Fluorescent proteins expressed in photoreceptors were imaged by the water-immersion technique as described previously [8 (link)]. Briefly, late pupae with GFP-positive RFP mosaic retina were attached to the slide glass using double-sided sticky tape, and the pupal cases around the heads were removed. The pupae were chilled on ice, embedded in 0.5% agarose, and observed using an FV1000 confocal microscope equipped with a LUMPlanFI water-immersion 40× objective (Olympus, Tokyo, Japan).
Lumplanfi water immersion 40 objective
Manufactured by Olympus
Sourced in Japan
The LUMPlanFI water-immersion 40× objective is a high-quality optical lens designed for use in microscopy applications. It has a magnification of 40× and is optimized for use with water-based samples. The objective provides a high numerical aperture and is suitable for a variety of microscopy techniques.
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Lab products found in correlation
4 protocols using lumplanfi water immersion 40 objective
1
Fluorescent Protein Imaging in Photoreceptors
2
Visualizing Rhodopsin Activation in Photoreceptors
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Fluorescent proteins expressed in photoreceptors were imaged by water-immersion technique.
y w ey-FLP;CG6750e02662FRT40A/ CyO y+ (KY114504) was mated with w;P3RFP FRT40A/SM1;Rh1-Arrestin2::GFP eye-FLP/TM6B (Satoh et al., 2013 (link)). Late pupae of the siblings with GFP-positive RFP mosaic retina were attached to the slide glass using double-sided sticky tape and the pupal cases around the heads were removed. The pupae were chilled on ice, embedded in 0.5% agarose, and observed using an FV1000 confocal microscope equipped with a LUMPlanFI water-immersion 40× objective (Olympus, Tokyo, Japan). Arrestin2::GFP specifically binds to activated rhodopsin (Satoh et al., 2010 (link)). Rh1 was activated by a 477 nm solid-state laser to bind Arr2:GFP and GFP. The wild-type marker P3RFP is DsRed gene under the control of three Pax3 binding sites and labels photoreceptors (Bischof et al., 2007 (link)).
y w ey-FLP;CG6750e02662FRT40A/ CyO y+ (KY114504) was mated with w;P3RFP FRT40A/SM1;Rh1-Arrestin2::GFP eye-FLP/TM6B (Satoh et al., 2013 (link)). Late pupae of the siblings with GFP-positive RFP mosaic retina were attached to the slide glass using double-sided sticky tape and the pupal cases around the heads were removed. The pupae were chilled on ice, embedded in 0.5% agarose, and observed using an FV1000 confocal microscope equipped with a LUMPlanFI water-immersion 40× objective (Olympus, Tokyo, Japan). Arrestin2::GFP specifically binds to activated rhodopsin (Satoh et al., 2010 (link)). Rh1 was activated by a 477 nm solid-state laser to bind Arr2:GFP and GFP. The wild-type marker P3RFP is DsRed gene under the control of three Pax3 binding sites and labels photoreceptors (Bischof et al., 2007 (link)).
3
Calcium Imaging with GCaMP5 Fluorescence
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Calcium imaging was performed using a moveable objective microscope (Sutter) equipped with a Chameleon titanium-sapphire laser tuned to 915 nm (Coherent), employing an Olympus LUMPlanFI 40× water immersion objective (NA 0.8). Emitted fluorescence was captured by the objective and filtered using an HQ 535/50GFP emission filter (Chroma Technology) before detection using Pho Image v.3.0 software3, run on a PC. Images were acquired at a resolution of 256 × 256 pixels. Image sequences were acquired at 1 ms per line using 256 × 256 or 128 × 128 pixels per frame and analyzed with Igor Pro v.6.10 or ImageJ v.1.46 (NIH). The regions of interest were defined by the standard deviations of image sequences. Scanning and image acquisition were controlled using scan normalized fluorescence responses calculated as follows: where F is the instantaneous GCaMP5 fluorescence, and F0 is the baseline GCaMP5 fluorescence.
4
Calcium Imaging of Human Retinal Organoids
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Calcium imaging was performed as previously described [51] . The hROs were incubated in DMEM/F12 without phenol red (Abcam Cat# 11-039-021) at room temperature. A moveable objective microscope (Sutter) equipped with a Chameleon titanium-sapphire laser tuned to 915 nm (Coherent) was used, and an Olympus LUM-PlanFI 40× water immersion objective (NA 0.8) was employed. The emitted fluorescence was captured by the objective and filtered using an HQ 535/50GFP emission filter (Chroma Technology) before detection using Pho Image v.3.0 software. Images were acquired at 1 ms per line using 256×256 per frame and further analyzed with Igor Pro v.6.10 and ImageJ v.1.53t. The regions of interest were defined by the semi-automatic analysis method described previously [52] . Signal acquisition were transferred as scan-normalized fluorescence (Z score), which was calculated as belowed: F/F=(F-F0)/F0, where F is the instantaneous GCaMP5G fluorescence and F0 is the baseline fluorescence of GCaMP5G.
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