To image LDs, cells were stained by 0.1 µg/ml Bodipy 493/503 followed by two washes in 50 mM Tris, pH 7.5, and subjected directly to fluorescence microscopy. All fluorescence images of cells were captured by using an Olympus IX81 inverted microscope equipped with a 100× Plan-Apochromat objective lens (NA = 1.4) and an ORCA Flash 4.0 cMos v2 camera (Hamamatsu) with MetaMorph software (Molecular Devices). The fluorescence filters used in this study were the GFP filter (excitation filter BP460-480, emission filter BP495-540), mCherry filter (excitation filter BP535-555, emission filter BA570-625), YFP filter (excitation filter BP490-500, emission filter BP515-560), and CFP filter (excitation filter BP425-445, emission filter BA460-510). Images were processed by using MetaMorph and/or Photoshop CS4 (Adobe).
Plan apochromat objective
Manufactured by Hamamatsu Photonics
Sourced in Germany
The Plan-Apochromat objective is a type of microscope objective lens designed to provide high-quality, distortion-free images across a wide field of view. It utilizes a combination of lens elements to minimize chromatic and spherical aberrations, resulting in sharp, color-corrected images.
Automatically generated - may contain errors
Lab products found in correlation
Metamorph, by Adobe (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Ix81 inverted microscope, by Olympus (1 mentions)
Axio scan z1, by Zeiss (1 mentions)
Orca flash 4.0 v2 digital cmos camera, by Zeiss (1 mentions)
Colibri 7 led light source, by Zeiss (1 mentions)
Axiovert 200m, by Zeiss (1 mentions)
Vt1000, by Leica (1 mentions)
Superfrost plus, by Thermo Fisher Scientific (1 mentions)
4 protocols using plan apochromat objective
1
Fluorescence Microscopy Imaging of Lipid Droplets
2
Whole-Slide Imaging with Zeiss Axio Scan
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The whole-slide imaging was performed using a Zeiss Axio Scan.Z1 with Zeiss Plan-Apochromat objective 20× (0.8 NA, M27), Hamamatsu ORCA-Flash 4.0 V2 Digital CMOS camera (16-bit; 0.325 μm/pixel resolution), and Zeiss Colibri.7 LED Light Source. DAPI, fluorescein isothiocyanate, Cy5, and Cy7 filters were used.
3
Fura-2 Fluorescence Imaging in Cardiomyocytes
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Fura-2 fluorescence in cardiomyocytes was measured using a Cell Observer fluorescent station based on an AxioVert 200 M motorized inverted microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a 10× PlanApochromat objective, an Orca-Flash R2 monochrome camera (Hamamatsu Photonics K.K., Iwata City, Japan), and a system for a high-speed change in excitatory filters Ludl MAC 5000 (Ludl Electronic Products, Hawthorne, NY, USA). Fura-2 fluorescence was excited with an HBO100 mercury lamp in two channels using a set of 21HE light filters (Carl Zeiss AG, Oberkochen, Germany): excitation 1—340 ± 15 nm (channel 340); excitation 2—387 ± 8 nm (channel 380); FT 409 beam splitter; and 510 ± 45 nm emission filter. The exposure time for each of the channels was 100 ms. The registration frequency was 1 frame per 5 s. The power of the light source was set to the minimum value that provided an acceptable signal-to-noise ratio. In control experiments with the selected recording parameters, no effect of illumination on the Fura-2 signal was detected for 30 min or more.
4
Histological Analysis and Cannula Placement
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For histological analysis mice were perfused transcardially with 4% paraformaldehyde (PFA) in phosphate buffer solution (PBS). After removing the brain they were left for 24 hours in 4% PFA solution in PBS, then transferred in 0.1% sodium azide solution in PBS. Brains were sliced in 50 um coronal sections on a vibratome (Leica VT 1000 S), collected in wells maintaining the anterior-posterior order, and finally mounted on microscope slides (Thermo scientific, superfrost plus), with mowiol.
Fluorescent images were acquired with an automated slide scanner (AxioScan Z1) equipped with a 10x, 0.45 NA PlanApochromat objective and a Hamamatsu OrcaFlash camera. Use of the appropriate filter combination allowed for DAPI and EYFP acquisition (Beam Splitter: 395, excitation: 330-375, emission: 430-470, and Beam splitter: 498, excitation: 453-485, emission: 507-546 respectively).
Optic cannula placement was determined using coronal sections of the prefrontal cortex through which the fiber tract was visible. We determined the position by locating the section with the broadest base of the cannula tract and comparing the DAPI staining with the Allen Mouse Brain Atlas (Lein et al., 2007 (link)) (Figure S2 ; Table S1 ).
Fluorescent images were acquired with an automated slide scanner (AxioScan Z1) equipped with a 10x, 0.45 NA PlanApochromat objective and a Hamamatsu OrcaFlash camera. Use of the appropriate filter combination allowed for DAPI and EYFP acquisition (Beam Splitter: 395, excitation: 330-375, emission: 430-470, and Beam splitter: 498, excitation: 453-485, emission: 507-546 respectively).
Optic cannula placement was determined using coronal sections of the prefrontal cortex through which the fiber tract was visible. We determined the position by locating the section with the broadest base of the cannula tract and comparing the DAPI staining with the Allen Mouse Brain Atlas (Lein et al., 2007 (link)) (
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