For bead experiments for color-classification (for both the training of the ANNs and the analysis of unknown samples), 40 nm dia. fluorescent beads from Invitrogen (F10720; yellow and orange FluoSpheres with emission peaks at 515 and 560 nm, respectively) were diluted in Dulbecco’s phosphate buffered saline (DPBS), mixed, and sealed between a glass slide and a pre-cleaned #1.5 thickness coverslip, and imaged with the above optical setup. The 488-nm and 561-nm lasers were used to excite the two types of beads to similar levels of brightness. To record images at different axial positions, the objective lens was scanned by the built-in motor over a range of −400 to + 400 nm of the focal plane in 50 nm steps. To compare the performance of the axial-localization ANN with cubic-spline MLE, 40 nm dia. fluorescent beads from Invitrogen (F8789; dark red FluoSphere with an emission peak at 680 nm) were similarly prepared as described above, excited with the 644-nm laser, and scanned from -400 nm to + 400 nm in 50 nm steps.
F10720
Manufactured by Thermo Fisher Scientific
The F10720 is a laboratory equipment product. It serves a core function within the scientific research and analysis workflow.
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Lab products found in correlation
3 protocols using f10720
1
Fluorescent Bead Axial Localization
2
Anterograde and Retrograde Tracing in ChAT-Cre Mice
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2-month-old ChAT-Cre mice were anesthetized with 2% isoflurane and placed into a stereotaxic apparatus. A Cre-dependent herpes simplex virus (HSV) anterograde transsynaptic tracer, HSV129(ΔTK)-loxP-STOP-loxP-tdtomato:2a:TK (H129ΔTK-TT) [33] (link) was bilaterally injected into the DMH of ChAT-Cre mice (1 × 1010 pfu, 200 nl per side, stereotaxic coordinates, bregma: AP: −1.95 mm, DV: −5.0 mm, ML: ±0.25 mm) with a Hamilton syringe. For retrograde monosynaptic tracing study, CTb (0.1%) was directly injected into the rRPa (AP: −6.0 mm, DV: −6.0 mm, ML: 0 mm). In some tracing experiments, Alexa Fluor 488 conjugated to wheat-germ agglutinin [22] (10 μg; Invitrogen, W11261) or fluorescent beads [23] (link) (100 μl, 0.04 μm diameter; Invitrogen, F10720) were directly injected into iBAT of ChAT-Cre::ChR2-YFP or ChAT-Cre::tdTomato mice 5 days prior to assays. To delete the Chat gene in DMH cholinergic neurons, we bilaterally injected AAV-hSyn-mCherry (control, 250 nl of 3 × 1012 pfu/ml per side) or AAV-hSyn-mCherry-Cre (250 nl of 3 × 1012 pfu/ml per side) viruses (UNC vector core) into the DMH of ChATF/F mice (The Jackson Lab).
3
Fluorescent Tracer Diffusion Assay
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Compartments were filled with 50
μL of either 5 μM fluorescein (F7137, Sigma), 62.5 μg/mL
70 kD fluorescein-labeled dextran (D1823, Invitrogen), or fluorescein-labeled
beads with 40 nm (0.025%, F10720 Invitrogen), respectively, 500 nm
diameter (0.008%, F8813, Invitrogen). Adjacent compartments were filled
with either equal volumes or twice the volume to establish a direct
flow. Plates were sealed and imaged for 72 h on an Infinite 500 plate
reader (Tecan) at 485/535 nm (excitation/emission).
μL of either 5 μM fluorescein (F7137, Sigma), 62.5 μg/mL
70 kD fluorescein-labeled dextran (D1823, Invitrogen), or fluorescein-labeled
beads with 40 nm (0.025%, F10720 Invitrogen), respectively, 500 nm
diameter (0.008%, F8813, Invitrogen). Adjacent compartments were filled
with either equal volumes or twice the volume to establish a direct
flow. Plates were sealed and imaged for 72 h on an Infinite 500 plate
reader (Tecan) at 485/535 nm (excitation/emission).
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