Figure 1. Live embryo single-molecule imaging and tracking of endogenous mEos3.2-Zld.

(A) First three columns are example images showing single molecules of mEos3.2-Zld tracked over at least five frames (white arrows and trajectories) at frame rates of 10, 100 and 500 ms. Cyan arrows indicate molecules that appear for only one frame and are thus detected but not tracked. For the 100 and 500 ms data, enough signal is present in the His2B-eGFP channel from the 405 nm activation laser to enable simultaneous imaging of chromatin. Last column shows all single-molecule trajectories acquired in each nucleus over 100 s, corresponding to 539, 263, and 186 trajectories over 10000, 1000, and 200 frames for the 10, 100 and 500 ms data, respectively. Dotted lines indicate the boundary of a nucleus. Contrast was manually adjusted for visualization. (B) Representative kymographs over 5 s of imaging, corresponding to 500, 50, and 10 frames for the 10, 100 and 500 ms frame rate data, respectively. Green arrows point to molecules that display relatively large motions, and white arrows to immobile molecules.

Figure 1—figure supplement 1. Overview of CRISPR-Cas9 genome editing strategy.

(A) Pools of homology repair template plasmids containing different protein tags were co-injected with a plasmid encoding an sgRNA targeting the N-terminus of the target coding sequence into embryos expressing transgenic Cas9. PCR genotyping and DNA sequencing were used to find chromosomes containing tag insertions and to determine the identity of the inserted protein. (B) Fusion proteins used in this study. Zelda and Bicoid fusion proteins were generated by inserting fluorescent proteins at the endogenous locus. To avoid potential problems associated with endogenous tagging of a multicopy locus, histone fusions were supplied as exogenous transgenes. (C) Western blots for Bcd and Zld in fluorescently tagged lines. OreR embryos are from a wild-type strain with untagged Bcd and Zld. The observed shifts in band size correspond to the size of fluorescently tagged Bcd or Zld protein. Expected sizes: Bcd = 54.4 kDa, mEos3.2-Bcd = 80.1 kDa, Zld = 170.1 kDa, mEos3.2-Zld = 195.8 kDa, mCherry-Zld = 198.9 kDa, mNeonGreen-Zld = 196.7 kDa.

Figure 1—figure supplement 2. Simplified Schematic of Lattice Light Sheet Microscope.

The schematic is organized to show the major modules of the microscope. The Laser Combiner module contains six lasers (three shown here) for excitation ranging from 405 to 639 nm, each of which are independently expanded and collimated by using a pair of lenses that serve as a beam expander (BE). The paths of each laser are combined and made collinear by using 1 mirror and 1 dichroic mirror (DC) per laser. The combined beams are then input into an Acousto-optic tunable filter (AOTF) for rapid switching between lasers and control of power. A beam dump (BD) is used to safely capture the light from lasers not being used. To achieve constant and controllable photoactivation the laser combiner was modified to include a 405 nm laser that bypasses the AOTF. A half-wave plate (HWP) is used to adjust the polarization of the input light to the Spatial Patterning module. For spatial patterning, a pair of cylindrical lenses (CL) is used to stretch the Gaussian beam output from the Laser Combiner module into a thin stripe, which illuminates the Spatial Light Modulator (SLM) after passing through a polarizing beam splitter (PBS) and a second HWP. A lens projects the Fourier transform of the plane of the SLM onto an annular mask which is used to confine the spatial frequencies of the patterned light to the desired minimum and maximum numerical apertures. In the scanning module, a pair of lenses de-magnifies and projects the annular mask plane onto first the z-galvo scanning mirror for moving the light sheet through the sample, and a second pair-of lenses relays the plane of the z-galvo onto the x-galvo for dithering the sheet for uniform illumination. Another pair of lenses is then used to project a magnified image of the galvo planes to the back focal plane of the excitation objective (EO) which focuses the light to project the lattice pattern through the sample. An orthogonally placed detection objective (DO) collects the emission light, and a tube-lens (TL) then forms an image at each cameras sensor plane. A dichroic mirror first splits the light into red (>560 nm) and green (<560 nm) channels, followed by a narrower bandpass emission filter (EMF) for further filtering before each camera. With the exception of the modifications to the laser combiner module and the use of two Hamamatsu sCMOS ORCA Flash 4.0 cameras for detection the design is identical to what was originally described by Chen et al., 2014a and Chen et al., 2014b.

Figure 1—figure supplement 3. Mean detections per nucleus per frame for each frame rate.

Detections per nucleus/frame characterized over 804245, 78281, 15165 frames of imaging and 169, 434, and 359 nuclei for 10 ms, 100 ms, and 500 ms, datasets respectively. Error bars show standard errors over all nuclei.

Figure 1—video 1. Movie illustrating ability to controllably photactiviate mEOS3.2.

Download video file ^{(503.3KB, mp4)}

DOI: 10.7554/eLife.40497.006

Related to Figure 1. Movie illustrating the ability to photo-activate mEos3.2 with the modified LLSM system. The power of the 405 nm laser was set to its highest value at approximately the 4 s mark after which a continuous increase in the signal can be observed reflecting the density of activated molecules.

Figure 1—video 2. Example movie of mEos3.2-Zld acquired at 10 ms frame rate.

Download video file ^{(28.2MB, mp4)}

DOI: 10.7554/eLife.40497.007

Related to Figure 1. White scale bar is 5 μm, images were gaussian filtered and inverted for display.

Figure 1—video 3. Example movie of mEos3.2-Zld (red) and His2B-EGFP (green) acquired at 100 ms frame rate.

Download video file ^{(1.7MB, mp4)}

DOI: 10.7554/eLife.40497.008

Related to Figure 1. White scale bar is 5 μm, a nucleus undergoing division is shown for illustrative purposes, data from mitotic nuclei were not used for single-molecule analysis in this work. Images were gaussian filtered for display.

Figure 1—video 4. Example movie of mEos3.2-Zld (red) and His2B-EGFP (green) acquired at 500 ms frame rate.

Download video file ^{(1.1MB, mp4)}

DOI: 10.7554/eLife.40497.009

Related to Figure 1. White scale bar is 5 μm, data from mitotic nuclei were not used for single-molecule analysis in this work. Images were gaussian filtered for display.

Figure 1—video 5. Example of a mobile molecule of mEos3.2-Zld tracked at 10 ms frame rate.

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DOI: 10.7554/eLife.40497.010

Related to Figure 1. White scale bar is 2 μm. Images were gaussian filtered and inverted for display.

Figure 1—video 6. Example of a immobile molecule of mEos3.2-Zld tracked at 10 ms frame rate.

Download video file ^{(135.9KB, mp4)}

DOI: 10.7554/eLife.40497.011

Related to Figure 1. White scale bar is 2 μm. Images were gaussian filtered and inverted for display.