Before CLSM analysis, TPU, BC TPUHST, and BC TPUHST/GFP were diced into small pieces (~1 × 1 × 1 mm3) and incubated in PBS, LB or CE at 37 °C under 150 rpm shaking overnight. 5 µg L−1 erythromycin was added to the media for BC TPUHST/GFP. The samples were then briefly rinsed with sterile PBS, then visualized using Leica SP8 confocal microscope equipped with white light laser (Leica Microsystems) at 100x magnification. The channel for GFP detection was set for excitation at 484 nm and the emission was collected at 497–594 nm. Additional bright-field images were recorded. Multiple images from independent regions of each sample were recorded. CLSM images were post-processed using ImageJ. GFP signals of all images were enhanced by uniformly adjusting brightness/contrast threshold at 0–100. Minimum values of brightness/contrast threshold for bright-field images were fixed at 50 and the maximum values varied from 100 to 150 depending on the samples.
White light laser
Manufactured by Leica
Sourced in Germany
The Leica white light laser is a high-performance light source that emits a broad spectrum of visible light. It is designed to provide consistent and reliable illumination for various applications in microscopy and imaging.
Automatically generated - may contain errors
Lab products found in correlation
Sp8 confocal microscope, by Leica (1 mentions)
Picoharp 300 tcspc module, by PicoQuant (1 mentions)
Tcs sp5 confocal laser scanning microscope, by Leica (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
Hyd detector, by Leica (1 mentions)
Tcs sp8 x, by Leica (1 mentions)
Andor neo, by Oxford Instruments (1 mentions)
X cite turbo, by Excelitas (1 mentions)
X cite xylis, by Excelitas (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
Hc pl apo cs2 63 1.40 oil immersion objective, by Leica (1 mentions)
Dapi 4 6 diamidino 2 fenilindol, by Merck Group (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Tcs sp5 x inverted microscope, by Leica (1 mentions)
Sp8x microscope, by Leica (1 mentions)
Csu x1 confocal scanner, by Yokogawa (1 mentions)
Sp8uv microscope, by Leica (1 mentions)
Ti microscope, by Nikon (1 mentions)
Axio vert a1 inverted microscope, by Zeiss (1 mentions)
Eukitt quick hardening mounting medium, by Merck Group (1 mentions)
13 protocols using white light laser
1
Confocal Microscopy Analysis of Engineered Bacterial Cells
2
Fluorescence Lifetime Imaging of Lipid Membranes
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Before and after peptide addition, 256 × 256 pixels FLIM images were acquired in the time domain using the Leica TCS SP5 microscope coupled with a PicoHarp 300 TCSPC Module (PicoQuant, Berlin, Germany). The fluorescence signal of CF and di-4-ANEPPDHQ was collected in the range of 500–650 nm using λexc = 470 nm. For Nile Red, λexc = 540 nm and its fluorescence signal were collected in the range of 580–700 nm. The pulsed and tunable White Light Laser (Leica Microsystem) source was used in these experiments.
3
Quantitative Analysis of Amyloid Aggregation
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Each sample was stained with 60 μM ThT, and 250 μL of samples were placed in microscope-chambered slides and imaged at 1024 × 1024 pixel resolution, using a Leica TCS SP5 confocal laser scanning microscope and a 63×/1.4 oil objective (Leica Microsystems, Germany). In these conditions, low protein/dye ratio rules out fluorescence quenching phenomena (47 (link)). ThT was excited using λex = 470 nm (white light laser, repetition rate 80 MHz (Leica Microsystem, Germany)) and the emission signal was collected in the range 485–600 nm. By employing a repetition rate of 80 MHz, we transmit pulses at intervals of 12.5 ns. In this way, we avoid potential critical issues associated with laser repetition rates, much faster than the fluorescence decay completion (56 (link),57 (link),58 (link),59 (link)). Fluorescence lifetime imaging measurements were acquired in the time domain by means of a picoHarp 300 standalone TCSPC module (Picoquant), and 256 × 256 pixel images were obtained at a scanning frequency of 400 Hz (pixel size 0.19 × 0.19 μm) using the aforementioned laser parameters.
4
Confocal and Epifluorescence Microscopy Protocols
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The confocal
fluorescence images
were acquired using a TCS SP8 X (Leica Microsystems, Germany), which
is a laser scanning confocal microscope equipped with a white-light
laser (Leica Microsystems, Germany). The fluorescent signals from
TRITC-DEX were obtained using a HyD detector (Leica Microsystems,
Germany). The confocal fluorescence images were analyzed using Fiji,
an image processing package of ImageJ. Additionally, the epifluorescence
images were obtained using an epifluorescence microscope (Eclipse
Ti2, Nikon or Olympus IX83, Olympus Co.) equipped with a sCMOS camera
(Zyla sCMOS or Andor neo, Andor Technology) and an LED light source
(X-Cite Turbo or X-cite XYLIS, Excelitas Technologies). The epifluorescence
images were acquired using Fiji (image processing package of ImageJ)
and custom-written macros.
fluorescence images
were acquired using a TCS SP8 X (Leica Microsystems, Germany), which
is a laser scanning confocal microscope equipped with a white-light
laser (Leica Microsystems, Germany). The fluorescent signals from
TRITC-DEX were obtained using a HyD detector (Leica Microsystems,
Germany). The confocal fluorescence images were analyzed using Fiji,
an image processing package of ImageJ. Additionally, the epifluorescence
images were obtained using an epifluorescence microscope (Eclipse
Ti2, Nikon or Olympus IX83, Olympus Co.) equipped with a sCMOS camera
(Zyla sCMOS or Andor neo, Andor Technology) and an LED light source
(X-Cite Turbo or X-cite XYLIS, Excelitas Technologies). The epifluorescence
images were acquired using Fiji (image processing package of ImageJ)
and custom-written macros.
5
Immunofluorescent Imaging of Mitochondrial Sirt3
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Immunofluorescence analysis was performed as described previously (17) . Briefly, cells were labelled with 200 nM MitoTracker Deep Red (ThermoFisher Scientific, USA), fixed with 2% paraformaldehyde, incubated with anti-Sirt3 primary polyclonal antibody (dilution 1:100, Santa Cruz, USA), followed by FITC-labeled Goat anti-Mouse IgG secondary antibody (dilution 1:100, Proteintech, USA). DAPI (4,6-diamidino-2-fenilindol, Sigma Aldrich, St. Louis, MO, USA) was used for nuclear staining. Confocal imaging was performed by sequential scanning using Leica TCS SP8 X laser scanning microscope, equipped with a HC PL APO CS2 63×/1.40 oil immersion objective and a white light laser (Leica Microsystems, Germany). The excitation wavelengths and emission detection ranges used were 405 nm and 420-477 nm for DAPI, 490 nm and 500-600 nm for FITC, 644 nm and 665-780 nm for MitoTracker Deep Red.
6
Cardiac Microtissue Imaging Workflow
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Immunofluorescence images were acquired using either the either the upright fluorescent microscope Axio Imager.Z2 (Zeiss) or the SP8 confocal microscope equipped with a White Light Laser (Leica). For the cardiac microtissues, 3–4 tiles and 20–34 Z-stacks were imaged per sample. Tiles were combined using the LeicaX confocal software. Z-stack projections and analysis were performed using Fiji version 1.0 and Imaris 8.4.1.
7
Confocal Microscopy Imaging and Deconvolution
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Imaging was conducted on a Leica SP5 confocal microscope. mTagBFP was excited by 405-nm laser diode, which was also used at higher power to induce UV injury [41 ]. eGFP was excited by an argon laser at 488 nm. mKO2-CAAX and mCherry-CAAX were excited by a Leica white-light laser at 552 and 587 nm, respectively. Imaging for Fig. 2 and Online Resources 1 and 6 was conducted with an 8 kHz Leica resonance scanner. Objectives used were a Leica 40x HCX APO L U-V-I water immersion and a Leica 63X HC APO U-V-I CS2 water immersion.
Imaging processing was conducted in ImageJ (1.51n). Deconvolution was applied for Figs.1 , 2 , 4 , 5 , 6 and 7 . Point spread functions were generated by the Richards and Wolf algorithm [51 (link)] as implemented in the PSF generator plugin for ImageJ [31 (link)]. Richardson–Lucy deconvolution was carried out in the DeconvolutionLab plugin for ImageJ [53 (link)]. A mean filter of radius 1 was applied to Figs. 4 and 6 .
Imaging processing was conducted in ImageJ (1.51n). Deconvolution was applied for Figs.
8
Multimodal Fluorescence Microscopy
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Imaging was performed on a Leica TCS SP5X inverted microscope equipped with HCX PL APO CS 10× NA 0.4 dry, 20× NA 0.7 dry and 40× NA 1.10 water-immersion objectives. All fluorophores were imaged using sequential scanning. NADH was excited using a Chameleon 2-photon laser (Coherent): λex = 725 nm, λem = 395–475 nm. All other fluorophores were excited using a Leica white light laser: λex = 495 nm, λem = 505–550 nm (DOPE-CF); λex = 555 nm, λem = 565–630 nm (DOPE-LRB); λex = 631 nm, λem = 650–750 nm (DOPE-Cy5). The image resolution was 1024 × 1024 pixels and scanning speed was 400 Hz. The pinhole was set to 1 Airy unit, except for the NADH channel where it was set to maximum to allow multiphoton imaging. Typically, 2–4× line averaging was applied.
9
Confocal Imaging of Fixed and Live Samples
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Confocal imaging of fixed samples was performed on an SP8UV microscope (Leica) equipped with a 561-nm DPSS laser, a 633-nm HeNe laser, a 405-nm laser diode, and a 488-nm argon laser. A 63× oil immersion objective (NA 1.4) was used, and images were taken by using the hybrid detector photon-counting mode. Confocal live imaging was performed on either an SP8X microscope (Leica) equipped with a white light laser (Leica) by using the 488-nm laser line or a Ti microscope (Nikon) equipped with a CSU-X1 confocal scanner (Yokogawa) and an Evolve back-illuminated EMCCD camera (Photometrics). 2D videos from the SP8X microscope were taken using a 63× oil immersion objective (NA 1.4) on photomultiplier tube detection mode and time intervals of 10 min. The Ti microscope 2D videos were taken using a 60× oil immersion objective (NA 1.4), an exposure time of 800 ms, and time intervals of 10 min. All images and videos were subsequently analyzed and processed by using Fiji/ImageJ software.
10
Live/Dead Cell Viability Evaluation of Bioprinted Scaffolds
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To determine
the cell viability following bioprinting, a live/dead analysis was
performed on the scaffolds after days 1, 21, and 35. The medium was
removed from the scaffolds and replaced with calcein AM (1 mg/mL in
DMSO) and diluted to 0.005 mg/mL using fresh medium. The scaffolds
were then incubated at 37 °C for 28 min. After this time, the
scaffolds were removed from the incubator, and propidium iodide (1
mg/mL) was added such that it was diluted to a final concentration
of 0.001 mg/mL. The scaffolds were then returned to the incubator
for 6 min. Following this, the medium was removed and 1 mL of PBS
was added. The scaffolds were then imaged using the Axiovert.A1 inverted
microscope [Zeiss] at ×2.5 and ×5 magnifications and processed
using Zen 3.0 (blue edition) and Fiji (ImageJ). In addition, day 1
scaffolds were stained as aforementioned and then mounted onto well
plates with glass coverslip bottoms with Eukitt Quick-hardening mounting
medium (Sigma). Images were taken with a FALCON SP8 confocal microscope,
equipped with a 405 nm UV laser and a white light laser (470–670
nm) (Leica) at ×10 magnification. Images were collected and analyzed
using Leica Application Suite X (LAS X) software and Fiji (ImageJ).
the cell viability following bioprinting, a live/dead analysis was
performed on the scaffolds after days 1, 21, and 35. The medium was
removed from the scaffolds and replaced with calcein AM (1 mg/mL in
DMSO) and diluted to 0.005 mg/mL using fresh medium. The scaffolds
were then incubated at 37 °C for 28 min. After this time, the
scaffolds were removed from the incubator, and propidium iodide (1
mg/mL) was added such that it was diluted to a final concentration
of 0.001 mg/mL. The scaffolds were then returned to the incubator
for 6 min. Following this, the medium was removed and 1 mL of PBS
was added. The scaffolds were then imaged using the Axiovert.A1 inverted
microscope [Zeiss] at ×2.5 and ×5 magnifications and processed
using Zen 3.0 (blue edition) and Fiji (ImageJ). In addition, day 1
scaffolds were stained as aforementioned and then mounted onto well
plates with glass coverslip bottoms with Eukitt Quick-hardening mounting
medium (Sigma). Images were taken with a FALCON SP8 confocal microscope,
equipped with a 405 nm UV laser and a white light laser (470–670
nm) (Leica) at ×10 magnification. Images were collected and analyzed
using Leica Application Suite X (LAS X) software and Fiji (ImageJ).
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