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Journal of the Optical Society of America

Journal of the Optical Society of America

  • Vol. 64, Iss. 7 — Jul. 1, 1974
  • pp: 903–918

Dichroic microspectrophotometer: A computer-assisted, rapid, wavelength-scanning photometer for measuring linear dichroism in single cells

Ferenc I. Hárosi and Edward F. MacNichol, Jr.  »View Author Affiliations


JOSA, Vol. 64, Issue 7, pp. 903-918 (1974)
http://dx.doi.org/10.1364/JOSA.64.000903


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Abstract

A novel microspectrophotometer is described, which simultaneously resolves cell absorption into two mutually orthogonal components allowing the determination of linear dichroism as a function of wavelength in the range of 325–695 nm. This instrument uses a single plane-polarized light beam and a small general-purpose digital computer, and is equipped with a photo-flash apparatus for rapid photolysis. Following visual pigment bleaching, it can detect changes occurring on a time scale of seconds in the orientation and spectral character of chromophores in isolated cells. The spectral scanning is performed in either single or multiple sweeps which may be unidrectional or bidirectional. The scanning rate is set to 500 nm/s. Spectral resolution is 5 nm. Its signal and data processing are discussed. Its performance is illustrated on subcellular organelles of retinal photoreceptors from turtle and frog. Rhodopsin and its photoproducts are shown to lend dichroism to frog rod outer segments. Metarhodopsin II, when formed, is transversely dichroic as rhodopsin. The late products (retinol, retainal oxime, etc.) show axial dichroism. The corrected specific optical density (transverse component) of frog rod outer segments (in hydroxylamine) is found to be 0.0182±0.002/ µm. The average absorption spectrum is presented for in situ rhodopsin.

© 1974 Optical Society of America

Citation
Ferenc I. Hárosi and Edward F. MacNichol, Jr., "Dichroic microspectrophotometer: A computer-assisted, rapid, wavelength-scanning photometer for measuring linear dichroism in single cells," J. Opt. Soc. Am. 64, 903-918 (1974)
http://www.opticsinfobase.org/josa/abstract.cfm?URI=josa-64-7-903


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References

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  25. With this statement, we imply that long-term fluctuations are either non-existent in the DMSP, or that their effects are detectable so that all records subject to drift may be discarded. Because of its permanent memory, the DMSP is well suited to test for slow drifts arising from any source in the instrument by computer comparison of reference transmittances recorded at different times during an experiment. The acceptance or rejection of records we decide by our selection rule (Ref. 33), which states that a spectral recording may be regarded free of distortions if the absorptance trace falls within about ±1% of the zero line, before as well as after photolysis, in spectral regions where no absorption is expected. This procedure sets also the limit on the error that may result from the reference being a clear area in the preparation instead of the cell when devoid of pigment. These issues are discussed in more detail in Ref. 33.
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  30. The effective bandwidth of 18 Hz is obtained with reference to a single-time-constant (τ) integrator (low-pass filter) for which the upper corner frequency (ƒc) is given by the relationship 2π ƒo = 1/τ. The DMSP is operated such that a 5-nm-wide spectral segment is swept in 10 ms. Since about 90% of this time is spent for summing repeated samples of the same signal (120 times on each channel), the integration time is about 9 ms. If τ=9×10-3 s, ƒc≅18 Hz. Note that a simple low-pass filter with τ=RC has a slow roll-off characteristic (20 dB/decade) and hence a broader bandwidth than the ƒc=1/2πτ formula indicates. Digital summation, on the other hand, approaches ideal integration by virtue of its high speed and the lack of phase distortion. Because of these, it is possible to process in the DMSP the separated fast modulation and the slowly varying average signal with essentially no phaseshift between them (except for a fixed lag of about 35 µs due to multiplexing the two analog channels).
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  35. According to H. Shichi, Biochem. 9, 1973 (1970), the density ratio of the β band peak to the α band peak is 0.29 for extracted, purified cattle rhodopsin. In the publication of H. Shichi, M. S. Lewis, F. Irreverre, and A. L. Stone, J. Biol. Chem. 244, 529 (1969), the ratio of molar extinction coefficients of the β band and the α band is 0.266 for pure cattle rhodopsin. On the other hand, the same ratio obtained from the same preparation by H. Shichi appears to be close to 0.21, in one of his more-recent records that he provided for us. Our choice of 0.25 for this figure represents not only an average to the above numbers, but also our best estimate of the density ratio of the β and α band peaks for in situ frog rhodopsin.
  36. The assumption, that the excess density measured at 370 nm over the expected density of rhodopsin is due to retinal oxime, should be valid only if other photoproducts do not persist long enough to interfere with the measurement. Although we can detect the formation of some Meta III, and the brief presence of some Meta II, at 5- and 10-mM hydroxylamine concentrations, at 75-mM concentration of this reagent the oxime formation appears to be faster than the time resolution of the DMSP.
  37. On the basis of a theoretical analysis by Hárosi and Malerba we derived an equation that relates the different absorbing states of the same cell in terms of molar extinction coefficients (∊′, 2245″), molecular extinctions (M′, M″), cellular optical density components (D11′, D11″), and dichroic ratios (R′=D˔′/D11′, R″=D˔″/D11″) as [Equation] Coefficient b is related to the numerical aperture (NA) of the microscope condenser (with aperture cone 2α) as b=tan2(α/2). For an oil-immersion-type condenser (n=1.455) of (NA)=0.4, b=0.0197, and thus [Equation] We use this equation to compute photoproduct densities from presumed molar extinction coefficients, or vice versa, and assume unchanging pigment concentrations and path lengths of measuring light within the cell.
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