PM sheets were imaged in a confocal setup Microtime 200 (PicoQuant) using an inverted microscope (IX73, Olympus) equipped with an oil objective lens (UPlanSApo 100 × 1.4 NA, Olympus). A fiber-coupled pulsed diode laser (LDH-D-C-485, PicoQuant) was used for 485 nm excitation at 10 MHz repetition rate. Fluorescence was separated from the excitation light by a dichroic mirror (Zt488/640rpc, Chroma) and split into four fractions by three 50:50 beam splitters (Beck Optronic Solutions Limited). Additional fluorescence filters (LP488, Semrock, and SP750, Chroma) were used in front of each of the four SPAD detectors (SPCM-AQRH-14-TR, Excelitas). Data were acquired with a time-correlated single-photon counting unit (HydraHarp 400, PicoQuant). Images were further analyzed with costum-written MATLAB routines (The Mathworks Inc).
Spcm aqrh 14 tr
Manufactured by Excelitas
Sourced in United States
The SPCM-AQRH-14-TR is a single-photon counting module manufactured by Excelitas. It is designed to detect single photons with high efficiency and low noise. The module features a high quantum efficiency, low dark count rate, and fast timing resolution. The core function of the SPCM-AQRH-14-TR is to enable the detection and counting of single photons in a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Ldh d c 485, by PicoQuant (2 mentions)
Matlab routine, by MathWorks (1 mentions)
Uplansapo 100 1.4 na, by Olympus (1 mentions)
Hydraharp 400, by PicoQuant (1 mentions)
Microtime 200, by PicoQuant (1 mentions)
Ldh p fa 530 b, by PicoQuant (1 mentions)
Ldh d c 640, by PicoQuant (1 mentions)
Microtime 200 confocal microscope, by PicoQuant (1 mentions)
Zt640rdc, by Chroma Technology (1 mentions)
Spcm cd3516h, by Excelitas (1 mentions)
Ix73 microscope, by Olympus (1 mentions)
Uplsapo60xw, by Olympus (1 mentions)
Immersol w 2010, by Zeiss (1 mentions)
4 protocols using spcm aqrh 14 tr
1
Confocal Microscopy with Pulsed Laser
2
Pulsed Interleaved Excitation for Solution-based smFRET
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pulsed Interleaved Excitation (PIE) and solution-based smFRET experiments were performed on a MicroTime 200 confocal microscope (PicoQuant, Berlin, Germany). Prior to the recording, microscope slides (170 µm thickness, No. 1.5 H precision cover slides, VWR Marienfeld, Leicestershire, Great Britain; LH26.1) were coated for at least one min with 1 mg/mL filtered (0.2 µm) bovine serum albumin (BSA) in 50 mM HEPES-K pH 7.0, after which the BSA solution was removed by pipetting and replaced by 150–200 µL of the sample.
The laser pulse rate was set at 40 MHz. Fluorophores were alternately excited, using a 532 nm (LDH-P-FA-530-B; PicoQuant, Berlin, Germany) and 638 nm (LDH-D-C-640; PicoQuant, Berlin, Germany) laser. The laser beam was focused 7 µm away from the glass-solution interface in the z-direction, by means of an oil-immersed objective lens (UPlanSApo 100x1.40 NA; Olympus, Tokyo, Japan). The emitted photons from the sample were coordinated through a 100 µm pinhole, separated through a laser beam-splitter (ZT640RDC; Chroma Technology, Bellows Falls, Vermont), filtered by either a HQ690/70 (Chroma Technology, Bellows Falls, Vermont) or a 582/75 (Semrock, Rochester, New York) emission filter, and recorded by two photon counting modules (donor photons: SPCM-AQRH-14-TR, acceptor photons: SPCM-CD-3516-H; Excelitas Technologies, Waltham, Massachusetts).
The laser pulse rate was set at 40 MHz. Fluorophores were alternately excited, using a 532 nm (LDH-P-FA-530-B; PicoQuant, Berlin, Germany) and 638 nm (LDH-D-C-640; PicoQuant, Berlin, Germany) laser. The laser beam was focused 7 µm away from the glass-solution interface in the z-direction, by means of an oil-immersed objective lens (UPlanSApo 100x1.40 NA; Olympus, Tokyo, Japan). The emitted photons from the sample were coordinated through a 100 µm pinhole, separated through a laser beam-splitter (ZT640RDC; Chroma Technology, Bellows Falls, Vermont), filtered by either a HQ690/70 (Chroma Technology, Bellows Falls, Vermont) or a 582/75 (Semrock, Rochester, New York) emission filter, and recorded by two photon counting modules (donor photons: SPCM-AQRH-14-TR, acceptor photons: SPCM-CD-3516-H; Excelitas Technologies, Waltham, Massachusetts).
3
Fluorescence Microscopy Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescence measurements were performed on a Microtime 200 device (PicoQuant) with an integrated laser line at 488 nm in pulsed mode (20 MHz), along with an Olympus IX73 microscope and a detection unit containing two single-photon avalanche diodes (SPCM-AQRH-14-TR; Excelitas). To detect signal from different flanks of the spectrum two bandpass filters at 520 nm (10 nm) and 530 nm (10 nm) were used in combination with a high numerical aperture (1.2), 60× water immersion objective (UPLSAPO60XW; Olympus). Instead of water an oil substitute with similar spectral properties as water was used (Immersol W2010; Zeiss) to avoid evaporation of the immersion fluid during the measurement cycle. For cell as well as vesicle measurements a chamber (FCS2; Bioptechs) was used which is closable by a coverslip containing the sample. The coverslip as part of the chamber was coupled to the immersion objective. The chamber was connected to a heating bath (Lauda Eco Silver). Outer pipes ensure a flow around the sample with heating bath fluids and allow temperature control of the sample. Temperature was measured with a thermometer (TMD-56; Amprobe) coupled to the chamber with an accuracy of 0.1 °C. However, absolute temperature values may be affected by the coupling of the chamber to the nonheated immersion objective depending on the extent of the deviation between sample and room temperature.
4
Fluorescence Lifetime Imaging of GFP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescence lifetime measurements were performed using a MicroTime200 optical setup. GFP samples were placed as drops on top of 175 µm glass slides (Precision Cover Glass No:1.5H, Marienfeld), mounted on an inverted microscope (IX83 inverted, Olympus) with a 60× water immersion objective (UPlanSApo, Superapochromat, Olympus). A 485 nm pulsed-interleaved excitation laser (LDH-D-C-485, PicoQuant) with a repetition rate of 20 MHz (50 ns) was directed via a dichroic mirror (ZT473/594rpc, Chroma) and focused ~10 µm into the sample. The fluorescence emission signal passed through a 50 µm pinhole and an emission filter (HC520/35, Semrock). Photons were focused into a single-photon avalanche diode (SPCM-AQRH-14-TR, Excelitas) coupled to a counting module (PicoHarp 400, PicoQuant), and time-correlated single-photon counting (TCSPC) histograms were generated. Each sample was measured for 1–5 min with laser intensities between 2–20 µW, adjusted using OD filters to reach a photon count rate of ~20 kHz. The profile for the instrument response function (IRF) was obtained by measuring scattered light from a mirror. The fluorescence decay curves were fitted with a bi-exponential fluorescence decay model by iterative IRF-reconvolution to extract the characteristic lifetimes and weights of the GFP designs.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!