2.2.多線TPLSM中通過成像檢測釋放光
在單光束TPLSM中，光電倍增管PMT或者雪崩二極管APD可以很方便地用于釋放光檢測，由于雙光子激發的原理，激發只發生在激光焦點處。因此，用于屏蔽離焦光線的共焦小孔變得不必要，并且可以使用NDD檢測。這意味著激發光不會被送回掃描鏡，而是直接進入位于靠近物鏡處的具有大的活性面積的PMT。但是，這種簡單檢測模式在多線PTLSM中卻很難實現，因為必須要分離出每一個激光焦點發出的熒光。因此，我們首先設想了一種使用多APD的檢測模式在非掃描模式（descanned mode）下操作。這種檢測模式的靈敏性是陽性的，在僅用一個激光束和單個APD的預實驗中(Kalb et al., 2004).但是我們抑制了安裝額外多個APD用于多線檢測的想法，因為檢測器的調整非常關鍵。取而代之的是，受到芯片信號放大的超靈敏度CCD（所謂電子增強CCD）的啟示，我們決定在non-descanned mode下使用成像設備。在這種模式下，使用同步CCD讀出光束一次或多次掃過樣品來獲取整幅圖像。這相對于使用PMT或APD的點掃描檢測，它們在每個激光焦點位置取一個值，圖像通過將這些值與激光的位置進行關聯來重構。在散射性組織中，使用成像檢測取代點掃描檢測在一定程度上不可避免地降低了空間分辨率。如我們所示，多線TPLSM也可以進行點掃描檢測，但只能以遠低于成像檢測的時間分辨率進行（見本段后面內容和section3對兩中檢測模式進行關鍵評估）。
Fig. 2. Schematic of multiline TPLSM. Top: principle of the mirror-based beam multiplexer as first described by Nielsen et al. (2001). A linear array of in this case eight laser beams is generated by repeated separation at a 50%-beamsplitter mirror (BS) and reflection at high-reflectivity mirrors (HR). Bottom: schematic of the components in the setup for multiline TPLSM. The laser beam of a tunable Ti:sapphire laser passes a shutter, an attenuator, a beam telescope to adjust the size of the beam, and a dispersion compensation consisting of a pair of prisms. The latter prevents elongation of laser pulses by compensating for the group velocity dispersion introduced by the glass in the optical path of the laser beam. The laser beam then enters the beam multiplexer which divides the laser beam into 64 beams. In the commercially available version of the beam multiplexer (LaVision BioTec, Bielefeld) used now in our setup the maximal number of beams can be reduced by software-controlled replacement of part of the beamsplitter mirror with a high-reflectivity mirror. However, in order to generate a beam array with widely separated laser foci (as used for the recording shown in Fig. 3B), manual blocking of some of the beams had to be used. Scan movements of the beam array are controlled by nonresonant xy-galvo-scan mirrors. Before entering the microscope (Olympus IX70), a second beam telescope is passed. The microscope long-distance water-immersion 40×/N.A. 0.8 objective was mounted on a z-piezo to control the focal plane for the acquisition of image stacks. The fly is fixed with bees wax at a small glass plate and mounted on the microscope stage to control its xy-position. Moving patterns are presented in the visual field of the fly at high contrast and fast update rate with a custom-built LED board. The microscope was not equipped with conventional epifluorescence illumination. However, delivery of excitation light (mercury lamp band-pass filtered at 420–480 nm) to the sample via a lightguide (not shown in the diagram) turned out more practical than TPLSM to localize the dye-filled neuron at the beginning of an experiment. In the first case, a 465 nm beamsplitter and a 515 nm long-pass emission filter was used. In the case of TPLSM a 680 nm beamsplitter in combination with a TP-emission filter (short pass 700 nm) was used. An EMCCD camera (Andor Ixon DV887BI) was installed for the detection of emission light.
由于技術限制，直到現在，TPLSM中的成像檢測主要限制在要么提供高水平的發射光，要么不要求高的時間分辨率(see e.g. Bewersdorf et al., 1998). 為了獲取更高的可能靈敏度（進而到時間分辨率），即使在正常動物原位成像過程中的暗淡激發光情況下，我們使用了一個背景照明CCD，在500-650nm處量子效率>90%（例如，在一個包含了大多數鈣離子染料的激發峰的范圍內）和高達1000的可變電子倍增增益因子(Andor iXon DV887BI)(Coates et al., 2004).