Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/21—Polarisation-affecting properties
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
  • G01J4/00—Measuring polarisation of light
  • G01J4/04—Polarimeters using electric detection means

Definitions

• LSM laser scanning microscope
• selected points (well defined volume units) of the tested material are irradiated by a focused laser beam in response of which information on the intensity of the transmitted, reflected or emitted light is obtained which can be stored generally in digital form.
• the signal of the laser scanning microscope during scanning a field of predetermined width and length is used to obtain a picture of high resolution for detailed analysis.
• the picture quality may be further increased by using the LSM in confocal mode, thereby substantially excluding the disturbing effect of the light emanating from points other than the focus point.
• the confocal mode is a basic feature but it may be used only for the reflected or emitted light (fluorescence).
• the confocal mode of the LSM provides for the nondestructive optical slicing of the sample and the reconstruction of three dimensional "images". Highly improved picture quality may be obtained by using a two or more photon laser excitation method which may be strictly limited to the tested area and thereby the disturbing effect of the background radiation (intensity) may be practically completely eliminated (A. Diaspro and M.Robello: Multi-Photon Excitation Microscopy to Study Biosystems, European Microscopy and Analysis, March 1999).
• Laser scanning microscopes - when compared to the conventional microscopes and methods - provide a high quality and high resolution information of the sample structure. Nevertheless these methods do not provide any information on the anisot- ropy and many other physical interactions of the sample that may only be examined with polarization spectroscopy methods.
• polarized light provides images of the sample comprising information on the anisotropic structure, e.g. the spatial arrangement of the transition dipoles, and the physical interaction between each other and the micro-environment.
• the anisotropic properties of the materials influence generally the polarization properties of the light emitted, reflected or transmitted by the materials in an anisotropic way, therefore the examination of the polarization properties of the light emitted, reflected or transmitted by the materials enables conclusions relating to the optical anisotropy and also the molecular order of the tested material.
• Measurements carried out with polarized light (LD: linear dichroism, CD: circular dichroism) are described by T.C.
• P degree of polarization
• one object of the invention is to provide a method and apparatus which combine the advantages of laser scanning microscopy and polarimetry with the combination resulting in more measured parameters with a single apparatus, or with different configurations of a single apparatus with special regard to the parameters of emitted fluorescence measured in confocal mode at a single or multiple wavelengths at substantially the same time, or with regard to the possibly most complete analysis of the polarization content of emitted, transmitted or reflected light.
• a further object of the invention is to eliminate or compensate the errors resulting from wavelength dependency and the polarization distortion of the optical devices during the measurements.
• the difference of the intensity signals of said two polarization components is generated during said signal processing and the difference is divided in accordance with the measurement carried out by the values 1, l ⁇ + ⁇ 2, l ⁇ + 212 or any other value assigned to an element of the Mueller matrix and determined by measurement or calculation.
• a laser light that allows spatial and/or temporal intensity contentration is used for illuminating the sample and for causing two or more photon excitation in the molecules of the sample virtually at the same time.
• This method automatically provides the advantage of confocal measuring. Using very short irradiation times of the individual pixels the sample may be scanned and measured for a longer period of time without being influenced adversely by the laser light.
• the invention provides also a method capable of determining the elements of the Mueller matrix in a transmitted or reflected laser beam of the microscope by changing the polarization state of the illuminating laser light in four steps (depolarized, linearly polarized at 0 (or 90 degree), linearly polarized at +45 (or -45 degree), and circularly polarized) while determining the Stokes parameters (I,Q,UN) in each of the said four states.
• the objectives of the invention are achieved by an apparatus for determining the polarization properties oflight emitted, reflected or transmitted by a material using a laser scanning microscope with a polarization state generator between the laser light source and the material being tested, and a detector in a light beam for determining the intensity oflight with a polarization state modified by the material or the intensity of light emitted by the material.
• a means for dividing different polarization components in space or time is used in front of the detector.
• the apparatus according to the invention may comprise an optical component in the light beam received from the material for dividing the polarization components in space according to their polarization state or according to a predetermined percentage of the intensity with a detector in the light beams divided according to the polarization state of the polarization components or with a detector and an analyzer in front of the detector for selectively transmitting one of the polarization components in the light beams divided according to a predetermined percentage of the intensity.
• a photoelastic modulator (PEM, Kerr cell, or the like) is comprised in the light beam received from the material for dividing the polarization components in time and a passive polarization analyzer is provided in front of the detector.
• PEM photoelastic modulator
• the apparatus according to the invention may comprise at least one wavelength selective beam splitter between the passive polarization analyzer and detector and further detectors may be provided in the light beams divided by the wavelength selective beam splitter.
• the apparatus may include a second photoelastic modulator (PEM) between the photoelastic modulator and the passive polarization analyzer with the second photoelastic modu- lat ⁇ r(PEM) having an optical axis rotated by 45° relative to the optical axis of the f ⁇ rst photoelastic modulator (PEM).
• PEM photoelastic modulator
• the apparatus may be provided with a polarization state generator comprising depolarizer, a linear or circular polarizer and an optical phase retarder or any combination thereof.
• the apparatus For converting and processing the measured signals and compensating the errors during measuring the apparatus according to the invention may be provided with a signal processing unit which is connected to the output of the detector(s).
• the signal processing unit comprises at least one lock-in amplifier, a low-pass filter and an ana- log-digital converter.
• a digital-analog converter is required in the signal processing unit.
• Fig. 1 is a schematic diagram of an apparatus according to the invention with two channels for working in emission or reflection mode,
• Fig.2 is a schematic diagram of the apparatus according to the invention with two channels for working in transmission mode
• Fig. 3 is a schematic diagram of the apparatus according to the invention with one channel and working according to the modulation principle
• Fig.4 is a schematic diagram of the apparatus according to the invention working according to the modulation principle for measuring at different wavelengths
• Fig. 5 is a schematic diagram of the apparatus according to the invention for measuring the Stokes parameters
• Fig. 6 is a schematic diagram of the apparatus according to the invention for determining the elements of the Mueller matrix
• Fig. 7 is a schematic diagram of a signal processing and imaging unit for displaying the measured values
• Fig. 8 is a schematic diagram of the signal processing unit used in the appa- ratus according to the invention.
• Fig. 9 is a schematic diagram of the apparatus according to the invention for determining the FDLD and FDCD values
• Fig. 10 is diagram illustrating the method for compensating the sinusoidal error of the photoelastic modulators.
• Fig. 1 depicts a two channel configuration with a sample 5 on object table 6 which is illuminated with a laser light of laser light source 1 with the laser light routed through polarization state setting unit 2.
• the reflected light or the fluorescence are deflected by beam splitter 4 (for example semitransparent mirror).
• beam splitter 4 for example semitransparent mirror
• This way two light beams are generated by a second beam splitter 8.
• a detector 16 and 17 producing an output signal proportional with the intensity oflight which is input to signal processing units 18.
• a semitransparent mirror can be used as a beam splitter 8. In this case the entering light beam will be separated in a given proportion of the intensity.
• a further mirror 9 may be placed for deflecting a selected light beam.
• Both of the divided (deflected) light beams contain all polarization components, therefore the polarization components to be measured must be selected by a passive polarization analyzer 12 and 13 located in front of the detectors 16 and 17.
• a beam splitter 8 also a polarization beam splitter (e.g. a Wollaston prism, or the like) can be used for transmitting only a first polarization component in the first divided beam and transmitting only a second polarization component in the second divided beam.
• Polarization beam splitters are commercially available from Melles Griot or Oriel. Special polarization beam splitters are also available from OZ Optics, Ltd.
• Measurements in a confocal mode include also the step of selecting the light component received from the focus point of the laser light on the sample and excluding the light components received from points other than the focus point. This enables the optical slicing of the sample.
• a phase retarder 7 quarter wavelength plate
• Fig.1 The arrangement of Fig.1 is one possible embodiment for measuring the intensity of two different polarization components at the same time received from the sample illuminated pixel by pixel by the laser scanning microscope.
• the analog signals proportional to the intensity values are input to the signal processing unit 18 where the necessary conversions, calculations and cor- rections will be carried out.
• the output signal On the basis of the two polarization components Ii and I 2 which are substantially orthogonal to each other the output signal will be calculated with the one of the following formula or one of the formula used to determine the elements of the Mueller matrix.
• Formula (3) is preferably used in case of measuring anisotropy of emission dipoles oriented in a cylindrical symmetry.
• the polarization components measured with the method according to the invention are orthogonal to each other.
• the resulting values of the above formulas are determined by the signal processing unit 18.
• One of the above calculations is carried out for each of the pixels (illuminated points of the sam- pie) and the result is stored in a storage means.
• the stored image information is displayed on a display means (screen, monitor, etc.) by a video signal processing unit and software.
• the output signal of the signal processing unit 18 is simply lead to the image signal input of the LSM.
• the synchron signal for scanning/rastering the laser light of the LSM and the output signal of the signal processing unit 18 is further processed by an electronic control unit, e.g. a computer COMP to an image signal and the image is then displayed on a monitor MON.
• the phase between the polarization components oflight beam received from the sample 5 is varied periodically according to a function with a predetermined frequency and shape (preferably according to a sinus function) between two end positions and the polarization components are measured subsequently through the same analyzer 25 with the same detector 27.
• a polarization modulator such as a photoelastic modulator (PEM), an elec- trooptical modulator (EOM), an optoacoustic modulator, a Kerr cell, a Pockels cell or the like.
• PEM photoelastic modulator
• EOM elec- trooptical modulator
• optoacoustic modulator a Kerr cell, a Pockels cell or the like.
• a photoelastic modulator 23 is used in front of a detector 27 for sensing the light intensity and providing an output signal proportional thereto.
• a passive polarization analyzer 25 is used between the detector 27 and the photoelastic modulator 23.
• a suitable photoelastic modulator is available from Hinds Instruments under the trade name I/FS50, but other types or means with the same effect may be used.
• the output of detector 27 is connected with an input of signal processing unit 28 in the same way as already seen in Figs. 1 and 2.
• a colour filter 16 may be used for rejecting the intensity of the undesired background light.
• Diaphragm 21 positioned in the light beam may be necessary for confocal imaging.
• a phase retarder 22 (quarter wavelength plate) may also be used for enabling the selection of the circularly polarized components even if only a linear polarization analyzer is used.
• the photoelastic modulator In its ground state the photoelastic modulator does not change the polarization properties of the transmitted light. If the maximal change of the width of the photoelastic modulator is lambda/4 (lambda is the wavelength of the light used) the linearly polarized incident light will be converted by the photoelastic modulator in its dO+A state into a cirularly left or right polarized light and in the dO-A state of the photoelastic modulator the output light is converted into a cirularly right or left polarized light.
• the linearly polarized incident light will be converted by the photoelastic modulator in its dO+A and dO-A state into a linearly polarized light with a polarization plane rotated at 90 degrees with respect to the polarization plane of the incident light. It should be noted that in this case when reaching the lambda/4 posi- tions circularly left of right polarized light is provided depending on the direction of change.
• the linear polarization analyzer in front of the detector transmits only substantially one selected linear polarized light having a polarization plane parallel to the po- ' lazation axis of the analyzer, which in return results in a maximum of the detector signal and all other polarized light components result in a smaller value of the detector signal.
• the linear polarized light with a polarization direction orthogonal to the polarization axis of the analyzer will provide a minimum of the detector signal.
• LSM systems also the LSM of Zeiss type 410) are suitable for measuring the intensity of the fluorescence emitted by the sample having components of different wavelength by separating each emission wavelength and measuring with a special detector (typically with a photoelectron multiplyer). The excitation may occur at a single frequency or at more frequencies.
• a special detector typically with a photoelectron multiplyer
• the excitation may occur at a single frequency or at more frequencies.
• dichroic mirrors may be used.
• the illumination of the sample 5 is the same as seen in and described in connection with Fig. 1.
• a photoelastic modulator 31 such as FS50 avalilable from Hinds
• a passive polarization analyzer 32 In the light beam received from the sample 5 and deflected by the semi- transparent mirror 4 there is a photoelastic modulator 31 (such as FS50 avalilable from Hinds) and a passive polarization analyzer 32.
• the analyzer is followed by dichroic mirrors or by dichroic beam splitters 33 for separating the light received from the sample according to the different wavelengths and an optical mirror 34 if neces- sary for deflecting a separated light beam.
• the photoelastic modulator 31 such as of Hinds type FS50
• all wavelength components of the fluorescence may be modulated at the same time.
• the modulation depth of the polarization state will be different due to the different wavelengths, it can be calculated and with a correction factor compensated. In this manner the method as described in connection with Fig. 3 can be extended to more wavelengths.
• the passive polarizer 32 may in some cases be omitted or replaced by dichroic mirrors.
• dichroic beam splitters that are in a large wavelength range insensitive to the polarization state oflight.
• the intensity is detected at the same time in detectors 37 and the detected signals are directed to a common signal processing unit 38 for performing a signal processing described above in detail.
• This configuration requires the application of multiple means or multichannel means for measuring.
• the signal processing is simplified by the fact that in this configuration the modulating frequency is the same in all channels.
• a diaphragm 35 positioned in the light beam may be necessary for confocal imaging and a colour filter 36 may be used for rejecting the intensity of the undesired background light.
• the signal processing unit 38 used with the configuration shown in Fig. 4 may be the same as shown in Fig. 8A and described in detail in connection with Fig. 3.
• the signal processing unit of Fig. 8 A may be tripled, or in case of sufficiently high processing speed it is also possible to connect alternately (multiplexing) the outputs of the detectors 37 of the three channels to the input of the signal processing unit 38.
• the measurement of I(+45°)-I(-45°) is performed in the same way as the measurement of I(90°)-I(0°) but the modulator is set to 0° and the analyzer to 45°. This may be especially important in measurements of anisotropy. During these measurements also a value proportional to the full intensity of the fluorescence may be determined and thus the differences in sensitivity may be compensated. This will be described in more detail in connection with Fig. 10. Using three channels all characteristic parameters, e.g. all components of the Stokes vector (I,Q,U,V) of the polarization state of the emitted fluorescence or reflectance may be determined and displayed as an image as described above.
• all characteristic parameters e.g. all components of the Stokes vector (I,Q,U,V) of the polarization state of the emitted fluorescence or reflectance may be determined and displayed as an image as described above.
• a passive polarization analyzer 45 (such as a Glann-Thompson prism) is used with an optical axis having an angle of 22,5 degrees with the same reference plane.
• the output of the detector 47 is connected to signal processing unit 48 similar to the embodiments described before.
• the signal processing unit 48 comprises three phase sensitive (lock-in) amplifiers FE1...FE3 and one low pass filter LP as shown in Fig. 8B.
• a digital low pass filter is used for compensating the error during measuring the intensity.
• a fourth phase sensitive (lock-in) amplifier FE4 is needed.
• the input signal may be multiplied by any periodical scalar signal before calculating an average, which means that it can not only measure the amplitude of a signal of a selected frequency in difference mode - like the conventional phase sensitive (lock-in) amplifiers - but it is also capable of measuring the same in addition mode.
• difference mode the output signal is responsive to the magnitude of the input signal at a phase ⁇ minus the magnitude of the input signal at a phase ⁇ + I.
• the output signal is responsive to the magnitude of the input signal at a phase ⁇ plus the magnitude of the input signal at a phase ⁇ + .
• the multiplier function for the addition mode is shown in Fig. 10B and the multiplier function for the difference mode is shown in Fig. IOC.
• the values of the Stokes vector are determined as described below.
• N circularly polarized component: signal of FE3 working in difference mode at a frequency f of PEM 43 or 44.
• the values measured may be - after conditioning - directed to a suitable input of the imaging unit (video signal processing board) of the LSM for displaying the differential polarization (DP) values as a picture similar to the intensity picture in "conventional" mode.
• DP differential polarization
• the synchron signals for scanning the pixels in x and y direction may be used directly by a suitable image processing circuit (Fig. 7).
• a suitable image processing circuit Fig. 7
• the advantage of this method when compared to the measurement according to Fig. 4 is that only one detecting channel is needed for the measurement and after relocating the detector it may be used as an external unit.
• the polarization image may be obtained without the further use of the LSM and that is independently of its hardware and software proper- ties.
• the elements of the Mueller matrix will be determined according to the invention with a method comprising the step of changing the polarization state of the illuminating laser light in four steps (depolarized, linearly polarized at 0 (or 90 degree), linearly polarized at +45 (or -45 degree), and circularly polarized) while determining the Stokes parameters (I,Q,U,N) in each of the said four states.
• a polarization state setting unit 2a-2d comprising depolarizer 51, a linear or circular polarizer 52, 53 and an optical phase retarder 54 or any combination thereof is used.
• Fig. 6A...6D four different polarization state setting units 2a...2d are shown which produces different but in the time constant polarization state laser light.
• the polarization state setting unit 2a comprises a depolarizer 51 for depolarizing the laser light which might be polarized and which illuminates the tested sample with a depolarized laser light.
• the polarization state setting unit 2b comprises a polarizer 52 which may be a linear or a circular polarizer as required.
• the polarization state setting unit 2b illuminates the tested sample with a linearly or circularly polarized laser light and may be used when the laser light was originally depolarized.
• the polarization state setting unit 2c comprises a linear polarizer 53 and a phase retarder 54 behind the linear polarizer 53 for illuminating the tested sample with a circularly polarized laser light and may be used when the laser light was originally depolarized.
• the polarization state setting unit 2d comprises a depolarizer 51 and a polarizer 52 behind the depolarizer 51.
• the polarizer 52 may be a linear or a circular polarizer as required.
• the polarization state setting unit 2d illuminates the tested sample with a linearly or circularly polarized laser light and may be used when the laser light was originally polarized.
• Fig. 9 depicts a further configuration according to the invention for measuring the fluorescence detected linear dichroism (FDLD) and the fluorescence detected cir- cular dichroism (FDCD).
• the polarization state setting unit 2 comprises a linear polarizer 81 and a photoelastic modulator 82 behind it. If the laser light source 1 provides a linearly polarized laser light the linear polarizer 81 may be omitted. With this arrangement the exciting laser light may be modulated to obtain differ- ent polarization states. The path of the luminescent light has not to be changed in the LSM. This provides an arrangement more simple than that seen in Fig. 1. The same elements "with the same function have been marked with the same reference sign as use in Fig. 1. The signal processing is performed with a demodulation method similar to the method described above. The value of FDLD is measured at a frequency 2f and the value of FDCD is measured at a frequency f and the image processing is also the same as describe above in detail.
• FDLD flu
• a high power laser light allowing spatial and/or temporal intensity concentration may also be used for illuminating the sample and for causing two or more photon excitation in the molecules of the sample virtually at the same time.
• two-photon-excitation falls off with the square of the distance from the focus a dramatic reduction of background fluorescence is obtained and thus the attainable spatial resolution is substantially improved, while the adverse effects of laser light can be minimized.
• Fig 10A shows the variation in width d of the photoelastic modulator (PEM) due to a sinusoidal alternating control voltage as a function of time.
• PEM photoelastic modulator
• FIG. 10D shows the intensity value measured by a detector behind a linear polarization analyzer as a function of time wherein the control of the PEM is identical with the control seen in Fig. 10 A.
• the analyzer is set to have an optical axis parallel to the polarization plane of a linearly polarized light passing through an unexcited PEM. This arrangement provides a maximal intensity I2 in each position dO. In the positions dO+A and dO-A the polarization plane of the linearly polarized light is orthogonal to the optical axis of the analyzer, therefore the intensity I j measured with the detector is minimal.
• I m has already been determined the compensated value of the intensity may be calculated according to
• the systematic errors of the DP measurements due to the different optical ele- ments affecting the polarization state may be compensated by correction methods, which may be performed during digital signal processing.
• the photose- lection may be eliminated and the signal to noise ratio may be improved.
• the laser light used in LSM is typically polarized. As the absorption and the emission is dependent on the polarization state of the illuminating light, the image obtained from a sample may vary depending on the polarization state of the light used. In the LSM images it produces undesired overexposures or underexposures or even diminishing of details. It is in anisotropic samples extremely significant. This effect may be eliminated. : by using a depolarizer in the polarization state setting unit as pro- posed in Fig. 6 A. This may be used in cases of transmission, fluorescence or reflectance as well. A passive depolarizer positioned in the exciting/illuminating light beam may however cause inhomogenities at microscopic level and the output is in many cases not satisfactory either.
• a PEM may be used for varying the polarization state of the inadiating light in time instead of a depolarizer. After a suitable gating or averaging of the detector signal the photoselection may be eliminated without any disturbing side effect as the illuminating (exciting) light comprises all polarization directions during the time the laser light is focused on one point and the averaging takes place.
• the method of modulating and demodulating may also be used for improving the signal to noise ratio if no polarization measurements are carried out.
• the polarization state generator comprises a photoelastic modulator (PEM) positioned between two passive linear polarizers with a polarization plane parallel or perpendicular to each other and the optical axis of the photoelastic modulator (PEM) set at 45° relative to the passive linear polarizers.
• a depolarizer behind the second linear polarizer may be used if necessary. This anangement provides a sinusoidal alternating intensity with a frequency 2f relative to the frequency f of the PEM.
• the demodulation may be carried out with a single phase sensitive (lock-in) amplifier at the frequency 2f. If the laser light source provides a distortion free linearly polarized laser light the linear polarizer on the side of the laser light source may be omitted.
• the depolarizer may be omitted. This method for improving the signal to noise ratio has the advantage when com- pared to other methods using high intensity modulating frequencies that the FDLD and FDCD measurements and the photoselection compensation may be performed with the same components.

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