Enhanced red and near infrared detection in flow cytometry using avalanche photodiodes.

Polychromatic flow cytometry enables detailed identification of cell phenotype using multiple fluorescent parameters. The photomultiplier tubes (PMTs) used to detect fluorescence in current instruments limit the sensitivity in the long wavelength spectral range. We demonstrate the flow cytometric applications of silicon avalanche photodiodes (APDs), which have improved red sensitivity and a working fluorescence detection range beyond 1,000 nm. A comparison of the wavelength-dependent performance of the APD and PMT was carried out using pulsed light-emitting diode sources, calibrated test beads, and biological samples. A breadboard flow cytometer test bench was constructed to compare the performance of PMTs and APD detectors. The APD used an additional amplifier stage to match the internal gain of the PMT. The resolution of the APD and PMT was compared for flow cytometry applications using a pulsed light-emitting diode source over the 500-1060 nm spectral range. These measurements showed the relative changes in the signal-to-noise performance of the APD and PMT over a broad spectral range. Both the APD and PMTs were used to measure the signal-to-noise response for a set of six peak calibration beads over the 530-800 nm wavelength range. CD4-positive cells labeled with antibody-conjugated phycoerythrin or 800 nm quantum dots were identified by simultaneous detection using the APD and the PMT. The ratios of the intensities of the CD4- and CD4+ populations were found to be similar for both detectors in the visible wavelengths, but only the APD was able to separate these populations at wavelengths above 800 nm. These measurements illustrate the differences in APD and PMT performance at different wavelengths and signal intensity levels. While the APD and PMT show similar signal-to-noise performance in the visible spectral range, the dark noise of the APD detector reduces the sensitivity at low signal levels. At wavelengths longer than 650 nm, the high quantum efficiency of the APD contributes to better signal-to-noise performance. The APD detector provides enhanced performance in the long wavelength region and may be used to extend the working range of the flow cytometer beyond 1,000 nm.

Performance of a novel piezoelectric motor at 4.7 T: applications and initial tests.

The focus of this report was to test the performance of a novel piezoelectric motor under high magnetic field strength conditions and to investigate its potential applications in small animal magnetic resonance imaging (MRI). The device is made entirely of nonferrous materials and consists of four piezoelectric ceramic plates connected to a threaded metal tube through which a screw migrates. Ultrasonic vibrations of the threads inherent to the tube result in rotational and translational motion of the screw. Potential applications of the piezoelectric motor were investigated at 4.7 T. Firstly, phantom studies showed the motor was capable of accurately delivering low injection volumes ( approximately 0.01 ml). Dynamic contrast-enhanced MRI (DCE-MRI) studies performed in vivo using serially acquired T1-weighted, spin-echo imaging demonstrated the ability of the motor to reliably administer MR contrast-enhancing agent into live tumor-bearing mice without the introduction of image artifacts. In a second set of experiments, the motor allowed for controlled, dynamic repositioning of an anatomic slice of interest in a live animal to magnetic field isocenter, which resulted in reduced geometric distortion and image artifact due to improved radiofrequency and gradient field homogeneity. In conclusion, piezoelectric motors are MR compatible and offer great potential for improving MRI efficiency and throughput, particularly in a preclinical setting. Further investigation into applications such as automated capacitor tuning and impedance matching for MR transceiver coils is warranted.

Flow cytometer in the infrared: inexpensive modifications to a commercial instrument.

BACKGROUND: The application of molecules that fluoresce in the infrared (IR) region to measure cell products would be enhanced by a flow cytometer capable of measuring them. To our knowledge, none exist at this time. Accordingly, we have developed such an instrument. METHODS: A Becton Dickinson LSR flow cytometer was modified to include a small 785-nm IR diode laser the size of a C cell battery with 44-mW output power. The instrument was modified further to accommodate this laser in addition to a 405-nm solid-state laser, a 488-nm air-cooled argon laser, and a 658-nm solid-state laser. Because the IR laser is dangerous to the eye, the laser beams were viewed for optical alignment using a CCD camera and video monitor. An avalanche photodiode was used in place of a photomultiplier tube because its detection sensitivity in the IR region is superior. RESULTS: To assess performance, scatter and fluorescence measurements were made using microspheres that fluoresce in the IR region, and human leukocytes were stained with CD45 biotin followed by a streptavidin conjugated with an IR dye. An avalanche photodiode was 2.3 to 2.8 times more sensitive than a photomultiplier tube for detecting IR fluorescence. Cells stained with CD45 biotin and avidin conjugated with an IR dye could easily be resolved and their fluorescence quantified; there was virtually no autofluorescence. In addition, a lipophilic membrane dye that emits in the IR region was studied. HL60 cells were stained with this dye and they exhibited bright fluorescence intensity. CONCLUSION: A commercial instrument could be modified to accommodate an IR laser for exciting dyes that fluoresce in the IR region. This new capability will extend the range of fluorescence that can be measured by flow cytometry.