The OLIS Polarization Toolbox is the sample compartment used on the
DSM 172, DSM 245, CPL Solo, and NIR CPL Solo
This sample compartment is unique in providing user selected positioning of the polarization hardware. This hardware – one or two polarizers and a modulator – can be positioned before or after the sample. In this way, one can configure a single sample compartment for circular dichroism and circularly polarized luminescence¹.
In both acquisition modes, a ‘direct subtractive method’ of data acquisition and processing is used. This DSM software allows use of a single detector and a polarizer fixed in position, obviating the need for G-factor correction for emission measurements. (It also obviating the need for electronic calibration in CD, but this is addressed in detail elsewhere.)
Rapid switching (50 kHz) between polarization states also supports kinetic studies such as stopped flow spectroscopy.
The light source is external to the Polarization Toolbox. The light source is a host spectrophotometer (i.e., lamp and scanning monochromator) or an LED.
For the DSM 172and 245, which support CD and CPL, the host spectrophotometer is a xenon arc lamp and a double monochromator.
For the CPL Solo, a wavelength specific LED is used. A well-chosen LED will have higher intensity and greater stability than the light from a xenon arc lamp through a double monochromator.
Polarizer Location One: At the entrance of the Polarization Toolbox, there is a shelf for a polarizer immediately after the entrance of the incoming light.
Circular dichroism is a measure using circularly polarized light, so the incoming light is polarized with this first polarizer. That is, with a polarizer here, the measurement light is polarized.
Circularly polarized luminescence is a measure of circularly polarized emitted light, so polarizing this exciting light is desirable only when polarized excitation is intentionally to be examined. Polarized excitation is not always the preference, because the polarizer does absorb some of the exciting light, ultimately reducing sensitivity, and because your sample’s emission might not be changed by polarization of the light. That is, the exciting light is non-polarized or polarized, as the user prefers for a given experiment.
Modulator: The means by which polarization is modulated left/ right or parallel/perpendicular
Circular dichroism employs a 50 kHz photoelastic modulator to produce the left circularly polarized and right circularly polarized light as the measurement beam. CD is equal to the difference in the absorbance by the sample of these two polarization states of the light, i.e., CD = abs(L) – abs(R). This switching of polarization states happens 50,000 times per second. The modulator is before the sample.
Circularly polarized luminescence employs the same photoelastic modulator. However, now the PEM is located after the sample to analyze the emitted light. The emission by the (chiral) sample is modulated to be linearly polarized. The modular is after the sample.
Polarizer Location Two: At the exit of the Polarization Toolbox, there is a shelf for a polarizer immediately before the exit port to the emission detection hardware.
Circular dichroism does not employ a polarizer after the sample. The light travels at 180 degrees from the sample directly into the (absorbance) detector. No polarizer is used in this position.
Circularly polarized luminescence employs the linear polarizer after the sample to analyze the polarization from the sample. CPL = emission(L) - emission(R). “CPL provides the differential emission intensity of right and left circularly polarized light, thereby providing information on the excited state properties of the chiral molecular systems.”² A polarizer is always in this position.
The detection hardware is external to the Polarization Toolbox.
Circular dichroism employs a high speed, high sensitivity photomultiplier tube, which sends its raw abs(L) and abs(R) tagged photons to our 16 bit A/D converter card and OLIS software.
Circularly polarized luminescence employs a high throughout single monochromator and an exquisitely sensitive gated photon counting detector for capturing and sorting the flu() and flu(R) signals, ultimately processed by the OLIS software. Because the polarization state of detected light is always horizontal, there is no effect of polarization (including Wood’s anomalies in the monochromator) on the intensity. Thus, no G-factor correction is required.
There are no moving parts in this module because neither polarizer has to be rotated during the experiment.
¹ CPL is a technique used to characterize transition metal ligand complexes (a signal is seen only when coordinated to a chiral ligand), trivalent lanthanide complexes, and chiral lactones. In addition, bio-macromolecular molecules have been probed by using Tb3+[ref], dansyl, acridine, and intrinsic tryptophan. These probes, particularly tryptophan, are sensitive to tertiary structure. Unfolded proteins exhibit zero CPL signal.
² https://pubs.acs.org/doi/10.1021/acs.jpclett.5b01452
Further Reading:
DEVICE FOR ENABLING SLOW AND DIRECT MEASUREMENT OF FLUORESCENCE POLARIZATION
US Patent 6,970.241 B1, assigned to Richard J. De Sa
Recording Anisotropy Spectrometer -- A Unique Application of Piezoelectric Birefringence Modulation
John E. Wampler and Richard J. De Sa, Analytical Chemistry, 46,563 (1974)
An On-Line Spectrofluorimeter System for Rapid Collection of Absolute Luminescence Spectra.
Wampler and De Sa, Applied Spectroscopy, 25, No. 6, 623-627 (1971).
