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Wave front engineering for microscopy of living cells

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A new method to perform simultaneously three dimensional optical sectioning and optical manipulation is presented. The system combines a multi trap optical tweezers with a video microscope to enable axial scanning of living cells while maintaining the trapping configuration at a fixed position. This is achieved compensating the axial movement of the objective by shaping the wave front of the trapping beam with properly diffractive optical elements displayed on a computer controlled spatial light modulator. Our method has been validated in three different experimental configurations. In the first, we decouple the position of a trapping plane from the axial movements of the objective and perform optical sectioning of a circle of beads kept on a fixed plane. In a second experiment, we extend the method to living cell microscopy by showing that mechanical constraints can be applied on the dorsal surface of a cell whilst performing its fluorescence optical sectioning. In the third experiment, we trapped beads in a three dimensional geometry and perform, always through the same objective, an axial scan of the volume delimited by the beads.

©2005 Optical Society of America

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Supplementary Material (3)

Media 1: AVI (1622 KB)     
Media 2: AVI (3146 KB)     
Media 3: AVI (876 KB)     

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Figures (6)

Fig. 1.
Fig. 1. (a) Schematic of the trapping (red) and the imaging (green) beam paths. L - laser, SLM - spatial light modulator, DM - infrared dichroic mirror, MO - microscope objective, TL - tube lens, T - telescope; (b) Calibration curve which relates the SLM focal length, fSLM, to the axial position, z, of the trapping plane.
Fig. 2.
Fig. 2. Transmission image of a 3D structure of beads trapped in three planes at an axial distance of 2 µm. The imaging plane is adjusted at z1=9.6µm, so that the intermediate plane of the structure results in focus.
Fig. 3.
Fig. 3. Relative positions (not in scale) of the trapping, imaging and objective planes with respect to the coverslip and the objective focal plane during an axial scan.
Fig. 4.
Fig. 4. (a)–(f) Selected sequence from a 3D DIC axial scan with a step of 200nm of 6 beads trapped by the laser. The imaging plane position, zIP , in respect to the coverslip and the trapping plane position, z, which compensate for the objective movements, are indicated in the figure. As a results of the compensation, the circle is kept at a fixed high, z 0=4.6 µm, from the coverslip. For the whole optical sectioning see Multimedia file 1. (1622 KB)
Fig. 5.
Fig. 5. Sequence of (a) transmission and (b) fluorescence images (step of 1µm) acquired during an axial scan where a circle of 6 beads is positioned and held on the dorsal cortex of a HeLa cell. The beads are kept at a fixed position while the objective scans a range of 8.2 µm, at step of 200 nm. Fluorescence images have been de-convoluted as explained in the text. For the whole fluorescence sectioning see Multimedia file 2 (3146 KB)
Fig. 6.
Fig. 6. (a)–(f) Selection of 6 cross sections, at step of 1 µm, of an optical sectioning of the 3D structure shown in Fig. 2. The positions, zIP , of the image plane in respect to the coverslip are indicated in the figure. In the original scan images are taken form zIP =12.6 µm to zIP =7.4 µm a step of 200 nm. For the whole optical sectioning see Multimedia file 3 (877 KB)

Equations (3)

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zz * = f MO 2
z * = f SLM + f MO d .
f SLM = d f MO f MO 2 z ,
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