assembly

With the laser turned on, The beam can be approximately aimed at the flow cell capillary usin the steering prisms and focuing optic, in front of the laser output.

The sheath fluid, sheath air, sample air and waste lines are then connected to the console and then the flow cell and the sheath and waste tanks. The sheath tank is filled with water, and the tank pressurized. Sheath fluid will then be pushed to the flow cell, resulting in dripping at the SIP tube. A sample tube containing Polyscience 6 micron beads is then inserted onto the SIP tube.

The sample or core stream containing fluorescent beads should then be hydrodynamically focused within the confined of the sheath flow. A ntoch filter blocking 488 nm laser light is then inserted in front of the camera, blocking 488 nm laser light but allowing the beads to be visualized. the cameraThe laser beam is then aligned to intercept the beam stream. If the beam is not focused, a wide vertical area will be iluminated. Focusing the beam by adjusting the focusing optic allows a small spot to be illuminated, maximizing resolution and excitation.

If the sheath pressure is turned off, hydrodynamic focusing is lost and the beads fill the entire flow cell. A dramatic example of the importance of sheath flow and hydrodynamic focusing!

With the laser roughly aligned, the dichroics and bandpass filters are then installed. This instrument has a side scatter detector in the orthagonal path, and two fluorescence detectors for green and orange fluorochromes. 510 nm and 560 nm longpass dichroics separate the signals, and 488/10 nm, 530/30 nm and 585/26 nm bandpass filters further refine the side scatter, green and orange fluorescent signals.

Schematic of instrument fluidics and air pressure, showing the sheath fluid line (blue), the sheath and sample air lines (green) and the waste line (orange).