Axiovert 35m microscope

For fluorescence microscopy of cell cultures, a Zeiss Axiovert 35M microscope equipped with fluorescence optics was used. For specific excitation of Venus a filter block with excitation of 450–490 nm and emission of 520–550 nm, for specific excitation of tdTomato a filter block with excitation of 530–570 nm and emission of 590–610 nm were used.
For imaging of tissue biopsies an Olympus SZ16 stereomicroscope with epifluorescence optics was used. For macroscopic imaging of the calves, either blue or green flood lights of light emitting diodes (LED; Eurolite) were used for excitation, and images were recorded with a digital camera and appropriate emission filters (Lee Filter)14 (link).

Recordings were performed on an inverted Zeiss Axiovert 35M microscope. Recordings were made in the whole-cell configuration using borosilicate glass pipettes (Harvard Apparatus GC150F-10) pulled with a Zeitz DMZ-Universal puller ranging from 2 to 6 MΩ. The pipette solution contained (in mM): 123 K-Gluconate, 7 KCl, 1 MgCl₂, 5 ATP_Na2, 10 EGTA, 10 HEPES (pH 7.4, KOH). The bath solution contained (in mM): 135 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, 10 HEPES (pH 7.4, NaOH). Signals were amplified with an Axopatch 200B Amplifiter, low pass filtered at 5 kHz and digitized at 50 kHz with an Axon Digidata 1440A. Acquisition and analysis was performed using pClamp software (Molecular Devices, Biberach, Germany). Membrane voltage was clamped at −60 mW and light pulses were supplied by two solid state lasers (Pusch Opto Tech GmbH, Wettenberg, Germany; λ=473 nm, λ2=593.5 nm), which were coupled to a 400 μm optic fiber. Light pulses were applied by a fast computer-controlled shutter (Uniblitz LS6ZM2, Vincent Associates, Rochester, NY, USA). YFP signals were observed using 473 nm excitation through a GFP filter set (#31001, Chroma Technology, Bellows Falls, VT, USA).

For fluorescence microscopy of cell cultures, a Zeiss Axiovert 35M microscope equipped with fluorescence optics was used. For specific excitation of tdTomato a filter block with excitation of 530–570 nm and emission of 590–610 nm were used. Alternatively, images were obtained by an Olympus BX 60 (Olympus, Hamburg, Germany) fluorescence microscope equipped with a high resolution digital camera (Olympus DP71).
For imaging of tissue biopsies an Olympus SZ16 stereomicroscope with epifluorescence optics was used. The light transmittance of lenses was also measures with the stereomicrosope. Therefore wildtype and transgenic lenses were isolated, and placed side by side under the stereozoom microscope. Normalized grey scale images were kept while illuminated from below. With the Olympus CellF software histograms (relative light transmission) of identically treated lenses (dotted lines in Fig 4A) were determined and plotted.