FRAP imaging by low-photobleaching light sheet microscopy.

<p>(A) Workflow for FRAP imaging by light sheet microscopy. After monitoring, the animals can be recovered to assess possible damage induced through the procedure. (B) Representative images of recovery at several time points after photobleaching IFE-2::GFP fluorescence in the anterior part of the animal (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127869#pone.0127869.s010" target="_blank">S6 Video</a>). The red arrow indicates a developing embryo within the parental gonad. Size bars correspond to 100μm. (C) Imaging of the anterior region after photobleaching and recovery time points of DCAP-1::dsRED fluorescence. The signal also recovers to the subcellular structures of P bodies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127869#pone.0127869.s011" target="_blank">S7 Video</a>). Size bars correspond to 100μm. (D) IFE-2::GFP and (E) DCAP-1::dsRED quantification of recovering fluorescent intensity of the animals in (B) and (C), respectively. The respective equations describing best-fit lines as well as r² values for each graph are shown. The line slopes correspond to the first derivative of fluorescent change within a time unit (df/dt) and determine the recovery rate.</p

A versatile imaging platform for light sheet microscopy and FRAP.

<p>(A) 3D scheme of the LSM and components (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127869#pone.0127869.s005" target="_blank">S1 Video</a> for full 3D overview of the setup and compare photographs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127869#pone.0127869.s001" target="_blank">S1 Fig</a>). The red line indicates the path of the laser beam. The laser beam is guided by a flexible mirror set to pass through the cylindrical lens, where a laser light sheet is created and focused on the sample, which is immersed in an index matching fluid bath. A 3D stage is used to move the sample along x-, y- and z-axis into position for imaging via the CCD camera with tube lens attachment, which is placed orthogonal to the light sheet. Components for FRAP performance are encircled and consist of a flip mirror and a focal lens to concentrate the beam for fluorescent photobleaching of the sample. A detailed description is provided in Materials and Methods. (B) Basic 2D scheme of the setup showing its essential components. LAS = laser, SH = shutter, FM = flip mirror, CL = cylindrical lens, FL1 = focal lens 1, FL2 = focal lens 2, OS = 3D stage, RBS = Refractive index matching fluid bath and sample, LED = white light LED, CCD = CCD camera, TL = tube lens, I = iris, F = fluorescent filter set, OL = objective lens.</p

Protein dynamics in a longitudinal ageing study in <i>C</i>. <i>elegans</i>.

<p>(A) Representative images of 3 single animals grown at 25°C at day 1, 3 and 5. Animals co-express p_<i>ife-2</i>IFE-2::GFP and p_{<i>dcap-1</i>}DCAP-1::dsRED (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127869#pone.0127869.s008" target="_blank">S4 Video</a>). Individuals show differences in protein localization during ageing. Size bars correspond to 100μm. (B) IFE-2::GFP and (C) DCAP-1::dsRED fluorescent intensity quantification of the 3 animals in (A).</p

Changes of protein localization upon starvation-induced stress.

<p>(A) Representative images of 3 starved animals grown at 25°C at day 1 and 3 in a group. Decrease of fluorescence for both reporters p_<i>ife-2</i>IFE-2::GFP and p_{<i>dcap-1</i>}DCAP-1::dsRED from day 1 to day 3 are visible, while the signal persists in some tissues, including pharynx, canal cells, muscles and the developing embryos (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127869#pone.0127869.s009" target="_blank">S5 Video</a>). Size bars correspond to 100μm. (B) IFE-2::GFP and (C) DCAP-1::dsRED fluorescent intensity quantification of the 3 animals in (A) individually and in the group.</p

Workflow for light sheet 3D microscopy to measure protein dynamics in <i>C</i>. <i>elegans</i>.

<p>Animals are maintained following standard procedures, collected and immobilized on agar pads, which are immersed in an oil bath. The laser beam for sample illumination passes through a cylindrical lens and forms a light sheet that is directed on the specimen. The recording lens is focused on the illuminated sheet and records stacks of the specimen, which is moved along the x-axis. Recorded data sets consist of image stacks and are combined into a 3D visualization via freeware, such as ImageJ. Anatomic landmarks (b_i1, b_i2, b_i3) are used to map and transform images via registration algorithms so that they can be aligned to a reference image A (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127869#sec002" target="_blank">Materials and Methods</a>).</p