DE19836183A1 - Device for tracking movement of microscopic and sub-microscopic objects in microscopic volumes - Google Patents

Abstract

The device uses two coherent beams which are crossed to form moving interference patterns. The frequencies of the beam in each pair are different, so that the scattered light intensity of a fixed object varies with the difference frequency of the beams, and each positional variation of the object perpendicular to the interference pattern can be detected as a phase change in the scattered light. A method for using the device is also claimed.

Description

State of the art with sites

In colloid chemistry, particle technology and biology, light scattering techniques have one widespread and are routinely used to characterize size, Size distribution and surface charge on suspensions or emulsions from Powders, pigments, colloids, polymers, vesicles, micelles and molecules are used (Berne, B.J., R. Pecora. 1976. Dynamic Light Scattering. Wiley, New York; Ostrowsky, N. 1993. Liposome size measurements by photon correlation spectroscopy. Chem. Phys. Lipids. 64: 45-56). In addition to these applications, light scattering methods are used in biology also applied to active movements of bacteria (Holz, M., S.-H. Chen. 1978. Tracking Bacterial Movements Using a One-Dimensional Fringe System. Optics Letters. 2: 109-111), sperm (Gottlieb, C., M. Bygdeman, P. Thyberg, B. Hellman, R. Rigler. 1991. Dynamic laser light scattering compared with video micrography for analysis of sperm velocity and sperm head rotation. Andrologia. 23: 1-5) epithelia, single flagella, protein movements or the dynamics of Membrane systems (Tishler; R.B., F.D. Carlson. 1993. A study of the dynamic properties of the human red blood cell membrane using quasi-elastic light-scattering spectroscopy biophys. J. 65: 2586-2600). For measuring inherent electrical properties of colloidal and biological particles and cells developed special light scattering methods, the alternating field-induced movements evaluate (Prüger, B., P. Eppmann, E. Donath, J. Gimsa. 1997. Measurement of inherent particle properties by dynamic light scattering - introducing electrorotational light scattering. Biophys. J. 72: 1414-1424; Prüger, B., P. Eppmann, J. Gimsa. 1998. Particle characterization by AC-electrokinetic phenomena: 3. New developments in electrorotational light scattering (ERLS). Colloids and Surfaces A. 136: 199-207, Gimsa, F. Eppmann, B. Prüger. 1997. Introducing phase analysis light scattering for dielectric character. Measurement of traveling wave pumping. Biophys. 73: 3309-3316).

The conventional dynamic light scattering (DLS), or "quasielastic Light scattering "is a single-beam method with homodyne or heterodyne detection. It allows the dynamics in particle ensembles to be recorded over many decades in the ns area. With this technique, the scattered light from the macroscopic sample, which consists of a large number of individual spreaders, at a greater distance to the sample volume and at a defined angle to the incident beam detected. The detector signal is caused by the interference of the scattered light signals of all Spreaders formed, their thermally or actively induced movements for superimposition of the relative phases of the stray field at the detection site. This creates temporal intensity fluctuations, which generally about the autocorrelation function of intensity be evaluated. So z. B. about the Stokes-Einstein relationship Determine particle radii. Superstructures with optical are also for the DLS Optical fibers are known (Dhadiwal, H. S. 1993. Integrated Fiber Optic Probe for Dynamic Light scanning. Applied optics. 32: 3901-3904).

Laser Doppler anemometry is a heterodyne two-beam method in which two coherent laser beams form an interference pattern in their intersection area that consists of elliptical, flat surfaces of high or low light intensity (Berne, B.J., R. Pecora. 1976. Dynamic Light Scattering. Wiley, New York). Particles that are  moving through the interference area generate a sinusoidal scattered light signal with Gaussian envelope (Doppler burst). Because the distance of the interference planes is known, the particle speed from the frequency of the sinusoidal signal to calculate. The description of the Doppler effect is physically equivalent: Particles that cross the intersection cross both rays in different angles. That is why they generate a scattered light signal with each beam slightly different frequency. The mixture of the simultaneously generated Doppler scattered light signals are generated directly by the particles according to the heterodyne principle in the crossover area. This results in the much lower differential frequency, the is identical to the frequency of the Doppler burst. In contrast to the DLS-Ein Beam method in which the fluctuation frequency of the scattered light at the detector location depends on the detection angle, the two-beam method is in principle independent of Detection angle. With the two-beam method there are several spatial directions components of the movement in the measurement volume can be detected simultaneously; two components, if the detectors are arranged behind polarization filters and one each horizontally and a vertically polarized pair of rays is used; three Components if the detection takes place behind color filters and three colored ones Beam pairs can be used. The direction of movement of the particles in the Interference area is determined by a wandering interference pattern is generated that a beam is changed in frequency. Does that move Interference pattern in or against the direction of particle movement, so reduced or the Doppler frequency increases accordingly. This method is used in engineering e.g. B. used for flow measurements.

Two-beam methods are also used to detect linear movements in microscopic Suitable volumes. However, for a fixed interference pattern, the Particle movement must be very fast so that the particles move through the generate entire detectable Doppler bursts. in the wandering interference pattern would in turn be at a high frequency difference Radiation pair (in technical applications in the range 40-120 MHz) and very slow particle movement, e.g. B. Brownian motion, no detectable Frequency shift expected. That is why in technology Migration rates of the interference pattern in the range of Particle speeds used, the z. B. generated by oscillating mirror can.

Phase analysis light scattering (PALS), a much more elegant method was originally developed for electrophoresis measurements in apolar media to record the smallest particle velocities (Schatzei, K., J. Merz. 1984. Measurement of small electrophoretic mobilities by light scattering and analysis of the amplitude weighted phase structure function. J. Chem. Phys. 81: 2482-2488, Schätzel, K. 1987. Correlation Techniques in Dynamic Light Scattering. Appl. Phys. B. 42: 193-213, Miller, JF , K. Schätzel, B. Vincent, 1991. The Determination of Very Small Electrophoretic Mobilities in Polar and Nonpolar Colloidal Dispersions Using Phase Analysis Light Scattering. J. Colloid Interface Sci. 143: 532-554). At the heart of the method are optoacoustic modulators that generate two laser beams with a very small frequency difference that is phase-locked within the measuring time (cf. also FIG. 1). For optoacoustic modulators such. B. use the Debye-Sears effect to frequency shift the beam 1 . Generate order that corresponds to the electronic control frequency. Since optoacoustic modulators are usually operated at frequencies around 30 to 150 MHz, both beams have to be frequency shifted by optoacoustic modulators in order to generate a smaller difference frequency. The frequency difference of the control signals then corresponds to the frequency difference of the emerging beams 1 . Order. These beams are crossed in the measuring volume and a wandering interference pattern is created. The scattered light intensity of a particle that is stationary in the measurement volume would be modulated exactly with the optical frequency difference. Each translation along an axis perpendicular to the planes of the interference pattern leads to a change in the phase relationship between the detected light and the electronic reference signal Δf, which can be evaluated extremely sensitively with a lock-in voltmeter (cf. also FIG. 1).

In the existing PALS method, the scattered light is created by the ensemble of Particles generated in the macroscopic measurement volume and the light intensities at the Locations where the individual particles are located have different ones Phase behavior. Therefore, the detected stray light reflects synchronous Translational movement of the ensemble is not directly reflected. To evaluate the Scattered light signal, the amplitude-weighted phase difference was introduced, the one statistically reliable measurement of the induced particle movements allowed (Miller, J.F., K. Schätzel, B. Vincent. 1991. The Determination of Very Small Electrophoretic Mobilities in Polar and Nonpolar Colloidal Dispersions Using Phase Analysis Light Scattering. J Colloid Interface Sci. 143: 532-554). Because the wandering interference pattern an evaluable scattered light signal even when the particles stand still (or pure diffusion) can generate much slower movements with PALS than with conventional two-beam methods can be measured. Compared to this, the sinks lower limit of the detectable particle speed by about two Orders of magnitude so that e.g. B. electrophoretic particle charges in the Order of magnitude of an elementary charge were detectable.

Devices with microscopic measurement volumes were used for the DLS (Blank, P.S., R.B. Tishler, F.D. Carlson. 1987. Quasielastic light scattering microscope spectrometer. Appl. Optics. 26: 351-356; Herbert, T.J., J.D. Acton. 1979. Photon correlation spectroscopy of light scattered from microscopic regions. Appl. Optics. 18: 588-590) as also for the laser Doppler method (Holz, Chen. 1978. Tracking Bacterial Movements Using a one-dimensional fringe system. Optics Letters. 2: 109-111. Cochrane, T., J.C. Earnshaw. 1978. Practical Laser Doppler microscopes. J. Phys. E; 11: 196) described. The reduction in the measuring volume was achieved by installing the Light scattering methods achieved in modified commercial microscopes. Blank et al. describe for a microscopic DLS spectrometer, depending on the one used Objective, diameter of the measuring volume between 2.5 and 50 µm. In their structure was the incident beam from above into the observed measurement volume under a certain angle reflected and the scattered light detected by the lens, with a detection angle error of 5.5 ° or more.

Kudo et al. (Kudo, S., Y. Mgariyama, S.-I. Aizawa. 1990. Abrupt changes in flagellar  rotation observed by laser dark-field microscopy. Nature. 346: 677-680) have one special laser dark field microscopy technology developed. This enables the Observation of the rotation of a single bacterial flagellum through a slit diaphragm with a temporal resolution of less than one ms. With the help of this slit diaphragm, which was oriented parallel to the flagellum, could be the tortuous shape of the flagellum be used for signal generation. Thus, despite its good structure, temporal, ultimately only a one-dimensional spatial resolution.

In principle, the "Localized DLS" does not have such a drastic limitation for the characterization of single particles below 1 µm in size with a temporal Resolution down to the ps range and a basic spatial resolution in the nm range was developed (Bar-Iv, R., A. Meller, T. Tlusty, E. Moses, J. Stavans, S.A. Safran. 1997. Localized dynamic light scattering: probing single particle dynamics at the nanoscale. Phys. Rev. Letters. 78: 154-157; Meller, A., R. Bar-Ziv, T. Tlusty, E. Moses, J. Stavans, S.A. Saffron. 1998. Localized dynamic light scattering: A new approach to dynamic measurements in optical microscopy Biophys. J. 74: 1541-1548). Disadvantageous for more general applications, however, is the previous structure: the particle is also localized with a pair of laser tweezers. A second, differently colored measuring laser beam is also used coupled through the microscope objective and into the center of the laser tweezers focused. The focus diameter can be below the light wavelength (Berns, M. W 1998. With laser tools into the cell interior. Spectrum of science (6) 56-61). The particle movement near the focus creates an intensity fluctuation of the scattered measuring laser light, which is evaluated via the autocorrelation analysis can be. This method also has no 3-dimensional resolution.

For microscopic two-beam laser Doppler methods, this is the usual technique Frequency shift of the beams greater than 30 MHz for the detection microscopic particle movements as well as devices with a fixed Interference pattern nonsensical. For the detection of z. B. Brownian movement of In the first case, proteins would not have a sufficient frequency difference. in the in the second case, very long detection times would be required. A number could do better of directional movements of biological objects, such as B. the light-induced Chloroplast movements, or protein movements during cell migration, can be detected. The forces for these directed movements are special Motor proteins, such as kinesin or dynein, are produced using laser tweezers examined (Ashkin, A., K. Schütze, J.M. Dziedzic, U. Euteneuer, M. Schliwa. 1990. Force generation of organic transport measured in vivo by an infrared laser trap. Nature. 348: 346-348; Svoboda, K., C.F. Schmidt, D. Branton, B.J. Schnapp, S.M. Block. 1993. Direct observation of kinesin stepping by optical trapping interferometry. Nature. 365: 721-727). The Atomic Force Microscopy and Scanning Optical Near Field Microscopy potentially reach molecular resolutions, but call one mechanical load on the test object or have a low Time resolution. The use of oscillating mirrors in the beam path for generation very little frequency difference is for accurate measurements of the change of location impractical.

Supplement to references (Relevant US patent applications) 4,986,659; 5,013,928; 5,404,218; 5,627,642 and 5,751,422.  

problem

The invention specified in claim 1 is based on the problem that So far, there is no method for continuous, three-dimensional tracking individual, microscopic or submicroscopic objects in microscopic Volumes with a temporal resolution in the ms range and a spatial resolution enabled in the nm range. Fast imaging procedures allow i. generally only one 2-dimensional resolution and require microscopic visibility of the objects. Scanning laser processes have a high spatial, but only a small one temporal resolution. For the measurement of molecular movements z. B. with the atomic force microscopy achieve a very high resolution, the measurement however requires mechanical contact with the object. This represents in such Microsystems represents an unacceptably large load on the system and leads to a Falsification of the measurement results.

solution

The problem is solved by the features listed in claim 1 with a localized phase analytical light scattering method solved. The invention allows microscopic or submicroscopic objects in microscopic volumes to track continuously.

Achieved advantages

The advantages achieved by the invention are in particular that a continuous tracking of microscopic or submicroscopic objects in microscopic volumes without mechanical stress with a temporal resolution at least in the ms range and parallel to a spatial resolution below the nm range is enabled. In parallel, the measurement volume can be microscopically be observed spectroscopically or otherwise. The method can be used Investigation of very different physical, chemical or biological Use phenomena and at the same time with a large number of physical, combine chemical or biological methods.

Further embodiment of the invention

An advantageous embodiment of the invention is specified in claim 2. The Use of 3 differently colored beam pairs, which are crossed in the measuring volume that the central axes of the interference pattern are orthogonal to each other Having components enables the detection of motion components in all 3 spatial directions. Although no absolute measurement of the location is possible, this allows However, proceeding to track the object relative to the starting location. This allows e.g. B. calculate the three-dimensional trajectory of an object.

The coupling of the beams into the measuring volume and the coupling of the scattering or Fluorescent light can be used in various ways, e.g. B. about a Microscope objective, over optical fibers or a combination of both. This enables precise localization of the measurement volume and a high signal  S / N ratio. At the same time, by limiting the measuring volume unambiguous assignment of the detected signal to a specific object possible.

Embodiments

An embodiment is shown in Fig. 1 (- main drawing -) and is described in more detail below. To improve the clarity of the representation, only the use of a single-color beam is shown in the exemplary embodiment, so that the object movement can only be traced in one dimension. However, the exemplary embodiment can be expanded very easily for the detection of the multidimensional movement.

All light paths in Fig. 1 are shown in dashed lines. The coherent light of a laser ( 1 ) is divided into two beams of the same intensity via the beam splitter ( 2 ), which are shifted in frequency by the frequency f ₁ or f ₂ by optoacoustic converters ( 3 a, 3 b). As an optoacoustic transducer ( 3 a, 3 b) z. B. Bragg cells can be used. The electrical control signals of the optoacoustic transducers ( 3 a, 3 b) are generated by the electronic drivers ( 16 a, 16 b). The difference frequency Δf is formed in the mixer ( 17 ) and used as a reference signal of the lock-in amplifier ( 15 ). The first-order frequency-shifted beams emerging from the optoacoustic transducers ( 3 a, 3 b), which have the frequency difference Δf to one another, are coupled into optical fibers ( 5 a, 5 b) by coupling optics ( 4 a, 4 b) and to the microscopic slide ( 8 ) directed. There, the rays through decoupling optics ( 6 a, 6 b), the z. B. can be designed as gradient index rods, focused in the center of the measurement volume ( 7 ), in which a wandering interference pattern is formed. The intensity of the scattered light from a stationary microscopic or submicroscopic object in the wandering interference pattern of the microscopic measurement volume ( 7 ) fluctuates with the frequency Δf and has a rigid phase relationship to the reference signal of the lock-in amplifier ( 15 ). Every change in location perpendicular to the interference pattern leads to a change in this phase relationship and can be converted directly and continuously into a speed component or a change of location relative to the starting location. The conversion of the scattered light intensity into an electronic signal with the frequency f _measurement is carried out by the photodetector ( 14 ), which, for. B. as a secondary electron multiplier (SEV) or avalanche photodiode. For detection, the scattered light is collected via the microscope objective ( 9 ) and guided via a mirror ( 10 ), the pinhole ( 12 ) to limit the measuring volume and the color or polarization filter ( 13 ). The mirror ( 10 ) can be semi-transparent or a folding mirror, so that a direct visual observation of the measurement volume or an observation via the camera ( 11 ) can take place parallel to the measurement.

Fig. 2 shows two different embodiments for optical fiber optics for generating the measurement volume ( 1 ) with a wandering interference pattern. To detect the movement component in the x direction, the interference pattern is formed by the two beam components x ₁ and x ₂ . A snapshot of the interference pattern is shown schematically in the measuring volume ( 1 ), which moves in the x-direction along the central axis ( 4 ). Fig. 2A shows optical fibers 2 or Gradientenindexstäbe (2, 3) from which unfocused rays (dashed limitation) leak. Nevertheless, the measurement volume ( 1 ) is limited to its overlap area. Fig. 2B shows 2 optical fibers ( 2 a, 3 a) with special optics, the z. B. from special collimation ( 2 b, 3 b) and focusing lenses ( 2 c, 3 c). This optics focuses the rays in the measuring volume ( 1 ) and thus limits it very strongly.

Fig. 3 shows two different embodiments for microscope optics for generating the measuring volume (1) with a changing interference pattern (see schematic snapshot of the interference pattern in the cut-out image in Fig. 3B). In both exemplary embodiments, the measurement volume ( 1 ) is located above the microscopic slide ( 2 ) and can be observed from below through the objective ( 3 ). The wandering interference pattern is formed by the two beam components with the frequencies f ₁ and f ₂ by frequency shift in the optoacoustic modulators ( 4 a, 4 b). The objective ( 3 ) focuses the beams into the measuring volume ( 1 ) and thus limits it very strongly. In Fig. 2A the detection with the photodetector ( 5 ), z. B. can be a secondary electron multiplier (SEV), by an optical fiber ( 7 ), which can be carried out with or without special optics ( 6 ). In Fig. 2B the detection is carried out by the microscope objective ( 3 ) with the photodetector ( 5 ).

Fig. 4 shows two different embodiments with optical fiber optics ( 2 a, 2 b, 3 a, 3 b, 4 a, 4 b) for generating a measuring volume ( 1 ) with several wandering interference patterns, for detecting the multidimensional movement components of the object. In the optical fiber optics shown ( 2 a, 2 b, 3 a, 3 b, 4 a, 4 b) it can be, for. B. are optical fibers with or without special coupling optics or gradient index rods from which unfocused or focused rays emerge. The beam path is shown in dashed lines. Fig. 4A shows 3 pairs of fiber optics (2 a- 2 b, 3 a-3 b, 4 a-4 b) for generating the measuring volume (1) with three differently colored, migratory interference patterns. The associated rays x ₁ -x ₂ , y ₁ -y ₂ and z ₁ -z ₂ each have the same color in pairs and can therefore be distinguished from one another. To detect a certain spatial direction, each pair generates a wandering interference pattern with central axes in the x, y or z direction. In order to enable the detection of the object movement in 3 spatial directions, the central axes do not necessarily have to be ideally orthogonal to one another, but in principle it is sufficient if they have orthogonal components. Deviations from an orthogonal arrangement can be offset when evaluating the trajectory of the object movement. In the orthogonal structure, the optical fiber optics 2 a, 2 b, 3 a, 3 b are arranged in one plane, the optical fiber optics 4 a and 4 b perpendicular to it. To detect the motion component in the x direction, the interference pattern is determined by the two beam components x ₁ and x ₂ (fibers 2 a and 2 b), and in the y direction by the two beam components y ₁ and y ₂ (fibers 3 a and 3 b) and for the z-direction by the two beam components z ₁ and z ₂ (fibers 4 a and 4 b). The detection of the scattered light from the measurement volume can be carried out using microscope optics (not shown), additional fiber optics (not shown) or one of the drawn optical fibers. Fig. 4B shows a structure with a reduced to 4 number of optical fiber optics ( 2 a, 2 b, 2 c, 2 d), which are arranged in two different, mutually perpendicular planes (parallel to the paper plane: 2 b and 2 c ; perpendicular to: 2 a and 2 d). The number of 4 fibers is sufficient to produce 3 wandering interference patterns in the measurement volume ( 1 ), the central axes of which have components orthogonal to one another. In this embodiment, too, the beams x ₁ -x ₂ , y ₁ -y ₂ and z ₁ -z ₂ each have the same color in pairs and can therefore be distinguished from one another. Each pair generates a wandering interference pattern to detect the object movement in a certain spatial direction. To detect the motion component in the x direction, the interference pattern is determined by the two beam components x ₁ and x ₂ (fibers 2 a and 2 b), and in the y direction by the two beam components y ₁ and y ₂ (fibers 2 b and 2 c) and formed for the z-direction by the two beam components z ₁ and z ₂ (fibers 2 a and 2 d). The detection of the scattered light from the measurement volume can be carried out using microscope optics (not shown), additional fiber optics (not shown) or one of the drawn optical fibers.

FIG. 5 shows an integrated optical fiber optic for generating two mutually orthogonal, traveling interference patterns in the measurement volume ( 1 ). An advantage of this embodiment is the compactness, which is achieved by using a common objective ( 2 ) for 4 optical fibers ( 3 a, 3 b, 4 a, 4 b). The structure shown is suitable for the detection of the two-dimensional movement components of the object if 2 differently colored or differently polarized beam pairs, the beams of a pair for generating a wandering interference pattern should have a small frequency difference, through the associated fiber pairs 3 a, 3 b and 4 a, 4 b can be focused into the measuring volume ( 1 ) via the common lens ( 2 ). Additional fiber or microscope optics are required for the detection of a third spatial component. As in the other exemplary embodiments, the scattered light can be detected from the measurement volume both via microscope optics (not shown), additional fiber optics (not shown) or also by one of the drawn optical fibers.

Claims (10)

1. Method and device for spatially and temporally resolved tracking of the movement of microscopic and submicroscopic objects in microscopic volumes, characterized in that the two coherent beams of one or more pairs of beams are crossed so that one or more wandering interference patterns are thereby formed in a microscopic measuring volume that the two beams of each pair differ in frequency in such a way that the scattered light intensity of a fixed object fluctuates with the difference frequency of the beams of each pair and every change in location of the object perpendicular to the respective interference pattern can be detected as a phase change in the scattered light relative to an independent reference. 2. The method and device according to claim 1, characterized in that for A beam pair is used for each direction of movement of the object, which is distinguishable from other beam pairs by its color or polarization. 3. The method and device according to claim 1 or 2, characterized in that the scattered light and / or fluorescent light of the Object is detected. 4. The method and device according to any one of the preceding claims, characterized characterized in that the difference frequency of the coherent rays of a pair is constant or variable, but is many orders of magnitude smaller than that Light frequency and the comparison of the fluctuation of the object detected Scattered light intensity with an independently generated difference frequency reference Measurement of the change in location and / or speed is used. 5. The method and device according to any one of the preceding claims, characterized characterized in that from the phase behavior of the scattered light intensity by each a pair of beams that is different from the other pairs of beams by its polarization or Color is distinguishable, the one- or multi-dimensional trajectory of the movement of the object or objects is calculated. 6. The method and device according to any one of the preceding claims, characterized characterized in that the spatial and temporal resolution of the movement of the or Objects in the microscopic measurement volume in the nm or ms range or better. 7. The method and device according to any one of the preceding claims, characterized characterized in that the coupling of the beams into the microscopic measurement volume, which is formed in the crossover area of the rays by means of optical fibers special decoupling optics and / or through optical fibers without such optics and / or through the microscope optics z. B. on collimators, mirrors, diaphragms and that Lens. 8. The method and device according to any one of the preceding claims, characterized characterized in that the detection of the scattered light signal of the object or objects, which are in the microscopic measurement volume with one or more photodetectors  about the microscope optics z. B. via a lens, mirror, aperture and color and / or Polarization filter, and / or via optical fibers with special coupling optics or by means of optical fibers without such coupling optics, the light being transmitted via Color and / or polarization filter is passed. 9. The method and device according to any one of the preceding claims, characterized characterized in that multiple use of optical fibers and / or lenses this means that rays of different color, frequency and / or polarization simultaneously through the same fiber or the same lens into the measuring volume be coupled in or scattered light from the measuring volume is guided to the photodetector becomes. 10. The method and device according to any one of the preceding claims, characterized characterized in that the method with microscopic observation of the Measuring volume is combined, which can be done visually or by a camera system.