Spinning disk confocal intravital microscopy was performed using an Olympus BX51WI (Olympus, Center Valley, PA) upright microscope equipped with a 20×/0.95 XLUM Plan Fl water immersion objective. The microscope was equipped with a confocal light path (WaveFx, Quorum, Guelph, ON) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation, Tokyo, Japan). Laser excitation at 488, 561 and 649nm (Cobalt, Stockholm, Sweden), was used in rapid succession and fluorescence in green, red and blue channels was visualized with the appropriate long pass filters (Semrock, Rochester, NY). Exposure time for all wavelengths was between 500 and 600ms. Sensitivity settings were maintained at the same level for all experiments. A 512×512 pixels back-thinned EMCCD camera (C9100–13, Hamamatsu, Bridgewater, NJ) was used for fluorescence detection. Volocity Acquisition software (Improvision Inc., Lexington, MA) was used to drive the confocal microscope. Images captured using the spinning disk were processed and analyzed in Volocity 6.0.1. NET area and NET number were quantified using the Volocity software.
Long pass filter
Manufactured by IDEX Corporation
Sourced in United States
Long pass filters are optical devices that selectively transmit light at wavelengths longer than a specific cutoff wavelength, while blocking shorter wavelengths. They are commonly used in various scientific and industrial applications to isolate specific regions of the electromagnetic spectrum.
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Lab products found in correlation
Csu 10 head, by Yokogawa (2 mentions)
Wavefx, by Quorum Technologies (2 mentions)
Bx51wi, by Olympus (1 mentions)
Wavefx, by Yokogawa (1 mentions)
C9100 13, by Hamamatsu Photonics (1 mentions)
Volocity acquisition software, by PerkinElmer (1 mentions)
Uplansapo 10 0, by Yokogawa (1 mentions)
Uplansapo 20 0.70 air, by Yokogawa (1 mentions)
Spcm aqrh 15, by Excelitas (1 mentions)
6 protocols using long pass filter
1
Intravital Spinning Disk Confocal Microscopy
2
Fluorescence Lifetime Measurement Technique
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For fluorescence lifetime measurements, a mode-locked Ti:sapphire laser (Coherent Libra, 1 kHz repetition rate, 100 fs pulse width) at 800 nm, 45 degree incident angle was used for excitation. Fluorescence of IR-140 was collected by a × 5 and 34-mm working distance Mitutoyo objective. The excitation laser and fluorescence signal were filtered by 800 nm bandpass and 820 nm longpass filters (Semrock), respectively. The filtered fluorescence was then coupled to a single-mode fibre and guided to the entrance slit of a spectrometer (Princeton Instrument SP2500). After being dispersed by a grating, the fluorescence was diverted to a second exit port of the spectrometer (∼1.5 nm bandwidth) and collected by a fast-timing avalanche photodiode (APD-PDM, Micro Photon Device). A TCSPC module (PicoHarp 300) with a time bin of 4 ps was used to analyse the number of photons as a function of time, when they arrived at the photodiode. Final lifetimes were obtained from fits to the data deconvolved with the instrument response function32 .
3
Spinning Disk Confocal Microscopy of Mice
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Spinning disk confocal microscopy was performed using an Olympus BX51 (Olympus, Center Valley, PA) upright microscope. The microscope used a confocal light path (WaveFx, Quorum) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation). Laser excitation at 488, 561 and 647 was used in rapid succession and fluorescence in green, red and far red channels was visualized with long pass filters (Semrock). Exposure time and sensitivity setting were uniformly maintained for each set of experiments. For imaging, velocity acquisition software (Improvision Inc. Lexington, KY) was used to drive the microscope. A 512×512 pixel back thinned EMCCD camera (C9100-13 Hamamatsu) was used for fluorescence detection. Mice were imaged using a 4×/0.16 air objective (Olympus, Center Valley, PA). Some mice were also imaged as required following 2 hours of infection with a 10×/0.30 numerical aperture air objective (Olympus, Center Valley, PA).
4
Confocal Microscopy of Liver Lobe
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The exposed liver lobe was visualized with an Olympus IX81 inverted microscope equipped with a confocal light path (Wave-Fx; Quorum) based on a modified Yokogawa CSU-X1 head (Yokogawa Electric Corporation) with a UPLANSAPO 10×/0.40 or UPLANSAPO 20×/0.70 air objective. Four laser excitation wavelengths (491, 561, 643, and 730 nm; Cobalt) were used in rapid succession and visualized with the appropriate long-pass filters (Semrock). Exposure times for excitation wavelengths were 400 ms for all lasers. A back-thinned EMCCD 512×512 pixel camera (C9100–13, Hamamatsu, Bridgewater, NJ) was used for fluorescence detection. Volocity acquisition software (Improvision) was used to drive the microscope.
5
Measuring TRPL of MPB SCBKs
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The TRPLs of MPB SCBKs with different applied biases were measured by means of a home-built confocal microscope. A pulsed supercontinuum laser (OYSL Photonics, SC-Pro, 150 ps pulse lengths) at a 2 MHz repetition rate was used as the laser source. The focused pump laser (the wavelength was 532 nm after laser lines filter) power through an objective lens N.A. = 0.4 was 0.132 μW. A long-pass filter with a 532 nm edge (Semrock) was used to filter out the pump scattered light from the pump laser to the detector. The photoluminescence from MPB SCBK was detected by a SPCM-AQRH single-photon counting module (SPCM-AQRH-15, Excelitas Technologies), and the lifetime module was TimeHarp 260P (PicoQuant).
6
All-fiber Optode for In-situ Temperature Sensing
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o confirm the performance of the fabricated optode, an all-fiber structure based PL measurement system was constructed as shown in Figure 4 . A specific single-multi mode fiber coupler (SMFC) was used for transmitting excitation light and collecting and transmitting fluorescence. The SMFC was coaxially aligned with the ORMOSILs-based optode. To reduce photobleaching of the Ruthenium complex, a pulse fiber pigtailed laser diode (635 nm, 5 mW, 3 KHz) (BWT Beijing, China) was used as the excited light source. The excitation light was delivered to the optode through a multi-mode fiber (MMF; NA = 0.22, id./od. = 580 μm/600 μm) of SMFC. The fluorescence signal from the optode was collected by the same MMF. A long-pass filter (Semrock, USA) at 450 nm was employed at the detector part to reject the fundamental excitation light that was elastically reflected back from the sample surface. Subsequently, the collected fluorescence was monitored by photodiodes through digital lock-in detection. Reducing optical components and unnecessary optical alignment ensures that the all-fiber optical sensing platform can be suitable for rapid in-situ temperature detection of the sample.
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