Video-rate volumetric neuronal imaging using 3D targeted illumination

Abstract

Fast volumetric microscopy is required to monitor large-scale neural ensembles with high spatio-temporal resolution. Widefield fluorescence microscopy can image large 2D fields of view at high resolution and speed while remaining simple and costeffective. A focal sweep add-on can further extend the capacity of widefield microscopy by enabling extended-depth-of-field (EDOF) imaging, but suffers from an inability to reject out-of-focus fluorescence background. Here, by using a digital micromirror device to target only in-focus sample features, we perform EDOF imaging with greatly enhanced contrast and signal-to-noise ratio, while reducing the light dosage delivered to the sample. Image quality is further improved by the application of a robust deconvolution algorithm. We demonstrate the advantages of our technique for in vivo calcium imaging in the mouse brain.

Figure 1

Experimental setup. f₁ = 150 mm, f₂ = 100 mm, f₃ = 150 mm, f₄ = 125 mm. Obj: objective. DM: dichromatic mirror. Additional excitation and emission filters are not shown.

Figure 2

(a) Schematic of our method. (c) Images of fluorescent tissue paper acquired at different depths with UI. (d) Images of the same sample acquired with TI at the same depths. The associated TI patterns are shown in (b). (e) is the summation of all the TI patterns from (e) over a full focal scan. (f) is single shot EDOF image obtained with uniform illumination. (g) is the single-shot EDOF image obtained with TI using the binary patterns shown in (b). Plots depict the intensity profiles along the yellow dashed line in each image. Scale bar, 50 μm.

Figure 3

Images of a 300 μm thick brain slice at z = 10 μm, 60 μm and 110 μm acquired with UI (a), TI (b) and confocal microscopy (c). (d) and (e) are EDOF images spanning a 150 μm axial range acquired in a single shot with 40 ms exposure time with UI and TI respectively. (f) is the confocal EDOF image spanning the same 3D FOV. (g) and (h) are the deconvolved images of (d) and (e). The yellow arrow indicates a neuron that is almost indistinguishable from background in the deconvolved UI-EDOF image, but is clearly apparent in the deconvolved TI-EDOF image. (i,j) Contrast and CNR comparisons of UI- and TI-EDOF images, before (blue dots) and after (red dots) deconvolution. Gray lines indicate when the two cases have equal contrast/CNR. Scale bar, 50 μm.

Figure 4

Influence of TI fill factor F on TI-EDOF images. From (a–e), TI-EDOF images when TI fill factor is 1.00, 0.51, 0.15, 0.03, 0.006 respectively. (f) is the confocal EDOF image spanning the same 3D FOV. (g) Contrast and (h) CNR of three cells as a function of TI fill factor. Cells marked in (d) with colored arrows are represented in (g,h) with the same colored lines. Markers on plots indicate measured data points. Scale bars in (a–f) are 50 μm.

Figure 5

One frame from the videos acquired with UI (a) and TI (c). (b) and (d) are the deconvolved images of (a) and (c), respectively. The imaging volume is approximately 500 × 500 × 200 μm. (e,f) Calcium traces of 40 neurons obtained from the deconvolved UI and TI videos. Arrows indicate times when motion artifacts occur (see Supplementary Information Fig. S8). (g) Expanded view of the calcium transients from the red shaded region in (e,f). Gray dashed line indicates ΔF/F = 0. (h,i) Comparison of ΔF/F peak signal (h) and SNR (i) acquired with UI and TI, without (blue dots) and with (red dots) deconvolution. (j) Maximum projection image of two neighboring neurons from the rectangular box region in (a–d). From top to bottom: UI, deconvolved UI, TI, deconvolved TI. The calcium traces of these two neurons are shown in (k,l), which correspond to the gray shaded calcium traces in (e,f). Gray shaded region in (k,l) illustrate when signal crosstalk present in deconvolved UI is reduced or eliminated with deconvolved TI. (m) Histogram of correlation coefficient between pairs of neurons in deconvolved UI-EDOF video (red) and deconvolved TI video (green). Scale bars in (a–d) are 50 μm. (The corresponding calcium traces in (e,f,k,l) and histogram of correlation coefficient in (m) before frame-by-frame deconvolution are shown in Supplementary Information).

Figure 6

One frame from a video acquired with (a) TI-EDOF and (b) UI-EDOF imaging across 500 × 500 × 100 μm FOV. (c) Deconvolved frame of (a). (d) Calcium traces of from the raw video (blue line) and frame-by-frame decon-volved video (red line). The positions of the associated neurons are indicated in (a) and (b). (e) 10 out of 20 TI patterns used for imaging. (f) Synchronization scheme. Scale bars in (a–c) are 50 μm.