We fixed the brains of awake mice and imaged them in a calcium fluorescence imaging system (see Supplement Fig. 1 ) as previously described71 (link). We used a frame rate of 30 Hz and a resolution of 512 × 512 with 4 × 4 binning. Illumination was provided by a high-power mercury lamp (UHGLGPS, Olympus; 130 W) through a liquid light guide (ULLG150/300, Olympus). The excitation beam, ~488 nm for GCaMP fluorescence, was created by an excitation filter (FF01-480/40-25, Semrock) and reflected onto the cortical surface with a dichroic mirror (FF495-Di0325 × 36, Semrock). Video was captured using an sCMOS camera (16 bits, 65 × 6.5 μm, Flash4.0V2C11440-22CU, Hamamatsu, Japan), with a dichroic mirror filtering out GCaMP6f excitation light. The objective lens was defocused down by ~400 μm to minimize vascular artifacts. Fluorescence imaging was performed in a darkened, soundproof chamber after 15 min of acclimation. We monitored the mice via a body camera throughout the imaging process. The fluorescent signals showed obvious changes, as demonstrated in Supplementary Fig. 1 . Under anesthesia, electroencephalogram (EEG) data were monitored using two stainless steel screws implanted into the prefrontal cortex.
Ff01 480 40 25
Manufactured by IDEX Corporation
The FF01-480/40-25 is a narrow bandpass filter that transmits light within a specific wavelength range. It has a center wavelength of 480 nm and a bandwidth of 40 nm. The filter is 25 mm in diameter.
Automatically generated - may contain errors
Lab products found in correlation
Ff495 di03 25 36, by IDEX Corporation (1 mentions)
U hglgps, by Olympus (1 mentions)
Blp01 488r 25, by IDEX Corporation (1 mentions)
Ff01 676 37 25, by IDEX Corporation (1 mentions)
Di01 r405 488 561 635, by IDEX Corporation (1 mentions)
Plan apo, by Nikon (1 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
Di01 405 488 532 635, by IDEX Corporation (1 mentions)
3 protocols using ff01 480 40 25
1
In Vivo Calcium Imaging of Awake Mouse Brains
2
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).
3
Colocalization Imaging via TIRF Microscopy
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Colocalization experiments were performed on a bespoke TIRF setup. Continuous-wave solid-state lasers operating at 488 nm and 641 nm were used for imaging. The beam power was controlled by attenuation through a neutral density filter, following which undesirable wavelengths were filtered out through excitation filters (LL01-488-25, FF01-640/14-25, Semrock). The beams were then circularly polarised by a quarter wave plate. Laser lines were combined by dichroic mirrors. The beams were then passed through a Köhler lens into the back aperture of an inverted microscope body (Olympus, IX73) and were reflected by a dichroic mirror (Di01-405/488/532/635, Semrock) through an oil immersion objective (Olympus, 60XOTIRF). Emitted radiation was collected through the same objective and passed through the dichroic before being filtered (FF01-480/40-25, LP02-647RU-25, Semrock). The fluorescence emission was then projected onto an EMCCD camera (Evolve Delta, Photometrics), each pixel was 275 nm in length. Data visualization was achieved through Micro-Manager and ImageJ software.
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