Gary C. Salzman

Automated single-cell manipulation and sorting by light trapping

Following the recently reported trapping of biological particles by finely focused laser beams, we report on the automated micromanipulation of cells and other microscopic particles by purely optical means as well as on a newly observed interaction between particles in the trapping beam. A simple instrument is described which allows single cells to be positioned with high accuracy, transported over several millimeters, and automatically sorted on the basis of their optical properties. These operations are performed inside a small enclosed chamber without mechanical contact or significant fluid flow. Potential applications of this technique in experimental cell biology are discussed.

Differential light scattering photometer for rapid analysis of single particles in flow

A differential light scattering photometer has been developed for rapid size analysis of single particles in flow. A fluid stream carrying individual particles in single file intersects a focused laser beam at the primary focal point of an annular strip of an ellipsoidal reflector situated in a scattering chamber. The light scattered from polar angles theta = 2.5-177.5 degrees at azimuthal angles phi = 0 and 180 degrees , spanning a circle of 355 degrees , is reflected onto a circular array of 60 photodiodes. The signal processing electronics and computer storage can accept 32 signals/particle at rates up to 1000 particles/sec. Photometer performance is tested by comparing measured responses from individual spherical particles with angular scattering patterns calculated for the particular detector geometry. These patterns exhibit the required symmetry in the two half scattering planes. Response measurements for eight samples with particle diameters of 1.1, 2.7, 5.0, 7.9, 10.0, 12.5, 15.6, and 19.5 microm are consistent with calculated size-response curves. The composition of a mixture of five components with particle diameters of 1.1, 5.0, 10.0, 15.6, and 19.5 Am is determined from an analysis of light scattering measurements at various forward-scattering angles.

Proposed new data file standard for flow cytometry, version FCS 3.0

In 1984, the first flow cytometry data file format was proposed as Flow Cytometry Standard 1.0 (FCS1.0). FCS 1.0 provided a uniform file format allowing data acquired on one computer to be correctly read and interpreted on other computers running a variety of operating systems. That standard was modified in 1990 and adopted by the Society of Analytical Cytology as FCS 2.0. Here, we report on an update of the FCS 2.0 standard which we propose to designate FCS 3.0. We have retained the basic four segment structure of earlier versions (HEADER, TEXT, DATA and ANALYSIS) in order to maintain analysis software compatibility, where possible. The changes described in this proposal include a method to collect files larger than 100 megabytes (not possible in earlier versions of the standard), the inclusion of international characters in the TEXT portions of the file, a method of verifying data integrity using a 16-bit cyclic redundancy check, and increased keyword support for cluster analysis and time acquisition. This report summarizes the work of the ISAC Data File Standards Committee. The complete and detailed FCS 3.0 standard is available through the ISAC office [Sherwood Group, 60 Revere Drive, Ste 500, Northbrook, IL 60062, phone: (847) 480-9080 ext. 231, fax: (847) 480-9282, E-mail: isac@sherwood-group.com] or through the internet at the ISAC WWW site, http://nucleus.immunol.washington.edu/ISAC.ht ml.

Evaluation of the scattering matrix of an arbitrary particle using the coupled dipole approximation

The coupled dipole approximation is applied to the calculation of the scattering matrix of an arbitrary particle. Both isotropic and anisotropic dipoles are used in the calculations. The forms of the matrices obtained for several types of scatterers are found to be in exact agreement with those predicted by symmetry. The method is tested quantitatively by comparison with Mie predictions for solid and coated spheres and good agreement is observed. This comparison is also used to establish appropriate magnitudes for anisotropic dipolar polarizabilities.

The scattering matrix for randomly oriented particles

An efficient numerical method is derived for evaluation of the scattering properties of randomly distributed particles described by the coupled dipole approximation. An exact analytic average for these properties is also derived. All elements of the scattering matrix for a collection of randomly oriented particles can be obtained by either method. The results are applicable to model calculations of the scattering matrix for realistic particles. The analytic average also allows qualitative interpretation of the dependence of the matrix elements on dipolar interactions.

Light Scatter: Detection and Usage

Light scatter is one of the most basic of measurements made in flow cytometry. This unit presents a short commentary on what light scatter is and how it is measured. First, the author describes the basic principles of the scattering of light from small particles such as cells. This is followed by a discussion of methods for detecting scattered light in a flow cytometer. The unit ends with a brief survey showing how light scattering is used in a broad range of flow cytometry applications.

Polarizabilities for light scattering from chiral particles

The coupled dipole method is used to calculate the circular intensity differential scattering (CIDS) from chiral particles. The particles are described by a collection of spherical or ellipsoidal dipoles. In the case of ellipsoidal dipoles, it is shown that triaxial polarizabilities have to be used to quantitatively describe the scattering matrix of the particle. For certain collections of ellipsoidal dipoles, the dipolar interactions may be neglected in calculating CIDS from the structure. The coupled dipole approximation also provides a convenient method for calculating CIDS from a one dimensional helical crystal.

Phase differential scattering from microspheres

Differential phase measurements on scattered light are possible using the two-frequency Zeeman effect laser. We refer to such measurements as phase differential scattering (PDS) in contrast to conventional intensity differential scattering measurements. PDS has certain significant experimental advantages for light scattering studies, most notably its simplicity. We find good agreement between experiment and theory for PDS from aqueous suspensions of polystyrene microspheres. The data show a strong dependence on concentration when the microspheres are larger than dipoles.

Circular intensity differential scattering of light by hierarchical molecular structures

We show that circular intensity differential scattering (CIDS) of light by macromolecules with different levels of chiral structure is the superposition of the CIDS from each individual level. As an example, we treat a model superhelix with anisotropic polarizability.

High speed single particle sizing by light scattering in a flow system

We present two approaches to rapid, single particle sizing for particles in the I to 20 μm diameter range. One method measures multiangle scattered light over a polar angular range of nearly 360 degrees. A second method is based on the analysis of the pulse shapes from small angle forward scattered light. In both cases the particles in liquid suspension are made to pass one at a time through a focused laser beam for analysis.