Orientation measurements of microsphere doublets and metaphase chromosomes in flow

Abstract

Obtaining information about the shape of particles from slit‐scan profiles is facilitated if the particles are oriented. Elongated particles orient in the nozzle of flow cytometers, but orientation may be disrupted before the particles get to the point of measurement. We have used our slit‐scan flow cytometer to investigate the orientation of microsphere doublets in a liquid jet in air, in flow across a glass surface, and in a 200‐μm‐square capillary tube as a function of distance from the flow chamber nozzle. Particles were classified as being oriented if there was a centrally located dip in the slit‐scan profile. Essentially all the doublets in the jet were oriented, and no disorientation was noted over the distances measured (up to 10 mm from the nozzle). Particle orientation was maintained for 80 μm in flow across a glass surface. In the capillary‐type flow chamber, essentially all of the particles were oriented at the tube entrance and for several millimeters into the tube. There then occurred a region where particle tumbling started and progressively fewer doublets met the orientation criteria. The distance to where tumbling began could be estimated by calculating the length required to establish the parabolic flow profile in the tube. Finally, the fraction of oriented particles reached a constant value that did not change with increased distance into the tube. When sample was injected off axis (i.e., half‐way between the chamber center and the chamber walls), particle tumbling began closer to the tube entrance.Orientation studies of Chinese hamster M3–1 metaphase chromosomes showed that over 95% of the larger chromosomes (1–5) and over 65% of the smaller chromosomes (9–11) were oriented near the entrance of the capillary tube.

Slit Scanning of Saccharomyces cerevisiae Cells: Quantification of Asymmetric Cell Division and Cell Cycle Progression in Asynchronous Culture

“…When the laser beam is positioned within this region, 80%-90% of the bead doublets are correctly oriented. This maximum percentage is consistent with that determined for other instruments (Lucas and Pinkel, 1986). Farther from the inlet, the orientation becomes random, giving a relatively constant fraction of correctly oriented doublets of about 50 % .…”

“…In fact, the value of slit scanning data would be restricted if one did not know the scanning axis of the object. Lucas and Pinkel (1986) have shown that a region exists in the flow cell of a flow cytometer in which the fluid flow is extensional, and in this region of accelerating fluid, objects with an aspect ratio greater than unity tend to align their longitudinal axis with the direction of flow. As the flow becomes fully developed, objects have a tendency t o tumble and thus take on a random orientation.…”

“…The first type of signal consisted of a unimodal signal that has been attributed to particles that are not oriented along their longitudinal axis. The second type of signal was a bimodal waveform characterized by the presence of a minimum between the two modes or by a shoulder with zero slope (Lucas and Pinkel, 1986). This type of signal is apparently due to doublets that are closely oriented along their longitudinal axis, and the fraction of bead doublets with this orientation was evaluated.…”

Instrument for real‐time pulse‐shape analysis of slit‐scan flow cytometry signals

“…In general, particles with dips in their fluorescence profiles have elongated shapes inducing them to align in the direction of flow (17). This process facilitates the slit-scanning analysis of particles such as sperm (3,111, cell aggregates (21), and chromosomes (1,2,5,6,9,12,18,19).…”

Flow cytometric analysis using digital signal processing

“…Previous reports of work in this area are quite limited perhaps as a consequence of the high cost and performance limitations of early DSP systems. Examples include the slit-scan flow cytometric analysis of chromosomes (3,15,(17)(18)(19)(20)28,29,32), microsphere doublets (19), and plankton (8,12). Calculations of the pulse waveform features have involved pulse width ( 16,20,24, 3 1 ), the area-based centromeric index (3,15,17,IS), the depth of the dip in a pulse waveform (19), the banding pattern contained within a pulse waveform ( 17,18), and the pulse integral and time of flight (8,12).…”