Models for low resolution slit scan measurements based on high resolution laser scanning image analysis: DNA and nuclear dimensions

doi:10.1016/0031-3203(81)90031-5

Pattern Recognition

Volume 13, Issue 1, 1981, Pages 49-55

https://doi.org/10.1016/0031-3203(81)90031-5 Get rights and content

Abstract

A laser scanning microscope has been developed and employed to make high precision measurements of cells from clinical cervical samples. These specimens have been stained with the DNA specific fluorochrome mithramycin and the fluorescent images analyzed to give information on nuclear size, DNA content and nuclear shape. This information has been used to develop low resolution parameters as measured with flow cytometers, thus permitting some comparisons between high resolution and low resolution features as measured from the same cells. The results indicate that both low resolution flow slit scan and high resolution pattern recognition techniques yield the same average cellular information. However, low resolution methods are insensitive to shape and the influence of shape on the broadening of low resolution features is now under investigation.

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Citation Excerpt :
Combining with image storage reduces the specimen irradiation to an absolute minimum. Scanning fluorescence microscopes have been reported by Mellors and Silver (1951), Loeser and West (1962), Slomba, Wasserman, Kaufman, and Nester (1972), and Mullaney et al. (1981). A point-source fluorescent microscope has the spatial frequency bandwidth of a conventional incoherent microscope operating at the primary wavelength.
Microscopy using a scanning approach is reviewed, with numerous references to previous work, as at 1987, before the confocal laser scanning microscope was widely available commercially. The advantages of microscope imaging using a scanning technique are described. The principles of confocal microscopy are introduced. Use of Bessel beam illumination in a confocal microscope is discussed. This combination can increase spatial resolution while retaining some confocal optical sectioning. The advantages of imaging using a scanning method with a point source are detailed. For example, differential phase contrast may be performed to obtain phase information. Use of a point source allows different types of spectroscopy to be performed in a spatially resolved fashion. An advantage of point-source systems for such applications is that imaging, performed with the incident radiation, is separated from wavelength selection and analysis of the emitted radiation, thus simplifying system design and resulting in superior performance. Use of a point source to excite carriers in a semiconducting sample allows the electronic properties of materials and devices to be investigated. Advantages of object scanning, rather than beam scanning, are discussed. Advantages of using a laser source are described, including the use of the heterodyne technique as a synthetic confocal pinhole, and nonlinear microscopy such as two-photon fluorescence and CARS (coherent anti-Stokes Raman spectroscopy). It should be noted that 1987, as well as being before commercial confocal microscopes were widely available, was before the special properties of Bessel beams were appreciated, and before two photon fluorescence microscopy was first experimentally demonstrated.
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^☆: This work performed at Battelle Institute while P. Mullaney was on sabbatical from LASL.

^†: Present address: Biology Division, Oak Ridge National Laboratory, Box Y, Oak Ridge, TN 37830, U.S.A.

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Models for low resolution slit scan measurements based on high resolution laser scanning image analysis: DNA and nuclear dimensions☆

Abstract

Rapid staining methods for analysis of DNA and protein in mammalian cells

J. Histochem. Cytochem.

Cell cycle analysis in 20 minutes

Science

Technique of impulse cytophotometry of cervical and vaginal smears

Signal analysis of slit scan contour data

J. Histochem. Cytochem.

Computer Analysis of cervical cells

Uniform lateral orientation, caused by flow forces of flat particles in flow-through systems

J. Histochem. Cytochem.

Laser stroboscopic photography: technique for cell orientation

Studies in Flow.

Imaging system for correlating fluorescence cell measurements in flow

SPIE

Improved acriflavine feulgen reagent for wuantitative DNA cytofluorometry

J. Histochem. Cytochem.

Estimation of cell size from pulse shape in flow cytophotometry

J. Histochem. Cytochem.