Multiphoton excitation spectra in biological samples
Multiphoton excitation spectra in biological samples
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DOI：
10.1117/1.1583734
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发表时间：
2003-07-01
影响因子：
3.5
通讯作者：
Fraser, SE
中科院分区：
文献类型：
--
作者：
Dickinson, ME;Simbuerger, E;Fraser, SE
多光子显微镜正在成为活细胞和固定细胞成像的流行模式。这种成像模式提供了几个优点，因为荧光染料激发是一种非线性事件，导致仅在焦平面处激发。多光子激发通过使用在近红外发射的超快激光来增强，提供更好的深度穿透以及有效的激发。因为这些激光器，如钛：蓝宝石激光器，提供可调谐输出，所以可以使用它们来收集多光子激发光谱。我们使用软件可调谐相干变色龙激光器耦合到蔡司LSM 510 Meta NLO在多个激发波长下获取生物样品的x-y图像，创建激发λ堆栈。相对于激发波长绘制的图像内像素的平均强度揭示了激发光谱。可以使用线性解混算法将激发λ叠层分离成对应于来自不同染料的信号的单独图像，其方式与使用Meta检测器可以使用发射指纹识别来从发射λ叠层生成无串扰通道的方式大致相同。我们展示了如何使用这种技术来消除自发荧光，并产生无串扰的图像，染料与非常接近的重叠，在他们的发射光谱，不能使用发射指纹分离。此外，激发指纹可以使用非去扫描检测器（NDD）进行，提供更多的灵活性，以消除自发荧光或荧光染料之间的串扰时，成像样品深处。因此，激发指纹识别补充并扩展了由Meta检测器和发射指纹识别提供的功能。我们纠正激光和显微镜传输的偏差，以获得现实的多光子激发光谱荧光染料细胞内使用显微镜，这使得单和多标记实验的激发波长的优化，并提供了一种手段，用于研究生物环境对非线性激发的影响。(C)2003年，由光学仪器工程师学会（Society of Photo-Optical Instrumentation Engineers）主办。
Multiphoton microscopy is becoming a popular mode of live and fixed cell imaging. This mode of imaging offers several advantages due to the fact that fluorochrome excitation is a nonlinear event resulting in excitation only at the plane of focus. Multiphoton excitation is enhanced by the use of ultrafast lasers emitting in the near IR, offering better depth penetration coupled with efficient excitation. Because these lasers, such as titanium:sapphire lasers, offer tunable output it is possible to use them to collect multiphoton excitation spectra. We use the software-tunable Coherent Chameleon laser coupled to the Zeiss LSM 510 META NLO to acquire x-y images of biological samples at multiple excitation wavelengths, creating excitation lambda stacks. The mean intensity of pixels within the image plotted versus excitation wavelength reveals the excitation spectra. Excitation lambda stacks can be separated into individual images corresponding to the signal from different dyes using linear unmixing algorithms in much the same way that emission fingerprinting can be used to generate crosstalk free channels from emission lambda stacks using the META detector. We show how this technique can be used to eliminate autofluorescence and to produce crosstalk-free images of dyes with very close overlap in their emission spectra that cannot be separated using emission fingerprinting. Moreover, excitation fingerprinting can be performed using nondescanned detectors (NDDs), offering more flexibility for eliminating autofluorescence or crosstalk between fluorochromes when imaging deep within the sample. Thus, excitation fingerprinting complements and extends the functions offered by the META detector and emission fingerprinting. We correct biases in the laser and microscope transmission to acquire realistic multiphoton excitation spectra for fluorochromes within cells using the microscope, which enables the optimization of the excitation wavelength for single and multilabel experiments and provides a means for studying the influence of the biological environment on nonlinear excitation. (C) 2003 Society of Photo-Optical Instrumentation Engineers.