For stimulated emission depletion (STED) microscopy and the corresponding confocal microscopy a LEICA SP5-STED microscope was used. The fluorophores ATTO647N (Activemotif) or MegaRed (Sigma) were excited with an 80 MHz pulsed diode laser at 635 nm or 531 nm respectively (Pico-Quant), (pulse width < 100 ps). STED depletion was achieved using a mode-locked titanium sapphire laser (Spectra Physics Mai Tai HP laser) operating at 770 nm with a repetition rate of 80 MHz. The delay between the excitation and STED pulses was adjusted electronically to optimize depletion. The excitation and the STED beams were focused by a 100× oil immersion objective (NA 1.4 PL APO STED, 100×; Leica Microsystems). The fluorescence signal was collected by the same objective and detected confocally between 650 and 690 nm for ATTO647N and between 540 and 595 nm for Megared with an APD detector. Using this set up, a resolution of ˜250 nm in the confocal images and 50–70 nm in the STED images was achieved. Except for contrast stretching and level adjustment no further image processing was applied.
Sp5 sted microscope
Manufactured by Leica
The Leica SP5-STED microscope is a high-performance imaging system designed for advanced fluorescence microscopy applications. It features a stimulated emission depletion (STED) capability, which can achieve super-resolution imaging beyond the diffraction limit of light. The SP5-STED provides researchers with the ability to visualize and analyze biological samples with improved spatial resolution and enhanced image quality.
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Lab products found in correlation
Fluo 4 am, by Thermo Fisher Scientific (3 mentions)
Maitai hp laser, by Spectra-Physics (1 mentions)
Atto 647n, by Active Motif (1 mentions)
Dmem media, by Thermo Fisher Scientific (1 mentions)
Tricaine, by Merck Group (1 mentions)
Lmd laser micro dissection 7000 microscope, by Leica (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
4 protocols using sp5 sted microscope
1
STED Microscopy Protocol and Setup
2
Multicolor STED Imaging of Cellular Structures
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More details on labelling and imaging are provided in supplementary methods and here we briefly summarise the experimental procedures. Cells were labelled with 2.5 μg/ml anti-dynamin mouse antibody, 10 μg/ml anti-arp2 rabbit antibody or 2 μg/ml anti-V-ATPase rabbit polyclonal antibody recognising all isoforms for 1 h in PBS containing 0.5% BSA. The secondary labeling was conducted for 1 h with anti-mouse or anti-rabbit alexa488 in PBS containing 0.5% BSA, along with 200 ng/ml phalloidin-Star635 and 10 ng/ml PicoGreen. To stain membranes we used 5 μg/ml of lipophilic DiI. The cells were then washed and embedded in mowiol. All images were acquired with a Leica SP5 STED microscope, equipped with pulsed Ti: Sapphire depletion laser MaiTai HP and 635 nm pulsed laser and an avalanche photo diode (APD) detector at 665–705 nm range. STED microscopy images were processed using DeconvolutionLab plugin60 (Richardson-Lucy with TV regularisation algorithm, regularisation parameter 0.005 and 20 iterations, for panel 3O only 4 iterations were used) for ImageJ with experimentally measured PSF.
3
Imaging Intracellular Calcium Oscillations
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The intracellular calcium (Ca2+) oscillations were studied using Fluo4-AM (Thermo Fisher Scientific). The cells were incubated with Fluo4-AM 2 µM for 5 minutes in DMEM media (GibcoTM), at room temperature. After incubation, the excess Fluo4-AM was removed by replacing the DMEM media. After incubation, the wells were mounted on a Leica SP5 STED microscope. The recordings were made at 37°C and 5% CO2. Fluo4-AM (Thermo Fisher Scientific) was excited at 488 nm and its fluorescence emission was collected at 525 nm using a 40x oil immersion objective. The images were sampled at a rate of 1 Hz for 2 minutes both for basal activity and after ATP bath application (10 µM). The images were stored as image stacks containing time-lapse images and analyzed using a custom Matlab script. The fluorescence traces were extracted from manually segmented regions of interest corresponding to cell bodies and were presented as relative changes in fluorescence (ΔF/F). Calcium fluctuations were measured by in house developed code kindly provided by Dr. Gertrudis Perea and Dr. Julio Esparza.31 (link)
4
Tol2-mediated Transgenesis in Zebrafish
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Exogenous genes were incorporated into embryo genomes at the one-cell stage using the Tol2 transposon system43 (link). DNA constructs (final concentration 25 ng/μl) were mixed with Tol2 mRNA (final concentration 100 ng/μl), and about 200 picoliters mixed solutions were micro-injected into embryos at the one-cell stage, following protocols described earlier44 (link). Alternatively, about 200 picoliters Cre-ERT2 mRNA (final concentration 50 ng/μl) was injected into the Tg(ubi:loxP-Eos-stop-loxP-kRASG12V-T2A-mTFP) embryos at the one-cell stage. After injection, embryos were incubated in E3 medium at 28 °C. Healthy, normal embryos were selected at 24 hpf and 3 dpf for further observation and analysis. Fluorescent imaging was performed on a Nikon Eclipse Ti microscope equipped with a C-HGFI Intensilight Fiber illuminator, and confocal imaging was acquired on a Leica SP5-STED microscope. Embryos and adult zebrafish were anesthetized first in tricaine (Sigma-Aldrich), and aligned in a small liquid drop on a cover slide for imaging. The fluorescence intensity of individual images was quantified with ImageJ64 after background subtraction. In situ hybridization and H&E samples were imaged on a Leica LMD (Laser Micro Dissection) 7000 Microscope.
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