C apochromat 40 1.2 na

Live cell imaging was performed using an inverted confocal laser scanning microscope (LSM510; Carl Zeiss, Jena, Germany). Confocal observations were performed at 25 °C. GFP was excited at 488 nm using a CW Ar⁺ laser through a water immersion objective lens (C-Apochromat, 40×, 1.2 NA; Carl Zeiss), with emission detected at 505–550 nm for single scanning experiments using cells expressing GFP and GFP-tagged proteins. The pinhole diameters for confocal imaging were adjusted to 70 mm for GFP.

Time-lapse LSM images for ccRICS analysis were acquired by a combination system consisting of an LSM510 and a ConfoCor3 (Zeiss, Jena, Germany) with a temperature control system. ATTO488 was excited by a 488-nm Ar-ion laser and ATTO647N was excited by a 633-nm He–Ne laser through a water-immersion objective (C-Apochromat, 40×, 1.2 NA; Zeiss). Two lasers simultaneously illuminated the observation volume at the same position. Emission signals were collected by photon counting at 505–610 nm for ATTO488 (green channel) and >650 nm for ATTO647N (red channel) with avalanche photodiodes (APDs). The pixel size was 22 nm, which is 5–10 times smaller than the focused laser spot, and the pixel dwell time was 5–50 μs per pixel.

All FCS experiments were carried out at 22 °C on a custom-built confocal fluorescence microscope with a Zeiss C-Apochromat 40× 1.2NA water-immersion objective using 100 mM PBS, 10 mM KCl, 10 mM MgCl₂ as a refolding buffer at pH 7.5. To diminish the surface interactions with the glass coverslip we added 0.1% Tween-20 (Sigma) to all the samples. The fluorescence intensity temporal fluctuations were analyzed with a hardware correlator (Flex02-12D/C correlator.com, Bridgewater NJ with 12.5 ns minimum channel width). All the experimental data were fitted using Eq. 1 by considering a single species and free Brownian 3D diffusion in the case of a Gaussian molecular detection efficiency:

where N is the average number of molecules in the focal volume, F is the total fluorescence signal, B is the background noise, n_T is the amplitude of the dark state population, τ_T is the dark state blinking time, is the mean diffusion time and s is the ratio of transversal to axial dimensions of the analysis volume. The molecular diffusion coefficient, D and hydrodynamic radius, R_H were calculated previously described in detail43 (link). We calibrate the parameter transversal waist in solvent viscosity, before each measurement on MreB samples by recording the FCS trace for Alexa Fluor 647 dyes which have a known hydrodynamic radius of 0.7 nm in pure water.

Co-labeling analysis was performed on pictures taken on at least three different sections using a LSM510 NLO multiphoton confocal microscope fitted on an Axiovert M200 inverted microscope equipped with C-Apochromat 40×/1.2 N.A. and 63×/1.2 N.A. water immersion objectives (Zeiss, Iena, Germany).
The 488 nm excitation wavelength of the Argon/2 laser, a main dichroic HFT 488 and a band-pass emission filter (BP500-550 nm) were used for selective detection of the green fluorochrome (Cy2, Alexa 488).
The 543 nm excitation wavelength of the HeNe1 laser, a main dichroic HFT 488/543/633 and a long-pass emission filter (BP565-615 nm) were used for selective detection of the red fluorochrome (Cy3).
Optical sections, two microns thick, 512 × 512 pixels, were collected sequentially for each fluorochrome. Z-stacks with a focus step of one micron were collected.
The data-sets generated were merged and displayed with the Zen software (Zeiss, 2009) and exported in LSM image format.
Counting was performed with the ImageJ 1.46a software (NIH, USA). Figures were prepared with Adobe Photoshop CS3 soft ware.

SYPRO® Red (Molecular Probes, Oregon) was prepared as a 50x stock solution in pre-filtered histidine-sucrose buffer and diluted to a final working concentration of 2.5x for fluorescence studies immediately prior to use (all solutions were prepared on the day of use) [28 (link)]. SYPRO® Red was added 15 min prior to visualization with confocal microscopy. A Zeiss 510 Confocor 2 (Zeiss, Jena, Germany) confocal microscope equipped with a c-Apochromat 40×/1.2NA water-immersion objective was used for image acquisition. Imaging was carried out by exciting the dye with a Helium-Neon laser at 543 nm and the emitted fluorescence collected above 585 nm (LP585 filter set). A confocal image time series of 1024 × 1024 pixel resolution was captured over 100 frames with a corresponding pixel dwell time of 6.4 μs. In-house RICS software (ManICS) was applied to analysis of images acquired using confocal microscopy. A full description of the RICS algorithm has been described elsewhere [28 (link),29 (link)]. The image time series were sub-divided into 32 × 32 pixel sub-regions and the diffusion coefficients (D) of each region of interest (ROI) was generated. The method is described in greater detail previously [28 (link)].

FCS measurements
were performed using a Zeiss 780 confocal laser scanning microscope
equipped for FCS and FCCS, with a Zeiss water immersion objective,
C-Apochromat 40×/1.2 NA. Samples labeled with Oregon Green were
excited at 488 nm and fluorescence emission was collected at 499–622
nm, while Alexa Fluor 647 samples were excited at 633 nm and fluorescence
was collected at 641–695 nm. HiLyte 488 (433 μm²/s) and HiLyte 647 (320 μm²/s)^{35 (link)} were used for calibration and yielded τ_Dg = 32 μs, ω_g = 0.24 μm, and τ_Dr = 62 μs, ω_r = 0.28 μm, respectively.
Thirty FCCS measurements of 10 s duration were carried out in the
measurement dish MatTek, 35 mm, 10 mm glass bottom, no. 1.5 glass.