Deep tissue imaging was performed on either an Olympus FV1000 or a Caliber ID RS-G4. While using the Olympus FV1000, either the confocal or two photon setting was used depending on which setting optimally excited the fluorophores present in the sample. On the Olympus FV1000, BrightZ setting was applied to increase signal as deeper areas of the sample were imaged. In all cases, a 25x objective (XLSLPLN25XGMP, Olympus USA; NA = 1.0 and WD = mm) was used on the Olympus FV1000. The Olympus FV1000 was equipped with filter set (460–500, 520–560, 525–625, 650–700 nm) and a mode-locked Ti:sapphire laser (Mai Tai DeepSee 690–1040 nm, Spectra-Physics). For the Caliber ID RS-G4, all images were acquired with a 20x objective. Imaging of 2D tissue slides was performed on an Olympus BX43 upright microscope for non-fluorescent histology and a Leica DM550 B inverted microscope for fluorescent histology. Gross imaging of whole brains was captured on a Leica M165 FC fluorescence stereoscope.
Fv1000
Manufactured by Olympus
Sourced in Japan, United States, Germany, China, France, United Kingdom, Canada
The FV1000 is a confocal laser scanning microscope designed for high-resolution imaging of biological samples. It features a modular design and advanced optics to provide clear, detailed images of cellular structures and processes.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (92 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (87 mentions)
Triton x 100, by Merck Group (42 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (39 mentions)
Fluoview software, by Olympus (37 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (37 mentions)
Bovine serum albumin (bsa), by Merck Group (29 mentions)
Hoechst 33342, by Thermo Fisher Scientific (28 mentions)
Vectashield, by Vector Laboratories (25 mentions)
Paraformaldehyde (pfa), by Merck Group (19 mentions)
Fv3000, by Olympus (17 mentions)
Hoechst 33342, by Merck Group (17 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (17 mentions)
Cm1950, by Leica (15 mentions)
Lsm 710, by Zeiss (15 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (15 mentions)
In situ cell death detection kit, by Roche (14 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (13 mentions)
Prolong gold, by Thermo Fisher Scientific (13 mentions)
Lsm 780, by Zeiss (13 mentions)
3 240 protocols using fv1000
1
Deep Tissue Imaging with Olympus and Caliber
2
Live Imaging of Oxidative Stress Response
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Four days post fertilization (dpf) wildtype larvae were treated with pentafluorobenzenesulfonyl-fluorescein (HPF) and 0.01% (3 mM) H2O2 or with HPF alone for 3 hours and static images were recorded using a FV1000 (Olympus) confocal microscope. Uninjured and amputated caudal fins of 3 dpf EPRE:GFP larvae were recorded in 12hr time-lapse movies (one stack every 30 min) on a FV1000 (Olympus) confocal microscope. Three dpf larvae of the Tg(NF-κB:EGFP) reporter strain59 (link) either untreated or treated with 0.01% (3 mM) H2O2 were imaged using a FV1000 (Olympus) confocal microscope. Both whole larvae (upper) and tail fins were imaged separately at the beginning and end (2hr post treatment) of an experiment. Quantitative analysis using relative mean fluoresces of the z-stack projected images using ImageJ were performed from three independent experiments using Surface Plot and Measurement tools.
3
Imaging Fluorescent Markers in Plant Gametes
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The accumulation of MpDUO1-Citrine was observed in isolated antheridia and sperm of MpDUO1-Citrineki plant. The Citrine fluorescence was detected in a range from 525 to 565 nm with confocal laser scanning microscopy (FV-1000; Olympus and LSM780; Zeiss) after excitation at 515 nm. An antheridiophore of the MpDUO1-Citrineki plant was hand-sectioned with a blade and immersed in 1 μg ml−1 4′,6-diamidino-2-phenylindole (DAPI) solution. Antheridia were isolated from the sections and observed immediately under a confocal microscope (FV-1000, Olympus) with the following setting: MpDUO1-Citrine was excited by 488-nm laser and detected by a GaAsP detector with 535−565-nm window; DAPI was excited by 405-nm laser and detected by a photomultiplier tube with 425−475-nm window.
Clover and mRuby2 in the transgenic Arabidopsis pollens were excited at 488 and 561 nm, respectively and detected in a range from 495 to 540 and from 566 to 600 nm, respectively, with confocal laser scanning microscopy (LSM780; Zeiss). In brief, 3-4 open flowers were collected in a microfuge tube containing 300 μl of the solution [0.1 M sodium phosphate (pH 7.0), 1 mM EDTA, 0.1% Triton X-100]. After brief vortexing and centrifugation, 15 μl of the pollen pellet was transferred to a microscope slide and imaged12 (link).
Clover and mRuby2 in the transgenic Arabidopsis pollens were excited at 488 and 561 nm, respectively and detected in a range from 495 to 540 and from 566 to 600 nm, respectively, with confocal laser scanning microscopy (LSM780; Zeiss). In brief, 3-4 open flowers were collected in a microfuge tube containing 300 μl of the solution [0.1 M sodium phosphate (pH 7.0), 1 mM EDTA, 0.1% Triton X-100]. After brief vortexing and centrifugation, 15 μl of the pollen pellet was transferred to a microscope slide and imaged12 (link).
4
Visualizing Autophagic Flux in HepG2 Cells
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HepG2 cells were transfected with a adenovirus expressing mCherry-GFP-LC3B fusion protein (Ad-mCherry-GFP-LC3B) (C3011, Beyotime Bio, China) according to the manufacturer’s instructions. After adenovirus transfection at a multiplicity of infection (MOI) of 20 for 24 h, the adenovirus-containing culture medium was removed and then the fresh complete culture medium was added to each well for another 24 h culture. The transfected cells were visualized using confocal microscope (FV1000, Olympus, Japan). LC3B punctate dots was calculated by using ImageJ 1.50b. For actin microfilaments in HepG2 cells with or without Ad-mCherry-GFP-LC3B transfection, cells were washed in PBS 2 times, fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.5% Triton X-100 for 3 min, washed 3 times in PBS, blocked with 1% BSA for 30 min, and stained with Texas Red®-X phalloidin (T7471, Invitrogen, USA) for 30 min and DAPI (4ʹ6′-diamidino-2-phenylindole, 760–4196, Roche, Germany) for 15 min at room temperature. Cells were sealed by 10% glycerol, kept in a dark place. The actin cytoskeleton (red) and nuclei (DAPI, blue) were visualized using confocal microscope (FV1000, Olympus, Japan). The average intensity of F-actin was measured by using ImageJ 1.50b.
5
Distinguishing Plum-expressing PFFs from Other Fluorescent Proteins
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To determine whether Plum-expressing PFFs could be distinguished from PFFs expressing different fluorescent proteins (e.g., EGFP and huKO), we carried out both in vitro fluorescence observations and flow cytometry analyses. PFFs that express EGFP [36 (link)] and huKO [22 (link)] were generated previously; we cocultured these PFFs with fibroblasts derived from Plum-expressing fetuses. The three cell types were inoculated onto a 35-mm dish (7.0 × 104 cells for each cell type) and observed by confocal microscopy (FV-1000, Olympus) at 24 h after the start of culture. To confirm that the three cell types could be fractionated using their different fluorescence colors, a cell mixture (1.0 × 106 cells of each cell type) was sorted using a BD FACSAria III cell sorter. For each cell type, 2.0 × 105 cells were sorted and inoculated onto a 35-mm dish; 24 h later, cells were
screened using a confocal microscope (FV-1000, Olympus). The excitation wavelengths and emission wavelengths of the three fluorescent proteins were as follows: EGFP, 488 nm/495–515 nm; huKO, 559 nm/561–610 nm; and Plum, 559 nm/ 650–750 nm.
screened using a confocal microscope (FV-1000, Olympus). The excitation wavelengths and emission wavelengths of the three fluorescent proteins were as follows: EGFP, 488 nm/495–515 nm; huKO, 559 nm/561–610 nm; and Plum, 559 nm/ 650–750 nm.
6
Collagen-Tumor Interactions in Murine Ovarian Cancer
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Female C57Bl/6 mice (Mus musculus) of varying ages (6–22mo) (n=12, Jackson Laboratories, Bar Harbor, ME) were injected intraperitoneally with 1–10×106 ID8-RFP murine EOC cells as previously described (49 (link)). Normal collagen was also examined in tumor free mice (n=30) as a component of a larger study. No randomization method was used. Animal procedures were carried out according to the regulations of the University of Notre Dame Animal Care and Use Committee. Mice were sacrificed by CO2 inhalation followed by cervical dislocation at 8–10 weeks post-injection. The parietal peritoneum was dissected, rinsed with PBS and placed between coverslips for imaging with the mesothelium side facing the objective (25X XLPlanN, 1.05na WATER) of the 2-Photon confocal microscope (Olympus FV1000, software FLUOVIEW FV1000). Using a Mai Tai DeepSee titanium-sapphire 690–1040nm laser, ID8 metastatic implants and peritoneal collagen were visualized by the RFP and SHG signals, respectively. The investigator was not blinded during animal study. Areas of peritoneum with collagen-tumor interactions present were specifically imaged. At 12% laser power, the 2-photon laser was set to 860 nm and emission was simultaneously collected at 425–465 nm and 575–625 nm for SHG and RFP, respectively.
7
Collagen-Tumor Interactions in Murine Ovarian Cancer
8
Retinal Histology and Imaging Protocol
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The retina was sectioned and IHC was performed as described previously.23 (link) GA was assessed by masked observers using the scoring system based on methods reported previously by Anderson et al.24 (link) A GA score of 4 or 5 indicated a high degree of GFAP, whereas a score of 1 to 3 indicated a low degree of GFAP staining. Apoptosis was evaluated by TUNEL. The number of TUNEL-positive cells (green) was recorded in three fields of each retina that covered 212 × 212 μm. Images were acquired with a confocal laser scanning microscope (FV1000, Olympus. Hamburg, Germany).
Microglial activation was assessed by ionized calcium-binding adapter molecule 1 (Iba-1) immunofluorescence. Retina sections were incubated overnight in a humid chamber at 4°C with goat anti-Iba-1 (Abcam, Madrid, Spain). Then, samples were washed and incubated for 60 minutes with anti-goat Alexa 488 (1:200; Molecular Probes, Eugene, OR, USA). After washing, sections were mounted in Vectashield (Vector Labs, Burlingame, CA, USA) mounting medium with 4′,6-diamidino-2-phenylendole (DAPI). Images were acquired with a confocal laser scanning microscope (FV1000; Olympus) with a ×60 objective. Images size were 1024 × 1024 pixels. Fluorescent values were obtained with the software Olimpus Fluoview (v.4.2) in the confocal images (oib) with an ROI plugin and with background substraction.
Microglial activation was assessed by ionized calcium-binding adapter molecule 1 (Iba-1) immunofluorescence. Retina sections were incubated overnight in a humid chamber at 4°C with goat anti-Iba-1 (Abcam, Madrid, Spain). Then, samples were washed and incubated for 60 minutes with anti-goat Alexa 488 (1:200; Molecular Probes, Eugene, OR, USA). After washing, sections were mounted in Vectashield (Vector Labs, Burlingame, CA, USA) mounting medium with 4′,6-diamidino-2-phenylendole (DAPI). Images were acquired with a confocal laser scanning microscope (FV1000; Olympus) with a ×60 objective. Images size were 1024 × 1024 pixels. Fluorescent values were obtained with the software Olimpus Fluoview (v.4.2) in the confocal images (oib) with an ROI plugin and with background substraction.
9
Characterizing Hydrolytic Compartments in Trypanosoma Epimastigotes
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For detection of hydrolytic compartments, Y-GFP strain epimastigotes previously treated in the absence (DMSO) or the presence of 10 µM carvedilol in Diamond (control) or starvation medium (Stv) for 24 h, were incubated with 10 μg/ml of DQ-BSA for the last 40 min, washed three times with PBS and then mounted on coverslips with Mowiol before examination. This compound emitted red fluorescence after BSA hydrolysis into small peptides in lysosomes, thus identifying lytic compartments. Data were represented using the mean values of the percentage of DQ-BSA positive parasites (parasites with more than 2 DQ-BSA positive vesicles) ± SE, observed in a confocal microscope (Olympus FV 1000). For Lysotracker studies, epimastigotes previously treated in the absence (DMSO) or the presence of 10 µM carvedilol in Diamond (control) or starvation medium (Stv) for 24 h, were incubated with 10 μg/ml of Lysotracker Red for the last 2 h, washed three times with PBS and then mounted on coverslips with Mowiol before examination in a confocal microscope (Olympus FV 1000). All parasites stained with Lysostracker were counted and expressed as percentage of Lysotracker positive parasites.
10
Immunostaining and Neuron Morphometrics
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Immunostaining was then performed as previously described [32, 33] . Mice were transcardially perfused with 4% paraformaldehyde (PFA), post xed at 4°C overnight, cryoprotected in 30% sucrose, and embedded in optimum cutting temperature (OCT) compound. Sections (25 μm thick) were obtained by a Leica cryostat (CM 3050S). Immunostaining was performed as previously described. Rabbit anticalretinin (Millipore, AB5054, 1:1000), mouse anti-RFP (Abcam, ab125244, 1:500) and mouse anti-vGlut2 (Synaptic System, 135311, 1:500) were used as primary antibodies, and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, A11008, 1:500) and Alexa Fluor 633 goat anti-mouse IgG (Molecular Probes, A21050, 1:500) were used as secondary antibodies. Before coverslips were applied, the slides were incubated with DAPI (Sigma, D9564, 1:1000) for 15 min. Pictures were captured by a confocal microscope (Olympus, FV1000).
Images of dendritic arbors were captured under a 40x objective lens with a confocal microscope (Olympus, FV1000) in Z-stack mode [34] . Neuron morphology was traced manually using the NeuronJ plugin in ImageJ software. Standard morphometric analysis (Sholl analysis) was conducted as described earlier. Signi cance was determined by a two-way repeated-measures analysis of variance (RM 2-ANOVA; genotype and circle radius as factors).
Images of dendritic arbors were captured under a 40x objective lens with a confocal microscope (Olympus, FV1000) in Z-stack mode [34] . Neuron morphology was traced manually using the NeuronJ plugin in ImageJ software. Standard morphometric analysis (Sholl analysis) was conducted as described earlier. Signi cance was determined by a two-way repeated-measures analysis of variance (RM 2-ANOVA; genotype and circle radius as factors).
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