Technical Field

The invention is related to the field of optical instrumentation design and development, specifically to the design of a spectropolarimeter for measuring circular dichroism (CD) in a wide spectral range. The key element of the spectropolarimeter of the current invention is the liquid crystal polarization diffraction grating (PDG). For CD measurement with high resolution of a few millidegrees, the difference in the absorption of left- and right- circularly polarized monochromatic light is used. A tunable monochromator coupled to a wide spectral bandwidth light source, or other monochromatic or narrow spectral bandwidth light source may be placed conventionally in front of the investigated sample to provide CD measurements within the required spectral range. The signal with a high signal/noise ratio is detected using two photodetectors placed at the output of the PDG, connected to downstream electronics and digital processing system.

Background Art

In the circular dichroism spectroscopy, circularly polarized light is being used to examine structural specifics of optically active chiral environment. CD measurements are indispensible in studying biological molecules, their structure and their interaction with other molecules. The absorption spectroscopy technique is being used to measure CD; this technique is based on recording the difference in the absorption strength of the left- and right- circularly polarized beams.

Conventional methods of CD measurements in a wide spectral range rely on application of spectropolarimeters described in the literature [1, 8-13]. Such devises measure polarization of the monochromatic light passing through the sample. Operation of the currently available spectropolarimeters is based on determining the difference in the intensities of the left- and right- circularly polarized beams passed through the sample at fixed wavelength; their main functional element is a photo-elastic modulator (PEM) [www.jascoinc.com, www.photophysics.com/tutorials/circular-dichroism-cd-spectro scopy/4-cd-spectrometer- operating-principles, www.sun-way.com.tw/Files/DownloadFile/J815_S.pdfJ. Conventionally, the left- and right- circularly polarized components of light passing through the sample are separated using PEM. This technique requires application of a sinusoidal high voltage of e.g. 50 kHz frequency to PEM. At the output of PEM, two temporally separated components with left- and right- circular polarizations are formed, which requires more time and real estate (downstream electronics) for additional data acquisition and processing of those two signals. Among the features of PEM-based system is a necessity to record intensities of the right- and left- polarized light at the maximum of the applied sinusoidal voltage. Thus, only a small part of light transmitted through PEM is used to record the signal by the photo receivers. To override this shortcoming, high power (100 Watts or higher) light sources are used in the conventional spectropolarimeters.

Thus, the spectropolarimeters based on application of PEM have at least two significant drawbacks: -first is a dramatically low efficiency due to inefficient usage of light energy and the second is a significant data acquisition and processing time required to obtain a high signal/noise ratio and, subsequently, to ensure high accuracy of measurements. Obviously, such devices cannot be used for studying of the fast running processes.

Real-time CD measurements became possible with the invention of a new generation optical element - a liquid crystal polarization diffraction grating (PDG) [2-5]. An important advantage of PDG is instantaneous and simultaneous formation of spatially separated spectral distributions of left- and right- circularly polarized components at the output of PDG. The other known in the industry optical devices that spatially separate incident light into the left- and right-hand circularly polarized beams may be used in place of PDG.

One of the closest prior arts for the apparatus of the current invention is the device for measuring circular dichroism described in [6]. Its main functional element is the liquid crystal polarization diffraction grating. The grating generates just only two diffracted fields of +1 and -1 orders. The intensities of these two fields are proportional to the intensities of the left- and right- circularly polarized components of light falling on the grating. Note that the light beam may be linearly polarized, not polarized, or randomly polarized. In the described prior art, the wide spectral bandwidth light beam passes through the analyzed sample and falls on PDG, which provides spectral decomposition of the incident light.

The described above device allows measuring CD in real time within a wide spectral range. This device provides a reasonably high signal/noise ratio allowing simultaneous recording of intensity distributions of the left- and right- circularly polarized components at different wavelengths using CCD/CMOS arrays or similar array detectors. However, this device does not provide high angular resolution, allowing the accuracy of the CD measurements in the order of a few hundreds of millidegrees (300 mdeg) [7]. Such low resolution of CD measurements is pre-determined by the used optical setup, in which the accuracy of the detector arrays' alignment is limited - to get high resolution the special alignment of the detector arrays in such systems has to be of the order of the wavelength of light. Any small spatial misalignment (close enough to the light's wavelength) of the two detector arrays in the direction of the impinging light or perpendicular to it, will produce a false circular dichroism signal.

However, measurements of CD with an accuracy of at least a few millidegrees are required in biology, medicine, and in other applications.

The present invention describes the device and methods that surpasses drawbacks of the conventional devices allowing measuring real-time circular dichroism spectra with high angular resolution and high signal/noise ratio, using the apparatus based on the liquid crystal polarization diffraction grating (LC PDG).

Summary of Invention

The principal objective/embodiment of the present invention is to propose a spectropolarimeter based on PDG and method for real-time measurements of circular dichroism, with high angular resolution and high signal/noise ratio. The other embodiments of the present invention include the spectral polarimeter based on PDG in which:

- A monochromatic or narrow spectral bandwidth light is used to probe the sample

- Alternatively, a broadband spectrum xenon lamp may be used as a light source; a fixed light wavelength (or a narrow spectral bandwidth beam) is selected using the monochromator placed between the light source and the investigated sample;

- CD measurements in the wide spectral range may be provided by scanning of the light wavelength using the monochromator;

- An investigated sample is placed in the pathway of light between the monochromatic/narrow spectral bandwidth beam or the wavelength selecting element and PDG;

- two high-sensitive photodetectors, each having single active area are used as the light receivers (detectors), which are placed separately at the output of PDG along the propagation directions of the right- and left-circularly polarized components of light; Alternatively, each of the two detectors may have multiple active areas, but the output of either all active areas or limited number of active areas of each of the two detectors may be connected in parallel to detect the signals;

- a downstream electronics and digitizing systems are connected to those receivers.

Unlike the conventional CD apparatus, in the proposed device:

- the intensities of diffracted beams are registered not by the arrays of detectors, but by high sensitive, single active-area photodetectors;

- the signal recording is realized on the fixed wavelength, allowing to circumvent the requirement for the accuracy of alignment of the two detector arrays, which requirement is typical for the conventional CD devices;

- CD measurements are achieved with the resolution of 3-5 millidegree or higher.

- CD measurements may be provided at a fixed wavelength, eliminating the need of a xenon lamp and monochromator. The possible version of such measurements may include light emitting diode (LED) emitting the required light wavelength or other monochromatic or narrow bandwidth light source known in the industry.

The present invention proposes the apparatus and method for measuring CD spectra, comprising the steps of irradiating the sample with a monochromatic or narrow spectral bandwidth light, dispersing/diffracting the light that passes through the sample into the right- and left- circular polarized components by using the polarization diffraction grating, detecting the light intensity of the two light beams by the two single active area detectors (one single-active area detector per beam), and then measuring a circular dichroism spectrum of the sample on the basis of a result of the detection by the two detectors. As mentioned above, in the apparatus and method according to the present invention, the CD measurements are virtually of a real-time type and the angular resolution (CD resolution) if not dependent on the alignment of the detectors, since the CD signal is measured by providing simultaneous readouts from the two single active area detectors. Therefore, the apparatus and method according to the present invention enables a wide variety of applications, in which high CD angular resolution, high signal-to-noise ratio, and real-time signal monitoring is important. In one of the embodiments, the monochromatic light beam from the output of a monochromator passes through the sample, and the said light beam successively passes through LC PDG. The two diffracted monochromatic beams are detected by the two high sensitive, single-active area photodetectors. The output currents from the two photodetectors are supplied to the inputs of differential logarithmic impedance amplifier. Alternatively, the output signals from the photodetectors can be recorded by any other downstream electronics, and may be further followed by digitizing.

In the other embodiments, the monochromatic light sources like lasers, known in the industry may be used as the source to irradiate the sample. In still other embodiments, either the narrow spectral bandwidth light emitting diodes (LED) or narrow bandwidth interference filters (or other spectral selective filters) may be used.

In some embodiments and by an example only, the important parameter for CD calculations is

where Ii and I ₂ are the intensities of the left- and right- circularly polarized beams at a fixed wavelength, recorded by the two photodetectors. CD is determined using the following formula:

where IOL and ½ are the reference values of the intensities of the left- and right- circularly polarized beams, when e.g. the distilled water or other known industry standard is used as a sample, and ITL and ITR are the intensities of the left- and right- circularly polarized components of beam passed through the sample and spatially separated by PDG. The output signal from the downstream electronic devices (e.g. the logarithmic amplifier) is digitized or processed using other appropriate methods, including e.g. specially designed software, and CD value is calculated using the intensity ratio of the two diffracted beams.

In some embodiments, a quartz cuvette is used for a sample placement. If the front and back walls of the cuvette are not perfectly parallel, the additional systematic error of CD measurement will be added. In order to take into account this error, the intensities of the left- and right- circularly polarized components of the beam passed through an empty cuvette and spatially separated by PDG are measured independently or in advance. In some embodiments, this systematic error is being accounted for using the software during data collection and processing. Brief Description of the Drawings

Fig. l. The optical block-schematics of the proposed devise in accord with one of the embodiments of the current invention, in which an optical element that spatially separates the optical beam into the left- and right-hand circularly polarized monochromatic or narrow spectral bandwidth components is used, and the light source is a monochromatic or narrow spectral- bandwidth beam.

Fig.2. A part of the optical block-schematics of the proposed devise in accord with the other embodiments of the current invention, in which a narrow spectral bandwidth LED is used as the light source.

Fig.3. A part of the optical block-schematics of the proposed devise in accord with the other embodiments of the current invention, in which a wide spectral bandwidth light source coupled to narrow band pass filters are used.

Fig.4. A part of the optical block-schematics of the proposed devise in accord with the other embodiments of the current invention, in which a monochromatic laser is used as the light source.

Modes for Carrying out the Invention

According to Fig.1 , a monochromatic or narrow spectral bandwidth light beam is formed from a wide spectral bandwidth light source, for example a xenon lamp (1), using a monochromator (2). This monochromatic or narrow spectral bandwidth light beam is being directed to a cuvette with the sample (3). The beam passing through the sample with CD is being spatially separated by the optical element (4) into the left- and right-hand circularly polarized monochromatic or narrow spectral bandwidth components. In some embodiments, the liquid crystal polarization diffraction grating (LC PDG) can serve as an optical element (4). The intensities of those components are being recorded by the two single-active area photoreceivers/photodetectors (5). The photocurrents from the two photoreceivers/photodetectors (5) are recorded by the downstream electronics (6) that as an example only may include a differential logarithmic impedance amplifier or other relevant amplification device. The amplified signal is sent to the computer (7) or an alternative processing unit, where further processing of the recorded data takes place using specially designed software. Fig.2, Fig.3, Fig.4 show few other types of light sources operating at certain specific wavelengths or within a narrow spectral bandwidth that can be used instead of the wide spectral bandwidth light source coupled to a monochromator in the current invention. Fig.2 shows the LED (8), the output beam from which enters the sample. Fig.3 shows a white light source (9) coupled to narrow spectral bandwidth filters (10). After passing the filters 10, light enters the sample. Various optical components known in the industry can be selected as the narrow spectral bandwidth filters - color glasses, multilayer dielectric filters, or other known in the industry spectral filters. Fig.4 shows a laser operating at the required light wavelengths (11). The light beam from laser enters the sample.

The proposed device and method therein allows recording the intensities of the right- and left- circularly polarized components of the light beam passed through the sample and spatially separated by PDG using sensitive photoreceivers at fixed wavelengths or within narrow spectral wavelengths intervals. The difference in photocurrents, which is proportional to sample's CD, is being measured using e.g. a differential logarithmic impedance amplifier. Measurement results are being processed using specially designed software. The wavelength is being changed by a monochromator, which is placed before the sample, in order to insure the required spectral range. Alternatively, any other known in the industry method to provide either monochromatic or narrow spectral bandwidth light could be used.

Application of the proposed apparatus and methods described herein provides the CD angular resolution of 3-5 millidegrees or better.

In the other embodiments, the proposed device and methods may be used for measuring CD at certain specific wavelengths or within a narrow spectral bandwidth. In this case, instead of the white source of light (e.g. a xenon lamp), LEDs of the necessary light wavelengths may be used as a light source, while the monochromator is eliminated from the optical scheme. In still other set of embodiments, various spectral filters (color glasses, multilayer dielectric filters, or other known in the industry spectral filters) may be used to create monochromatic or narrow spectral bandwidth light source for CD measurements using the apparatus and methods of the present invention. References

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