van Munster E B, Goedhart J, Kremers G J, Manders E M M, Gadella T W J
Section Molecular Cytology, Swammerdam Institute for Life Sciences, Centre for Advanced Microscopy, University of Amsterdam, Kruislaan 316, 1098 SM, Amsterdam, The Netherlands.
Cytometry A. 2007 Apr;71(4):207-14. doi: 10.1002/cyto.a.20379.
Wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM) is an established technique to determine fluorescence lifetimes. Disadvantage of wide-field imaging is that measurements are compromised by out-of-focus blur. Conventional scanning confocal typically means long acquisition times and more photo bleaching. An alternative is spinning-disc confocal whereby samples are scanned simultaneously by thousands of pinholes, resulting in a virtually instantaneous image with more than tenfold reduced photo bleaching.
A spinning disc unit was integrated into an existing FLIM system. Measurements were made of fluorescent beads with a lifetime of 2.2 ns against a 5.3 ns fluorescent background outside the focal plane. In addition, living HeLa cells were imaged with different lifetimes in the cytosol and the plasma membrane.
In spinning-disc mode, a lifetime of the beads of 2.8 ns was measured, whereas in wide field a lifetime of 4.1 ns was measured. Lifetime contrast within living HeLa cells could be resolved with the spinning-disc unit, where this was impossible in wide field.
Integration of a spinning-disc unit into a frequency-domain FLIM instrument considerably reduces artifacts, while maintaining the advantages of wide field. For FLIM on objects with 3D lifetime structure, spinning-disc is by far preferable over wide-field measurements.
宽场频域荧光寿命成像显微镜（FLIM）是一种用于确定荧光寿命的成熟技术。宽场成像的缺点是测量会受到离焦模糊的影响。传统的扫描共聚焦通常意味着采集时间长且光漂白更严重。另一种选择是转盘共聚焦，通过数千个针孔同时对样品进行扫描，从而得到几乎瞬间的图像，且光漂白减少了十倍以上。
将一个转盘单元集成到现有的FLIM系统中。针对焦平面外5.3 ns的荧光背景，对寿命为2.2 ns的荧光珠进行测量。此外，对胞质溶胶和质膜中具有不同寿命的活HeLa细胞进行成像。
在转盘模式下，测得珠子的寿命为2.8 ns，而在宽场模式下测得的寿命为4.1 ns。利用转盘单元可以分辨活HeLa细胞内的寿命对比度，而在宽场模式下则无法做到。
将转盘单元集成到频域FLIM仪器中可显著减少伪影，同时保留宽场的优点。对于具有三维寿命结构的物体进行FLIM成像，转盘模式远比宽场测量更可取。
