Confocal scanning fluorescence microscope system was employed to measure the fluorescence intensity and lifetime of single QDs53 (link)55 (link). The system was equipped with a picosecond pulsed diode laser (PDL800-D PicoQuant) with a central wavelength of 635 nm, output pulse width of 55 ps, and repetition frequency of 80 MHz. The laser light was led to an inversion microscope (Nikon ECLIPSE TE2000-U) through a single mode polarization maintain fiber. A λ/2 and a λ/4 wave-plate were used to change the linearly polarized laser into circular polarization light. An oil immersion objective (Nikon, 100×, 1.3 NA) was used to focus laser light onto the sample and collect fluorescence simultaneously. The fluorescence, passing through a dichroic mirror (BrightLine, Semrock), an emission filter (BrightLine, Semrock), and a notch filter (BrightLine, Semrock), was focused into a 100 μm pinhole for spatial filtering to reject out-of-focus photons. Finally, the fluorescence was collected by a single photon detector (PerkinElmer, SPCM-AQR-15). A piezo-scan stage (Tritor 200/20 SG) with an active x-y-z feedback loop mounted on the inversion microscope was used to scan the sample over the focused excitation spot. A time-to-amplitude converter (TAC, ORTEC) and a multi-channel analyzer (MCA, ORTEC) were used to measure the fluorescence decay curves to obtain fluorescence lifetime of QDs.
Brightline
Manufactured by IDEX Corporation
Sourced in Japan, United States
BrightLine is a piece of lab equipment designed for fluid analysis applications. It provides high-quality illumination to enhance visibility and clarity during sample observation and testing.
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Lab products found in correlation
Csu x1, by Yokogawa (4 mentions)
Ix81 microscope, by Olympus (2 mentions)
Fp1500urt, by Thorlabs (2 mentions)
Mvx stereo macroscope, by IDEX Corporation (2 mentions)
Orca flash4.0 v2, by Hamamatsu Photonics (2 mentions)
Labview, by National Instruments (2 mentions)
Revolution xd, by Oxford Instruments (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Acton spectrometer, by Teledyne (1 mentions)
Ldh d c 640, by PicoQuant (1 mentions)
Uplansapo 60x 1.2w, by Olympus (1 mentions)
Tau spad, by PicoQuant (1 mentions)
Hydraharp 400, by PicoQuant (1 mentions)
Fv1200, by Olympus (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Bx51w, by Olympus (1 mentions)
Pdl 800 d, by PicoQuant (1 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
Spcm aqr 15, by PerkinElmer (1 mentions)
Brightline single band bandpass filter, by IDEX Corporation (1 mentions)
22 protocols using brightline
1
Confocal Fluorescence Microscopy of Single QDs
2
Fluorescence Microscopy Imaging with Ibidi Slides
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Optical and fluorescence microscopy images were recorded on an Olympus UIS2 microscope, equipped with a motorized stage (Prior, Optiscan II). Fluorescent images were recorded with an EMCCD camera (Andor, iXon), using illumination from a mercury lamp, an excitation filter of 482/18 nm (Semrock BrightLine) and an emission filter of 525/45 nm (Semrock BrightLine).
Samples were loaded into the wells of PLL-g-PEG-functionalized Ibidi μ-slides and closed with a lid (microscopy chambers).
Samples were loaded into the wells of PLL-g-PEG-functionalized Ibidi μ-slides and closed with a lid (microscopy chambers).
3
Fluorescence Microscopy Imaging Protocol
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Images were recorded on an Olympus UIS2 microscope, equipped with a motorized stage (Prior, Optiscan II). Fluorescent images were recorded with an EMCCD camera (Andor, iXon), using illumination from a mercury lamp, an excitation filter of 482/18 nm (Semrock BrightLine) and an emission filter of 525/45 nm (Semrock BrightLine). Images were analyzed and prepared for presentation in ImageJ.
4
Polarization Measurement Protocol
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For polarization measurements, the prepared sample on a mica substrate is excited with a Coherent 405 nm continuous wave laser via a Nikon 0.80 numerical aperture 20× objective. For measuring emission polarization, the incoming laser light (which is naturally strongly linearly polarized) is circularly polarized with a quarter-wave plate (AQWP05M-600) to ensure uniform excitation. The emission is filtered with a Semrock BrightLine 409-nm longpass filter to remove the laser light, and then sample emission passes through a half-wave plate (HWP) (AHWP05M-600) and a wire-grid polarizer (WP25M-VIS). For acquiring energy spectra, the light was directed into a Princeton Instruments Acton spectrometer and a Princeton Instruments ProEM 512 × 512 charge-coupled device array. As the HWP is rotated, the change intensity is recorded by integrating the entirety of the spectral range (roughly 533 to 700 nm), as it passes through the parallel (perpendicular) polarizer, and the degree of polarization at each detection angle is calculated using Eq. 5 (6 (link)) where I|| and I⊥ are the parallel and perpendicular intensities, respectively.
5
Optical Characterization of hBN Emitters
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Optical measurements were performed in a setup as shown in Supplementary Note 10 . Broad band emission in hBN and single emitters were excited by a continuous-wave 532 nm laser (Gem 532, Laser Quantum Ltd.). The laser beam passed a polarizer and a half waveplate, and was focused using a high numerical aperture objective lens (NA = 0.9, Nikon). The acquired signal from the sample was collected using the same objective lens, then separated from excitation by a dichroic mirror (BrightLine, Semrock). After the dichroic mirror, further spectral filtering was achieved using a tuneable 20 nm bandpass filter. A flip mirror guided the light either to a Hanburry-Brown and Twiss (HBT) Interferometer or a Spectrometer (Acton SpectraPro, Princeton Instrument Inc.). The HBT setup consists of two avalanche photodiodes (APD, Excelitas Technologies), with a 100 ns delay time induced in one of the APDs connected to a time-correlation card (PicoHarp 300).
6
Confocal FCS of Cy5 in Aqueous Solution
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FCS measurements were performed on a commercial, epi-illuminated, confocal laser scanning microscope (Olympus, Tokyo, Japan, FV1200). Cy5 in aqueous solution was excited with a focused beam (338 nm, 1/e2 radius) of a 638 nm laser (LDH-D-C-640 from PicoQuant GmbH, Berlin, Germany) in continuous wave. The emitted fluorescence was collected back through the microscope objective (UPlanSApo 60x/1.2 w, Olympus, Tokyo, Japan), passed through a dichroic mirror (ZT405/488/635rpc-UF2, Chroma), an emission filter (HQ720/150, Chroma, 680 nm blocking edge Brightline, Semrock, in combination with last mentioned filter or 710/40 Brightline, Semrock), and focused onto a pinhole (50 µm diameter) in the back focal plane. The fluorescence signal was finally split and directed on two avalanche photodiodes (Tau-SPAD, PicoQuant GmbH, Berlin, Germany), whose signals were collected with a data acquisition card (Hydraharp 400, Picoquant, Berlin, Germany).
7
Whole-Brain Light Field Microscopy
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Whole brain recordings were performed using light field microscopy as described in detail elsewhere (Aimon et al., 2019 (link)). In short, a modified upright Olympus BX51W with a 20x NA 1.0 XLUMPlanFL (Olympus) was used. An adequate microlens array (RPC Photonics) positioned at the image plane and two relay lenses (50 mm f/1.4 NIKKOR-S Auto from Nikon) projected the image onto the sensor of a scientific CMOS camera (Hamamatsu ORCA-Flash 4.0). A 490 nm LED (pE100 CoolLED) at approximately 10% of its full power was used for excitation. As filter set we used a 482/25 bandpass filter, a 495-nm dichroic beam splitter, and a 520/35 bandpass emission filter (BrightLine, Semrock). The recording was performed at a frame rate of 200 Hz. The whole brain volume was reconstruction from the light field image as described in Aimon et al. (2019) (link).
8
Visualizing Actin-Tropomyosin Interactions
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Actin-tropomyosin interactions were observed by using total internal reflection fluorescence (TIRF) inverted microscope (TILL Photonics) equipped with a Zeiss 100× Plan-apochromat NA 1.46 oil objective and Andor iXon 897U EMCCD cameras. Lasers used were 488 nm and 640 nm (Toptica photonics AG) with 525/50-25 and 697/75-25-D single-band filters, respectively (BrightLine, Semrock). Time-lapse images were acquired typically at 10 Hz for Tpm association and 1 Hz for dissociation. The laser power was 0.44 mW and 0.30 mW for the 488 nm and 640 nm lasers, respectively. The exposure time was 20 ms and the multiplication gain was 300 for both channels.
9
Ratiometric pH Imaging Protocol
10
Multimodal Surgical Microscope for FD-FLIM and Spectroscopy
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Frequency-domain fluorescence lifetime imaging (FD-FLIM) and spectroscopic measurements were performed with a multimodal surgical microscope as recently described (15 (link), 16 (link)). We acquired FD-FLIM images via raster-scanning at a working distance of 200 mm. The field of view was 6.5 x 6.5 mm2. Fluorescence was filtered in the range of flavin emission from 500 nm to 580 nm (BrightLine HC Semrock, Rochester, New York) for FD-FLIM. Fluorescence spectra were acquired on spatially correlated areas (0.6 x 0.6 mm2) from 430 nm to 740 nm. Our system employed a diode laser (phoxX-405, Omicron Laserage, Rodgau, Germany) at 405 nm excitation with 6 mW of laser power at the sample plane. Post-processing routines for reconstructing the fluorescence lifetime and intensity images as well as for processing the acquired spectra were implemented in Python (RRID : SCR_008394) as described previously (16 (link)).
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