing for analysis in the depth direction without, for example, the need to cut the sample.

[0052] In confocal microscopy, a point light source, preferably a laser, is imaged onto a single point in the sample. This pixel is then, preferably by the same optical system, focused onto a pinhole in front of the detector. The size of the pinhole aperture must be matched to the diffraction-limited image of the illumination image. This image is generated by scanning the measurement points on the sample, i.e., scanning the sample point by point. This type of imaging achieves a significant increase in image contrast because only the focal plane of the lens contributes to the imaging. Furthermore, the resolution can be reduced by a factor of √2 to approximately λ / 3 due to the convolution of the diffraction point and the pinhole aperture. Moreover, a three-dimensional image of the sample structure can be obtained with a distance resolution of approximately one wavelength.

[0053] Regarding confocal microscopy, for example, German Patent Application Publication No. 199 02 234(A1) is cited.

[0054] Figure 1a shows a possible structure of a confocal Raman microscope, for example, the Alpha 300R microscope from WITec GmbH, located at Ulm, D-89081, Germany. In the confocal Raman microscope 1, light from a first light source 10 is directed onto a sample table 18 in the direction of the sample 16 at a beam splitter mirror 12 behind a beam expander 14. The light from the first light source, specifically the light from a first laser, is preferably excitation light for Raman or fluorescence emission and is in the wavelength range of 350 nm to 1000 nm, preferably 500 nm to 1000 nm. The refracted light beam 19 of the excitation light is focused by a suitable optical system 21 into a basically point-like region 20 on the sample 16. The light from the first light source, specifically the laser light source 10, interacts with the material of the sample 16. Meanwhile, backscattered Lowry light of the same wavelength as the incident light is produced. This light is refracted through the beam splitter 12 towards the edge filter or notch filter 13 and does not reach the detection optical system.

[0055] Light emitted by the sample, having a frequency different from or multiple frequencies from the Lowry light, i.e., Raman light, passes through the beam splitter 12. Behind the beam splitter 12, the Raman light is marked with reference numeral 22. The Raman light 22 is injected into the optical fiber 30 through a pinhole (not shown) and reaches the spectrometer 40. Inside the spectrometer 40, the Raman light beam is spread again by a suitable optical system to produce a beam 42, which strikes a diffraction grating spectral filter 44. The diffraction grating spectral filter 44 diffracts the light in different directions depending on its wavelength, thereby allowing the spectral signal to be captured on the CCD chip 50 in a position-dependent manner. For example, the CCD chip 50 has 1024 channels, and as a result, a total of 1024 channels on the CCD chip can absorb light of different wavelengths.

[0056] The image of the sample is created by scanning in the x / y plane in the direction of arrow 130.

[0057] Light from the white light source 120 can also be injected onto the sample 16 for adjustment or observation.

[0058] The confocal Raman microscope 1 further includes a second light source 80. The second light source 80 exists in addition to the first light source of the confocal Raman microscope 1. The second light source has its own independent beam path, as shown in Figure 1a, which is coupled into the optical system 21 of the lens via a mirror 82. The beam path of the second light source strikes the sample via the same optical system 21 as the excitation light beam for Raman and / or fluorescence measurements of the first laser light source, and is directed to a beam splitter via the mirror 82. The beam splitter allows the light from the second light source to pass in the direction of the mirror 82 and the optical system 21, and the optical system 21 separates the light reflected from the sample surface so that the light reflected from the sample surface is directed to the photodiode 86. The reflected signal is then detected and provided to an evaluation unit (not shown). From the reflected signal, the evaluation unit determines the phase position of the maximum of the photodiode signal relative to the excitation signal of the electrically focusing lens 84. This phase position is a measure of the height change of the sample surface. Depending on the phase position, the sample table is then moved so that the sample is continuously positioned within the focal plane of Raman and / or fluorescence microscopy. The movement of the sample table is controlled or adjusted according to the phase position.

[0059] Furthermore, an electrically focusing lens 84 is inserted into the beam path after the second light source 80, which works to change the focal position of the second light source 80. The electrically focusing lens can be, for example, the electrically adjustable, i.e., focusing lens EL-16-40TC from Optotune (Switzerland AG, Bernstrasse 388, CH-8953 Dietikon), which allows detuning within the range of -2 to +3 diopters at 30°C. This lens can also be used to adjust the lens to a desired position. The aperture of the electrically focusing lens EL-16-40TC is 16 mm, and the transmittance in the wavelength range of 450 nm to 950 nm is over 90%. The electrically adjustable lenses described are illustrative and not limiting. The disclosures in the technical data sheet of Optotune Swiss AG regarding electrically adjustable lenses are included in the disclosures of this application. The electrically focusing lens is preferably excited periodically, particularly at a frequency in the range of 200-500 Hz. Other frequencies are also possible. The focal position of the second light source changes periodically due to the periodic excitation of the electrically focusing lens. Whenever the focal plane remains within / above the sample, a signal is detected in the photodiode 86. From this signal, the phase position of the photodiode signal relative to the excitation signal of the electrically focusing lens can be determined and thus inferred from the sample topography. Depending on the phase position, the sample table is then moved so that the surface topography of the sample is balanced and the sample is always within the focal plane for Raman and / or fluorescence microscopy. This is described in detail in Figures 2a and 2b.

[0060] Figure 1b shows the main structure of a confocal Raman microscope, where light from the first light source 2010, i.e., a laser, for Raman measurements is guided in parallel with light from the second light source 2080 for topographic measurements. The same components as in the previous Figure 1a are marked with reference numerals increased by 2000. In the Raman microscope 2001 shown in Figure 1b, both light from the first light source 2010 for exciting the Raman effect and light from the second light source 2080 are focused on essentially the same lateral region 2020 of the sample 2016 by the same optical system in 2029. The focal position for Raman measurements, i.e., the confocal point of the excitation laser beam of the first light source 2010, is selected for exciting the Raman effect. Light from the first light source 2010 is injected into the sample 2016 by a beam splitter 2012.1. The light beam 2019 is redirected towards the sample 2016 within beam splitter 2012.1 and then passes through another beam splitter 2012.2. The Raman light generated by interaction with the sample passes through both beam splitters 2012.1 and 2012.2, indicated by reference number 2022 behind beam splitter 2012.2. Behind beam splitter 2012.2, the light beam 2022 is focused over a pinhole 2013 in front of a detector (not shown). The optical path of the second light source is marked by reference number 2092. An electrically focusing lens 2094 is positioned in the optical path from the second light source 2080 to the sample 2016.

[0061] In addition to the light from light source 2010 used to excite the Raman effect within the sample, another beam splitter 2012.2 directs light 2092 from a second light source 2080 onto sample 2016 through the same optical system 2029 as the light used to excite the Raman effect. This light beam is marked with reference numeral 2019. The light from the second light source 2080 irradiating the sample is reflected by the sample. This reflected light 2089 is directed again over another beam splitter 2012.2, and from there onto diode 2096. The signal recorded by diode 2096 is provided to evaluation unit 2100. Evaluation unit 2100 also acts as a scanner tuner, and the scanner is moved in the z direction by the signal from the photodiode.

[0062] The diode 2096 that captures the optical signal is, for example, an InGaAs diode.

[0063] To allow both the light for exciting the Raman effect and the light from the second light source to pass through the same optical system, it is advantageous to use different spectral ranges or time-division multiplexing equipment. For example, the light from the second light source could be in the wavelength range of 1000 nm to 2000 nm, and the wavelength of the light for exciting the Raman effect could be 532 nm, i.e., in the range of 350 nm to 1000 nm. Such an arrangement would typically allow recording of Raman spectra above 532 nm. Of course, other wavelength choices could also be considered.

[0064] The structure shown in Figure 1b allows the sample surface to be moved within the confocal plane of the Raman measurement by a calibration method using a second light source 2080 and a focusing lens 2094, particularly an electrically focusing lens. Scanner calibration and / or control can be performed by a calibration and / or control unit.

[0065] In addition to adjustments / controls for Raman measurements, pure topographic measurements alone are also possible. Non-contact topographic measurements are particularly suitable for fragile samples, samples whose topography is already too high (>5 μm) for AFM, or samples whose lateral structure is much larger than the typical scanning range of a piezo scanner, e.g., 100 μm.

[0066] Figure 2a shows the sinusoidal modulation of the lens focus of the second light source. In this example, the selected modulation frequency is 400 Hz. Due to the sinusoidal modulation of the lens focus, the focal position of the second light source also progresses sinusoidally, as shown in Figure 2a. In Figure 2a, two heights of the investigated surface are drawn within the sinusoidally modulated path. The first height is marked as H1 and the second height as H2. When the sinusoidal focus of the second light source is on the surface of the sample at height H1, the confocal condition is met, and signal 1000, shown in Figure 2b, is detected in the diode. The phase position of signal 1000 is approximately 15°. If the height of the sample increases, for example, due to thermal expansion, the height changes from H1 to H2. Now, signal 1100 is detected instead of signal 1000. As shown in Figure 2b, signal 1100 shifts to the right relative to signal 1000, with a phase position of 30°, and a phase difference of 15° between the first and second signals. Signal 1100 is detected again when the focal point of the second light source is at the surface at height H2. Thus, the phase shift of the second light source signal detected in the diode becomes a measure of how the sample height changes and can be used as an adjustment or control variable to control or adjust the sample to compensate for differences in sample height and to move the sample to the focal plane of the first light source for Raman and / or fluorescence microscopy. As shown in Figure 2b, the signal in the photodiode moves from 1000 to 1100, to the right (signal group 1) and to the left (signal group 2) during the height change from H1 to H2. However, only movement in one direction is used for adjustment, in which case the movement to the right by signal group 1 is used.

[0067] Figure 2c shows the height H 試料 A sample having height H試料 is the height H of the focus of Raman microscopy 焦点 shows a sample lower than that. The focus of the second light source is indicated by reference numeral 4000 and is sinusoidally modulated within a capture range, for example, between 4100 and 4200. This makes it possible to determine the height H 試料 of the sample from the focal position of the second light source. As in the illustrated example, for example, when H 試料 deepens behind H 焦点 and the height decreases, the focus 5000 of the Raman measurement will be outside the sample. Due to the sinusoidal modulation between the limits 4100 and 4200 of the focal position of the second light source, the height H 試料 is detected and, depending on the phase position, the sample is returned to the focus 5000 of the Raman measurement by control or adjustment. The movement of the sample from H 試料 to H 焦点 is marked with reference numeral 10000.

[0068] Figure 3 shows the topography of the sample. This sample has a bulge of 5000 μm in the x direction. In the z direction, the sample is curved at a depth of ±30 μm.

[0069] Figure 4 is an image of the sample having the height profile shown in Figure 3 by confocal Raman microscopy, showing an image where the sample was not moved and no adjustment of the sample into the confocal plane was performed. It is clearly seen that only the narrow edge region of the sample is within the focus of the Raman measurement and as a result only this region is clearly imaged. The imaged clear region is a bright region and the blurred region is dark.

[0070] Figure 5 shows the Raman measurement of the surface shown in Figure 3, where, in contrast to Figure 4, the sample was moved into the confocal plane in a manner controlled by the focus of the second light source. The result is a completely clear and bright image.

[0071] Figure 6 illustrates the effect of the adjustment according to the present invention. In Figure 6, the path of the sample surface is indicated by the reference numeral 2000. The sample surface has a depth or z deviation of 100 μm over an x-expansion of 7000 μm. Curve 2100 represents the Raman intensity without adjustment. As can be seen from curve 2100, the Raman signal is produced only when the sample is within the confocal plane of the Raman measurement. This is precisely the case for two surface values. Higher or lower regions of the surface are outside the confocal plane and do not show a signal. However, by using a second light source, it is possible to keep the surface always within the confocal plane of the Raman and / or fluorescence measurement by evaluating the focal position of the second light source with the adjustment according to the present invention, so that the Raman signal 2200 is detected throughout the entire sample.

[0072] As shown in the figure, the Raman signal 2200 obtained by the adjustment is as strong as the two peaks 2100 detected when the sample is moved within the focus without adjustment. This means that the signal corresponding to the sample within the focus is obtained throughout the entire sample during adjustment, despite the surface path of 2000.

[0073] Firstly, the present invention provides an instrument that enables rapid and easy acquisition of information regarding the surface of a sample using confocal microscopy. Specifically, this is achieved using a second excitation and detection pathway, which allows the confocal primary measurement to be continuously kept in focus, even with the most challenging samples. Surface topography is preferably determined by adjusting a focusable lens that affects only the second excitation and detection pathway.

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