Imaging was carried out using a home-built total internal reflection fluorescence (TIRF) microscope consisting of a Ti-E Eclipse inverted microscope (Nikon) with an oil-immersion objective (UPlanSApo, 100×, 1.49 NA; Olympus) and a perfect focus system. AF647 was excited with a 638 nm laser (iBeam-Smart, Toptica) and coupled into the sample by a dichroic mirror (Di01-R405/488/561/635, Semrock). Emission was collected by the objective lens and separated from excitation light using a dichroic mirror (Di01-R405/488/561/635; Semrock) and passed through appropriate emission filters (FF01-692/40-25; Semrock). The emitted fluorescence was then focused onto an air-cooled EMCCD camera (Photometrics Evolve, EVO-512-M-FW-16-AC-110). Image stacks of 50 frames were acquired with an exposure time of 50 ms. To minimize any bias associated with the ROI selection, an automated script (Micro-Manager) was used to collect images in a grid. Images were analyzed using a custom-written ImageJ script analyzing the maxima in each image.
Di01 r405 488 561 635
Manufactured by IDEX Corporation
Sourced in United States
The Di01-R405/488/561/635 is a multi-line laser combiner that combines up to four distinct laser wavelengths into a single collimated output beam. It features individual laser input channels for 405 nm, 488 nm, 561 nm, and 635 nm wavelengths.
Automatically generated - may contain errors
Lab products found in correlation
Ibeam smart, by Toptica (3 mentions)
Ilas2 motorized tirf illuminator, by Teledyne (3 mentions)
Diaphot 200, by Oxford Instruments (2 mentions)
Apon60xo tirf, by Olympus (2 mentions)
Ff01 692 40 25, by IDEX Corporation (2 mentions)
Ff01 446 523 600 677, by IDEX Corporation (2 mentions)
Uplansapo 100 1.4 na, by Olympus (1 mentions)
Lp02 514re, by IDEX Corporation (1 mentions)
Ff560 fdi01, by IDEX Corporation (1 mentions)
Nf03 561e, by IDEX Corporation (1 mentions)
Ixon du 897, by Oxford Instruments (1 mentions)
Eclipse ti e microscope, by Nikon (1 mentions)
Du897, by Oxford Instruments (1 mentions)
Eclipse ti e inverted microscope, by Nikon (1 mentions)
Blp01 488r 25, by IDEX Corporation (1 mentions)
Ff01 480 40 25, by IDEX Corporation (1 mentions)
Ff01 676 37 25, by IDEX Corporation (1 mentions)
Plan apo, by Nikon (1 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
Ti inverted microscope, by Nikon (1 mentions)
17 protocols using di01 r405 488 561 635
1
TIRF Microscopy for Single-Molecule Imaging
2
Superlocalization Imaging of Single Proteins
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Caulobacter cells are around 0.5-µm diameter by 2- to 4-µm long, which is on the same order of magnitude as the diffraction limit (∼250 nm). To accurately track movements of single proteins in live cells, we use superlocalization to pinpoint the location of single molecules where photobleaching was used to reduce the emitting concentration (44 (link), 48 (link), 79 (link)). Live cells were immobilized on agarose pad that was bleached overnight before the experiment. Single-molecule imaging experiments were performed on a custom epifluorescence microscope (Nikon; Diaphot 200) equipped with a Si EMCCD camera (Andor; iXon DU-897) and a high numerical aperture (N.A.) oil-immersion objective (Olympus; UPlanSapo, 100×/1.4 N.A.). Molecules were excited with either 514-nm, 1-W continuous-wave laser or 642-nm, 1-W continuous-wave laser (MPB Communications) and photoactivated by a 405-nm Obis laser at 0.8 kW/cm2, 0.65 kW/cm2, and 0.1 to 1 W/cm2, respectively. The emission from fluorescent molecules was collected through a four-pass dichroic mirror (Semrock; Di01-R405/488/561/635) and filtered by a 514-nm long-pass filter (Semrock; LP02-514RE), a 560-nm dichroic beam splitter (Semrock; FF560-FDi01), a 561-nm notch filter (Semrock; NF03-561E), and a bandpass filter (Semrock; FF01-532/610) as previously described (48 (link), 79 (link)).
3
Dual TIRF-SRIC Microscopy Technique
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The same setup as above was used except that a mercury lamp (X-Cite 200DC, 200 W, Excelitas, Waltham, MA) was used as the light source. The mercury lamp had a spectrum output at wavelengths between 340 and 800 nm. A bandpass filter (609/54; Semrock) and neutral density filters (ND4) were used. Near-parallel rays produced by closing the aperture diaphragm obliquely illuminated the sample through the TIRF-CFI objective (100X N.A. 1.49, Nikon). Reflected light rays from two interfaces (between coverslip and solution and between solution and plasma membrane) interfered with each other, creating a bright or dark patch when they were constructive or destructive, respectively. In order to perform sequential TIRF/SRIC with fast frame rate, mechanical changes of the optical parts were reduced to a minimum level. A 30/70 beam splitter was used at the back port of the L-shape fluo-illuminator of the Nikon microscope to combine the white light source (mercury lamp used for SRIC) and the lasers (used for TIRF). The same quad-bandpass dichroic cube (Di01-R405/488/561/635, Semrock) used for TIRF was used for SRIC, which allows an estimated 2% of incident light (609/54) to be reflected during the SRIC acquisition. Similar results were obtained using a Nikon SRIC cube composed of excitation filter 535/50, dichroic mirror DM400, and neutral density filter ND16.
4
TIRF Microscopy of α-Synuclein Fibrillization
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Imaging was performed using a homebuilt total internal reflection fluorescence microscope11 (link). This imaging mode restricts detectable fluorescence signal to within 200 nm from the sample slide. For imaging of WT α-synuclein in the presence of 5 μM ThT, the output from laser operating at 405 nm (Oxxius LaserBoxx, LBX-405-100-CIR-PP) was aligned and directed parallel to the optical axis at the edge of a 1.49 NA TIRF objective (APON60XO TIRF, Olympus, N2709400), mounted on an inverted Nikon Eclipse TiE microscope. Fluorescence was collected by the same objective and was separated from the returning TIR beam by a dichroic (Di01-R405/488/561/635, Semrock), and passed through an emission filter (BLP01-488R). The images were recorded on an EMCCD camera (Evolve 512, Photometrics) operating in frame transfer mode (EMGain of 11.5 e–/ADU and 250 ADU/photon). Each pixel was 160 nm in length. For each data set, 3 × 3 image grids were measured in three different regions of the cover slide. The distance between the nine images measured in each grid was set to 350 μm, and was automated (bean-shell script, micromanager) to prevent user bias. Images were recorded at 50 frames s−1 for 100 frames with 405 nm illumination (150–200 W/c2).
5
Quantitative 3D Imaging of Fluorescent Molecules
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Cells were imaged on a custom epifluorescence microscope using a Nikon Diaphot 200 as core, equipped with an Andor Ixon DU-897 electron-multiplying charge-coupled device camera, a high-NA oil-immersion objective (UPlanSapo 100×/1.40 NA; Olympus), a motorized xy-stage (M26821LOJ; Physik Instrumente), and a xyz-pizeo stage (P-545.3C7; Physik Instrumente). Molecules were excited with a 642-nm, 1-W continuous-wave laser (MPB Communications Inc.). The emission was passed through a quadpass dichroic mirror (Di01-R405/488/561/635; Semrock) and filtered using a ZET642 notch filter (Chroma) and a 670/90 bandpass filter (Chroma). For 3D imaging, DH (Double Helix Optics) and Tetra6 phase masks (described in ref. 42 (link)) were inserted into the 4f-system of the microscope as described previously (43 (link)).
6
Imaging Amyloid Aggregation Using TIRF Microscopy
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Imaging experiments were carried out with bespoke TIRF inverted microscope (Eclipse TE2000-U; Nikon) fitted with a Perfect Focus unit. Excitation of ThT and AF647 was achieved with either a 405-nm laser (LBX-405-50-CIR-PP; Oxxius) or 641-nm laser (Cube, 1150205; Coherent), respectively. The beams were aligned parallel to the optical axis and directed into an oil immersion objective lens (1.49 numerical aperture [N.A.], 60×, Plan Apo, TIRF; Nikon) above the critical angle to ensure TIR at the coverslip/sample (glass/water) interface. Fluorescence emission was also collected by the same objective and selected by the presence of a dichroic (Di01-R405/488/561/635; Semrock) and subsequently passed through appropriate emission filters (BLP01-488R-25, FF01-480/40-25, and FF01-676/37-25; Semrock). Image stacks of the AF647 and ThT emission channels were collected by sequential excitation of AF647 followed by ThT. Images were recorded by an electron multiplying charge-coupled device (Evolve delta 512; Photometrics) with an electron multiplication gain of 250 analog-to-digital units per photon running in frame transfer, clear presequence mode. Each pixel on the image was 237 nm. Images from 27 different fields of view were recorded at 50 ms for 200 frames in each emission channel using a custom beanshell script through Micromanager software (v. 1.4).
7
Live Imaging of Mitochondria-Lysosome Interactions
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The R18-labeled LEs (LE-R18) were isolated from HUVECs treated with 20 μM R18 dye. The MitoTracker Green-labeled Mito were isolated from HUVECs without R18 treatment. Purified LE (Red, R18 lipid dye labeled) and Mito (labeled by MitoTracker Green) were mixed in PBS in a poly-d -lysine (PDL)-coated Petri dish. Their direct interaction was examined by TIRFM performed using a Nikon Ti inverted microscope with three laser lines (491, 561 and 642 nm). The microscope was equipped with an iLAS2 motorized TIRF illuminator (Roper Scientific GmbH) and with a Prime 95B sCMOS camera (Photometrics, 16 bit, pixel size 11 μm). All TIRF images were acquired using Nikon objectives (Apo TIRF 100×, N.A. 1.49 oil). Samples were imaged in two channels by sequential excitation with the laser at 491 and 561 nm through a quad-bandpass filter (Di01-R405/488/561/635, Semrock, Rochester, NY, USA) for both channels, a 520/35 filter (Semrock) for 491 nm channel, and a 641/75 filter (Semrock) for 561 nm channel. All images were acquired at 1 Hz. The microscope was controlled by Metamorph 7.10 software (Universal Imaging, Bedford Hills, NY, USA). Time-lapse images were taken per second for 10 min. All images were acquired at 37 °C throughout the experiment. The kinetic curve of the fluorescent intensity of the red (R18) and green (MitoTracker Green) channels was generated by ImageJ.
8
Quantifying Ca2+ Influx in Vesicles Using TIRFM
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The measurements of Ca2+ influx into individual vesicles was performed using a home-built total internal reflection fluorescence microscope (TIRFM) based on an inverted Olympus IX-71 microscope. A 488-nm laser (Toptica, iBeam smart, 200 mW, Germany) beam was expanded and focused on the back focal plane of the 60×, 1.49 NA oil-immersion objective lens (APON60XO TIRF, Olympus, N2709400) to excite the Cal-520 dye incorporated into the vesicle. The emerging fluorescence was collected by the same objective. The emission beam was separated from the excitation beam by a dichroic (Di01-R405/488/561/635, Semrock), passed through a set of filters (BLP01-488R, Semrock and FF01-520/44-25, Semrock), and imaged onto an air-cooled EMCCD camera (Photometrics Evolve, EVO-512-M-FW- 16-AC-110). Excitation power density was fixed to ∼10 W/cm2 for 50 frames with a scan speed of 20 Hz and bit depth of 16 bits.
9
Live Cell Imaging of Axon Degeneration
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Images were obtained using a confocal microscope system (FV1000, Olympus) equipped with a 60× oil immersion lens (NA 1.42, Olympus) and 488, 561, 640 nm lasers. Images were collected at an additional electronic zoom factor of 7× (DiI and immunostaining) or 5× (GFP and PSD-95-TagRFP) and multiple optical slices with 0.5–1 µm in z-steps and the total thickness of 5–10 µm.
Live cells were placed in a custom-made chamber at 37°C with a continuous flow of 5% CO2 to maintain pH of the medium. To prevent evaporation of the medium, dishes were covered by a custom-made lid which can pass air and CO2. Live cell imaging was performed with a custom-made fluorescent microscope system based on an inverted microscope (IX81, Olympus), equipped with a 100× oil immersion lens (NA 1.49, Olympus), a Z-Drift Compensation System (Olympus), a motorized XY stage and an EMCCD camera (iXon3, Andor). MetaMorph software (Universal Imaging) was used to control filter wheels and z-axis controller. Light from a metal halide lamp (PhotoFluor II, Chroma) was passed through single band exciters, reflected by a dichroic mirror (Di01-R405/488/561/635, Semrock). Fluorescence signal was detected by the camera operated with EM Gain. Single XY images were obtained with intervals of 5 min from 3 to 15 h after axon cutting.
Live cells were placed in a custom-made chamber at 37°C with a continuous flow of 5% CO2 to maintain pH of the medium. To prevent evaporation of the medium, dishes were covered by a custom-made lid which can pass air and CO2. Live cell imaging was performed with a custom-made fluorescent microscope system based on an inverted microscope (IX81, Olympus), equipped with a 100× oil immersion lens (NA 1.49, Olympus), a Z-Drift Compensation System (Olympus), a motorized XY stage and an EMCCD camera (iXon3, Andor). MetaMorph software (Universal Imaging) was used to control filter wheels and z-axis controller. Light from a metal halide lamp (PhotoFluor II, Chroma) was passed through single band exciters, reflected by a dichroic mirror (Di01-R405/488/561/635, Semrock). Fluorescence signal was detected by the camera operated with EM Gain. Single XY images were obtained with intervals of 5 min from 3 to 15 h after axon cutting.
10
Multimodal Microscopy Imaging Protocol
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Epi-fluorescent images were acquired using a Nikon inverted Ti-S microscope equipped with a 100× oil immersion objective (CFI Plan Fluor ADH, NA 1.30; Nikon). Samples were illuminated using a light engine (Lumencor), and images were acquired using a charge-coupled device (CCD) camera (Coolsnap HQ; Photometrics). TIRF time-lapse images were acquired using a Nikon inverted Ti-S microscope equipped with a 100× oil immersion objective (CFI Apochromat TIRF, NA 1.49; Nikon) and an electron-multiplying CCD camera (Evolve 512; Photometrics) or sCOMS camera (Prime95B; Photometrics). Excitation laser (iLas2; Roper Scientific) was supplied at wavelengths of 488 nm (100 mW), 561 nm (100 mW), or 642 nm (100 mW) reflected from a multi-band dichroic mirror (Di01-R405/488/561/635; Semrock). The emitted light was filtered through multi-band emission filter (FF01-446/523/600/677; Semrock).
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