Walsh Alex J, Sharick Joe T, Skala Melissa C, Beier Hope T
National Research Council, JBSA Fort Sam Houston, Texas, 78234, USA; 711th Human Performance Wing, Human Effectiveness Directorate, Bioeffects Division, Optical Radiation Bioeffects Branch, Air Force Research Lab, JBSA Fort Sam Houston, Texas, 78234, USA.
Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee, 37235, USA.
Biomed Opt Express. 2016 Mar 18;7(4):1385-99. doi: 10.1364/BOE.7.001385. eCollection 2016 Apr 1.
Time-correlated single photon counting (TCSPC) enables acquisition of fluorescence lifetime decays with high temporal resolution within the fluorescence decay. However, many thousands of photons per pixel are required for accurate lifetime decay curve representation, instrument response deconvolution, and lifetime estimation, particularly for two-component lifetimes. TCSPC imaging speed is inherently limited due to the single photon per laser pulse nature and low fluorescence event efficiencies (<10%) required to reduce bias towards short lifetimes. Here, simulated fluorescence lifetime decays are analyzed by SPCImage and SLIM Curve software to determine the limiting lifetime parameters and photon requirements of fluorescence lifetime decays that can be accurately fit. Data analysis techniques to improve fitting accuracy for low photon count data were evaluated. Temporal binning of the decays from 256 time bins to 42 time bins significantly (p<0.0001) improved fit accuracy in SPCImage and enabled accurate fits with low photon counts (as low as 700 photons/decay), a 6-fold reduction in required photons and therefore improvement in imaging speed. Additionally, reducing the number of free parameters in the fitting algorithm by fixing the lifetimes to known values significantly reduced the lifetime component error from 27.3% to 3.2% in SPCImage (p<0.0001) and from 50.6% to 4.2% in SLIM Curve (p<0.0001). Analysis of nicotinamide adenine dinucleotide-lactate dehydrogenase (NADH-LDH) solutions confirmed temporal binning of TCSPC data and a reduced number of free parameters improves exponential decay fit accuracy in SPCImage. Altogether, temporal binning (in SPCImage) and reduced free parameters are data analysis techniques that enable accurate lifetime estimation from low photon count data and enable TCSPC imaging speeds up to 6x and 300x faster, respectively, than traditional TCSPC analysis.
时间相关单光子计数（TCSPC）能够在荧光衰减过程中以高时间分辨率获取荧光寿命衰减。然而，要准确表示寿命衰减曲线、进行仪器响应解卷积和寿命估计，每个像素需要数千个光子，特别是对于双组分寿命而言。由于每个激光脉冲产生单个光子的特性以及为减少对短寿命的偏差所需的低荧光事件效率（<10%），TCSPC成像速度本质上受到限制。在此，通过SPCImage和SLIM Curve软件分析模拟的荧光寿命衰减，以确定能够被准确拟合的荧光寿命衰减的极限寿命参数和光子需求。评估了提高低光子计数数据拟合精度的数据分析技术。将衰减的时间分箱从256个时间箱减少到42个时间箱，在SPCImage中显著（p<0.0001）提高了拟合精度，并能够在低光子计数（低至700个光子/衰减）下进行准确拟合，所需光子数量减少了6倍，从而提高了成像速度。此外，通过将寿命固定为已知值来减少拟合算法中的自由参数数量，在SPCImage中显著将寿命分量误差从27.3%降低到3.2%（p<0.0001），在SLIM Curve中从50.6%降低到4.2%（p<0.0001）。烟酰胺腺嘌呤二核苷酸 - 乳酸脱氢酶（NADH - LDH）溶液的分析证实了TCSPC数据的时间分箱以及减少自由参数数量可提高SPCImage中指数衰减拟合的精度。总之，时间分箱（在SPCImage中）和减少自由参数是数据分析技术，能够从低光子计数数据中准确估计寿命，并使TCSPC成像速度分别比传统TCSPC分析快6倍和300倍。
