We used an Axiovert 200 (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) and replaced the microscope stage with a custom-built stage, which allowed illumination perpendicular to the detection axis. The lateral position of the specimen chamber was adjusted by micrometer screws (BM11.16, Newport, Darmstadt, Germany), and axially by a motorized stage (M105.1B translation stage with DC-Mike linear actuator M232-17 from PI, Karlsruhe, Germany). The specimen chamber was made of BK7 glass and was especially manufactured for our purposes (Hellma, Müllheim, Germany). The internal dimensions of the chamber were 4 mm×20 mm×2 mm. The wall thickness was 2.5 mm and the bottom had standard cover slip thickness, 0.17 mm.
For fluorescence excitation three laser lines were used: A 488 nm DPSS laser (Sapphire-100, Coherent, Germany), a 532 nm solid state laser (LaNova50 Green, Lasos, Germany) and a 638 nm laser diode module (Cube635-25C, Coherent, Germany). All three laser lines were guided with dichroic beam splitters to an acousto-optical tunable filter (AOTF.nC 1001, Opto-Electronics, France). The AOTF selected laser lines, and defined illumination durations and intensity. After the AOTF the light was coupled into a mono-mode fiber (kineFlex, Point Source, Hamble, UK) and guided onto the optical table. Here the elliptical Gaussian illumination beam was formed by a cylindrical Galilean beam expander. It consisted of a convex cylindrical lens with a focal length of f
250 mm and a concave lens with a focal length of f
−38.1 mm (CKX540-C and CKV522-C, Newport, Darmstadt, Germany). As illumination objective we used a plan apochromat 10×, NA 0.28 long working distance objective lens (Mitutoyo, Japan).
Fluorescence signals were collected with a 40×, NA 1.2 water immersion objective lens (C-Apochromat, Carl Zeiss MicroImaging GmbH, Göttingen, Germany), or a 10×, NA 0.3 objective lens (EC Plan-Neofluar, Zeiss). In the emission beam path respective narrow bandwidth notch filters were employed (Semrock, Rochester, USA). For imaging an EMCCD camera with 128×128 pixels was used (iXon BI DV-860, pixel size 24 µm, Andor Technologies, Belfast, Ireland). A 4× magnifier (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) was added in front of the camera, which resulted in an objective field pixel size of 150 nm for the 40×, NA 1.2 objective lens. The dimensions of the light sheet were measured with a slow-scan CCD camera (Axiocam MRm, Carl Zeiss MicroImaging GmbH, Göttingen, Germany) with a pixel size of 645 nm for the 10×, NA 0.3 objective lens.
Determination of light sheet thickness
ATTO647N (ATTO-TEC GmbH, Siegen, Germany) was diluted in buffer at a concentration of 100 µM and filled into the specimen chamber. Upon laser excitation a homogenous image of the light sheet was created (). In standard configuration the extension in the y-direction of the light sheet was imaged. Rotating the cylindrical lenses by 90° rotated the elliptical illumination beam, and therefore the z-width of the light sheet could be imaged. Images were taken for all excitation laser lines. Excitation of the ATTO647N by 488 and 532 nm occurred due to a small absorption band in the blue-green range of the dye spectrum. The light sheet was imaged by a 10× NA 0.3 objective (EC Plan-Neofluar, Zeiss) and detected by an Axiocam MRm (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). For both y- and z-directions the FWHM of every vertical pixel line in the image was plotted versus the position on the illumination axis, which allowed the determination of the light sheet waists ().
Single molecule imaging and analysis
For imaging of single molecules in solution the molecules were diluted to a final concentration of 100 pM in transport buffer. Fluorescence signals were detected with a 40×, NA 1.2 water immersion objective lens at room temperature. All movies were recorded at a frame rate of 483.09 Hz corresponding to an exposure time of 2 ms per frame. Fluorescence was excited by the 638 nm laser line.
Before tracking a background subtraction and 3×3 Gaussian filtering was performed on the image stacks. Single molecule signals were identified by the threshold algorithm of the tracking software Diatrack v3.03 (Semasopht), which was also used for trajectory definition. To exclude occasionally occurring aggregates of ovalbumin or oligonucleotides from the analysis unusually bright signals and unusually slow moving particles were excluded from the analysis.
From the resulting trajectories the mean-square-displacement (MSD) was calculated and plotted versus time t. The data were fitted according to <x2
4Dt to determine the diffusion coefficient D. This was done with Origin 8.0 PRO (OriginLab Corporation, Northampton, USA).
To calculate the theoretical diffusion coefficient we used the Stokes-Einstein equation 
is the diffusion coefficient, kB
the Boltzmann constant, T
the temperature and f
the frictional drag coefficient. For a sphere with radius r such as the 500 kDa dextran and ovalbumin, f
is defined as 
For a randomly moving ellipsoid, such as the 30b oligonucleotide, it is defined as:η
is the viscosity of the medium, a and b the semi-major and -minor axes of a prolate ellipsoid of revolution. The according radii were obtained from the literature (500 kDa dextran 
and oligonucleotide 
) or calculated according to the equation 
is the molar mass, NA
the Avogadro constant and ρ
refers to the protein density (1.2 g cm−3
The contrast C
of image sequences of 500 kDa dextran molecules was determined according to this definition.Imax
designates the intensity of a molecule in focus above background Imin
was determined by fitting a 2D-Gaussian to the signals. Imin
was determined by measuring the mean intensity in areas where no molecules were seen. All image sequences were recorded with an exposure time of 10 ms, which equates to frame rate of 98.91 Hz.
In vivo imaging and analysis
Imaging of the 500kDa dextran molecules and the labeled mRNP particles in salivary gland cell nuclei of C. tentans larvae were performed with a 40×, NA 1.2 water immersion objective lens at room temperature. Image integration time was 20 ms corresponding to a frame rate of 49.46 Hz. Fluorescence was excited by the 638 nm laser line.
All image stacks were analyzed with the tracking software Diatrack v3.03 (Semasopht). Background subtraction and Gaussian filtering was performed before signal identification. To localize the particle, every particle signal was fitted by a 2D Gaussian. Sequences of single, localized events were combined to trajectories.
The determination of the 2D-localization precision of moving BR mRNPs in the salivary gland cells was achieved using the equation
is the standard deviation of the point-spread function, a
the pixel size, b
the standard deviation of the background and N
the total number of photons contributing to a signal. To determine the latter value the average number of the integrated intensity values for a signal was determined. Finally the total number of photons per signal was calculated considering the count-to-photoelectron conversion factor for the used iXon camera 
Out of the trajectories the jump distance from one frame to the next frame was calculated for every trajectory. These jump distances were plotted in a normalized histogram () and fitted according to the following equation 
This equation represents the theoretical jump distance probability density function for i
diffusive species. Ai
is the fractional amount of a single species and Di
the respective diffusion coefficient. r
is the jump distance covered in the time interval Δt. In a complex medium like a cell nucleus a single, distinct diffusion coefficient was not expected and the equation above allowed a multi-component analysis of the in vivo
data. The calculations and data fitting were performed by Origin 8.0 PRO (OriginLab Corporation).
We measured a reference diffusion coefficient in aqueous solution (1 cP) and also the diffusion coefficient in the nucleoplasm. For the calculation of the viscosity of the nucleoplasm we used Eq. 1.
Microinjection of C.tentans salivary glands
were raised as described 
. Salivary glands were isolated from fourth instar larvae and microinjected with an Eppendorf injection and micromanipulation setup using a holding pressure of 25 hPa and manual injection procedure.
Buffer and reagents
Phosphate buffered saline (PBS) was prepared from a commercially available stock solution (Biochrom AG, Berlin, Germany). Transport buffer contained 20 mM HEPES/KOH, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA, and 2 mM DTT. Amino-derivatized dextran (molecular mass 500 kDa; Invitrogen, Germany) was dissolved in 0.1 M NaHCO3
, pH 8, and fluorescence labeled with a 5-fold excess of ATTO647 succidinimidyl ester (ATTO-TEC GmbH, Siegen, Germany). Chicken egg Ovalbumin (molecular mass ~43 kDa; Sigma-Aldrich, Germany) was solved in PBS containing 1mM TCEP (tris(2-carboxyethyl)phosphine; Sigma-Aldrich, Germany) and labeled with ATTO647N maleimide (ATTO-TEC GmbH, Siegen, Germany). Preparation of ATTO647N-labeled hrp36 was according to 
. All labeling reactions were set up at room temperature for 2 hours and free dye was removed by gel filtration on a BioRad-P6 desalting column (MW cut off 6 kDa; BioRad, Munich, Germany). Labeled probes were finally size-fractionated on a Superose 12 column to remove aggregates and smaller fragments. The 2′-O-methyl RNA oligonucleotide homologue to the repetitive portion of the BR 2.1 mRNA was obtained from IBA BioTAGnologies (Göttingen, Germany). It comprised 30 bases (ACT TGG CTT GCT GTG TTT GCT TGG TTT GCT
) and contained a 5′ fluorescence label (ATTO647N). To check for purity the oligo was resolved on a 15% polyacrylamide gel prior to use.
Fluorescence correlation spectroscopy
FCS measurements were performed using a Zeiss Confocor I microscope setup. For calibration of the beam width ATTO647N-maleimide dye molecules (MW 870 Da; ATTO-TEC GmbH, Siegen, Germany) in buffer were used. The diffusion coefficient of ATTO655-maleimide dye (MW 810 Da) has been measured previously with high precision 
, and a diffusion coefficient of 400 µm2
/s was determined. Since both dyes were comparable in molecular weight, we assumed a diffusion coefficient of 400 µm2
/s for ATTO647N-maleimide as well. This assumption was corroborated by the characteristic diffusion times for the two dyes of 63 and 62 µs, respectively. Calibration of the beam width was done prior to the measurements. Before a single FCS run, a z-scan was recorded to ensure that the measurement was performed in the solution not close to the coverslip. Data analysis was performed using FCS ACCESS (Evotec, Hamburg, Germany). The theoretical autocorrelation curve for 3D diffusion of up to 3 mobility components is,
are the fractions corresponding to the different diffusion times τD.j
is the total number of fluorescent molecules, T
is the ratio of triplet state, τt
is the triplet time and κ
is the axial e−2
beam radius of the laser beam divided by the lateral e−2
beam radius. κ
was usually close to 5. τD
is related to the diffusion coefficient by
is the lateral e−2
beam radius of the laser spot. Laser illumination was performed with 633 nm light.
ATTO647-labeled 500 kDa dextran molecules were diluted 1
1000 from the stock in PBS, followed by centrifugation at 22000×g for 45 minutes. Before measuring, a MatTek dish (MatTek Corp., Ashland, USA) was coated with bovine serum albumine (10g/L). After removing the coating solution, 300 µL dextran solution was added and covered with a coverslip to prevent evaporation. Single FCS runs with 10 to 30 seconds were performed and average values were calculated. ATTO647N-labeled ovalbumin was diluted in transport buffer 1
1000 from stock solution, and centrifuged at 22000×g for 30 minutes. Single FCS runs of 60 seconds length were performed and average values were calculated.
ATTO647N-labeled Oligonucleotides were diluted 1
from stock concentration (1 nmol/µL) with PBS, and measured as described for ovalbumin.