Lawrence William G, Varadi Gyula, Entine Gerald, Podniesinski Edward, Wallace Paul K
Department of Research, Radiation Monitoring Devices Inc., 44 Hunt St., Watertown, Massachusetts 02472, USA.
Cytometry A. 2008 Aug;73(8):767-76. doi: 10.1002/cyto.a.20595.
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.
多色流式细胞术能够利用多个荧光参数详细鉴定细胞表型。当前仪器中用于检测荧光的光电倍增管（PMT）限制了长波长光谱范围内的灵敏度。我们展示了硅雪崩光电二极管（APD）在流式细胞术方面的应用，其具有更高的红色灵敏度以及超过1000nm的有效荧光检测范围。使用脉冲发光二极管光源、校准测试微珠和生物样本对APD和PMT的波长依赖性性能进行了比较。构建了一个简易流式细胞仪测试平台来比较PMT和APD探测器的性能。APD使用了一个额外的放大级来匹配PMT的内部增益。在500 - 1060nm光谱范围内，使用脉冲发光二极管光源比较了APD和PMT在流式细胞术应用中的分辨率。这些测量结果显示了APD和PMT在宽光谱范围内信噪比性能的相对变化。在530 - 800nm波长范围内，使用APD和PMT同时检测标记有抗体偶联藻红蛋白或800nm量子点的CD4阳性细胞。在可见光波长下，两种探测器检测到的CD4阴性和CD4阳性群体强度之比相似，但只有APD能够在800nm以上波长分离这些群体。这些测量结果说明了APD和PMT在不同波长和信号强度水平下的性能差异。虽然APD和PMT在可见光谱范围内显示出相似的信噪比性能，但APD探测器的暗噪声在低信号水平下会降低灵敏度。在波长大于650nm时，APD的高量子效率有助于获得更好的信噪比性能。APD探测器在长波长区域提供了增强的性能，可用于将流式细胞仪的工作范围扩展到1000nm以上。
