Fig. 1 Schematic drawing of the SPIM sample chamber. The sample chamber can contain both, illumination and detection, objectives as shown here. In our case only the water dipping detection objective was contained in the sample chamber while the light sheet from the illumination objective was coupled over a thin glass window into the sample chamber. The particular setup can be adapted to the NA required for the application. The sample chamber itself is filled with aqueous solution which is chosen according to the nature of the sample. The yellow 3D specimen, held in agarose by a capillary coming from the top (capillary is not shown for clarity), is positioned in the excitation light sheet (cyan). The emitted light (green) from the illuminated plane is collected by the detection objective and imaged over a tube lens on an EMCCD camera (camera not shown).

Fig. 2 (A) Comparison of the light sheet profiles of the 10x/0.2 and 20x/0.4 illumination objective lenses. An image of the light sheet was recorded with the EMCCD camera by placing a mirror at an angle of 45° into the focal plane of the detection objective (100x/1.0 W). The full widths at half maximum (FWHM) of the light sheets are 2.4 µm and 1.4 μm, respectively. The 1/e² radii are 1.9 µm and 1.4 μm, respectively. (B) Comparison of the normalized autocorrelation functions obtained with three different combinations of illumination and detection objectives. The measurement with the 10x/0.2 illumination objective was taken at an exposure time of 1 ms. The two measurements with the 20x/0.4 objective were taken with an exposure time of 5 ms. All three measurements result in similar diffusion coefficients between 0.9 μm²/s and 1.5 μm²/s. Using the same detection objective (100x/1.0 W) but different illumination objectives (10x/0.2 or 20x/0.4), the autocorrelation functions show little difference since diffusion along the z-direction contribute less significantly to the autocorrelation function than along the xy-directions. However, for different detection objectives (100x/1.0 W or 40x/0.75 W), but the same illumination objective (20x/0.4), the differences are significant since the detection objective influences the xy resolutions of the system. (C) The 0.2 μm bead sample is homogeneous. All 1024 ACFs are very similar. (D) The ACFs of the 1.0 μm bead sample allow us to distinguish single microspheres as well as aggregates. (E) Examples of the different ACFs for the 1.0 μm bead sample (solid gray lines) including their fits (black lines). (F) Comparison of ACFs for single 0.2 µm and 1.0 μm microspheres quantitate the different ACFs. The average diffusion coefficients are 1.1 ± 0.5 μm²/s and 0.28 ± 0.18 μm²/s for the two bead populations.

Fig. 3 Diffusion measurement of 0.2 μm microspheres at a water/agarose border. During the course of the experiment, the microspheres were injected into the aqueous medium. (A) Intensity image averaged over 10,000 frames. (B) Particle number N extracted from the single fits to each ACF of the 1343 pixels. (C) The diffusion coefficient D extracted from the fits to each ACF. (D) Diffusion coefficients of all 17 lines (each with 79 pixels). The diffusion coefficient D within the aqueous phase sample agree well while D decreases towards the water/agarose border (from left to right). Beyond the border (pixel position ~50), no consistent diffusion coefficients can be found due to a lack of correlations. (E-G) Depicted are all experimental ACFs for areas from position 0-20 (solution, E), 21-54 transition region from solution to agarose (F), and 55-79 (agarose, G). The average D for position 1-5 (solution) is D = 1.2 ± 0.1 μm²/s, while the average D for position 30-35 (transition region) is D = 0.23 ± 0.02 μm²/s due to the decrease in diffusion towards the water/agarose border. Beyond position 55 no correlations are discernible.

Fig. 4 SPIM-FCS measurements within a live zebrafish 48 hpf. Microspheres with adiameter of 0.2 μm were injected into the blood circulation 68 seconds prior to the start of the experiment. (A) Fast blood flow within a blood vessel. The ACFs are narrower and steeper compared to solution measurements since flow transports the molecules with a speed of about 60-170 μm/s through the observation volumes. (B) This figure shows cross-correlation functions (CCFs) between the central pixel of the 20 × 20 pixel ROI and the surrounding pixels at a distance of 3 pixels (same experiments as A). A prominent peak in the CCF confirms the transport of particles from the central pixel to the surrounding pixels. The development of the peak is direction dependent, with the flow going from the green marked pixels to the central pixel in the direction of the red marked pixels, giving the blood flow profile. (C) The ACFs were been recorded close to the heart and show dominant peaks due to the heart beat, which is about 3 per second in this example as observed from the ACFs. They show little transport since flow is slow in the large vessels. (D) Comparison between measurements of 0.2 μm microspheres in solution and at different parts in the zebrafish.