Tunable Dynamic Speckle Generation for Random Illumination Microscopy

Abstract

Speckled illumination enhances widefield fluorescence microscopy by enabling optical sectioning and super resolution. In random illumination microscopy, sequences of speckled illumination patterns are used to excite fluorescent samples and images are reconstructed based on a statistical analysis of the intensity fluctuations. Although random illumination microscopy has been shown to give excellent performance, its widespread implementation is hindered by the high cost and complexity of the generation of suitable speckled illumination patterns, which is achieved using digital micro-mirror devices or spatial light modulators. Here, we present a zwitterion-doped liquid crystal (LC) device capable of generating independent, high-contrast speckle patterns with a tunable decorrelation time in the 0.1 s to 0.1 ms range under visible laser illumination. This LC-based dynamic speckle generator is applied to widefield random illumination fluorescence microscopy of tissue and cell samples, where it enables optical sectioning with a 2 micron axial resolution, and a 1.5-fold improvement in lateral spatial resolution. Owing to its low cost and simplicity, this LC speckle generator offers an attractive alternative to digital micro-mirror and spatial light modulator devices for implementing widefield random illumination microscopy.

Figures (6)

• Figure 1: Liquid crystal (LC) dynamic speckle generator.a) Images of the LC device in its OFF (no voltage applied) and ON (voltage applied) state. The active area is 10-by-10 mm. b) Experimental configuration used for imaging LC texture between crossed polarisers whilst applying an electric field across the LC layer. Additional details can be found in the experimental section. c) Images of the LC textures for varying strength and frequency of the alternating electric field. The arrows represent the orientation of the crossed polarisers. The mean domain size $\bar{r}$ was determined from the computed autocorrelation functions as detailed in the methods. All scale bars represent 25 µm.
• Figure 2: Dynamics of speckled illumination for widefield microscopy.a) Experimental setup. Speckled illumination is generated by a continuous laser ($\uplambda$=532 nm) which passes through the LC device followed by a thin diffuser which eliminates the zero-order. The LC is imaged on the back focal plane of the objective, generating a widefield speckled illumination. Fluorescence intensity is collected in the epi-configuration and captured by a scientific camera. b) Zoomed images of a thin, uniform fluorescent film under dynamic speckle illumination whilst operating the LC at 9 Vµm for two different frequencies over time. Scale bars are 2 µm. c) Decorrelation curves of speckle patterns at 9 Vµm for various values of $f$. The correlation coefficient $\gamma$ is computed between the first image and an image taken at time $t$. Black dashed lines indicate an exponential decay fit whose characteristic decay time is the decorrelation time $\tau$. d) Decorrelation time $\tau$ as a function of the applied electric field frequency and amplitude.
• Figure 3: Optical sectioning by dynamic speckle imaging (DSI)a) Schematic illustration of the excitation speckle field in green and the detection point spread function (PSF) in red. The two dashed lines indicate the focal plane (orange) and an out-of-focus plane (blue). b) Normalized images of speckle generated on a fluorescent thin film of the out-of-focus and in-focus planes. Scale bars are 5 µm. c) Demonstration of optical sectioning by acquiring image stacks at different z-positions. For each stack, we compute the average intensity image $\bar{I}$ and the standard deviation image $\sigma(I)$ and plot their spatial averages (indicated by $\langle$$\rangle$) for each z-position. The full width half maximum (FWHM) of $\langle\sigma(I)\rangle$ is indicated in grey.
• Figure 4: Demonstration of optical sectioning using DSI in a mouse intestinal jejunum labeled with rhodamine. A stack of images with different speckle illumination patterns is acquired (left column) with an exposure time $t_{exp} =$ 50 ms, whilst operating the LC at $\tau \approx$ 55 ms. A conventional widefield image is reconstructed by computing the average intensity $\bar{I}$ image of the stack (middle column). The standard deviation $\sigma(I)$ provides an optically sectioned image with improved contrast and resolution (right column). Scale bars are 50 µm for the large FOV and 20 µm for the zoomed images.
• Figure 5: High speed DSI imaging of fluorescent beads. Images are acquired at 1400 FPS, with $t_{exp} =$ 700 µm and $\tau \approx$ 2 ms. a) Standard deviation $\sigma(I)$ images based on a stacks with a different number frames $N$. Scale bars are 2.5 µm. b) Signal to noise ratio (SNR) of the generated $\sigma(I)$ image based on $N$ frames. The SNR is approximated as the ratio between the mean value and spatial standard deviation of each $\sigma(I)$ image. Dashed lines correspond to the images in a).