The IF protocol was explained elsewhere in detail (Özkan et al., 2021 (link)). The primary antibodies used in this study were Mouse anti-myelin-basic protein (anti-MBP) (1:200, Ab62631), Rabbit anti-collagen 1 (1:500, Ab34710), Rabbit anti-collagen 3 (1:500, Ab7778), Mouse anti-smooth muscle actin-FITC conjugated (1:100, F3777), and Rabbit anti-fibronectin (1:500, F3648). Sections were mounted with 4′ 6-diamidino-2-phenylindole (DAPI, Abcam, ab104139). We divided the spinal cord into five parts axially and at least 2 sections per part were taken for each animal. The image analysis was conducted for n = 14–90 per group. All photos obtained within each experiment were subjected to the same acquisition parameters with Fluorescent (DMI8; Leica) or confocal (DMI8/SP8; Leica) microscopes. The images were exported with LASX software and analyzed with ImageJ software as previously described (Özkan et al., 2023 (link)).
Dmi8 sp8
Manufactured by Leica
Sourced in United States
The DMi8/SP8 is a modular inverted microscope system designed for advanced fluorescence imaging. It features a motorized and encoded stand, allowing for precise and reproducible positioning of the sample. The system is compatible with a wide range of objective lenses and can be equipped with various imaging modules, including confocal scanning, widefield, and superresolution capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Ab104139, by Abcam (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)
Alexa fluor 555 conjugated donkey antimouse secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 dm6000 cs, by Leica (1 mentions)
9 protocols using dmi8 sp8
1
Multimodal Spinal Cord Analysis
2
Microfluidic Cell Imaging Protocol
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Microfluidic chips were
imaged using a laser scanning confocal microscope (Leica DMi8/SP8)
to determine the location of live cells and dextran fluorescence.
During the cell culture process, a small proportion of cells did not
successfully adhere to the top of the channel and continued adhered
culture on the bottom of the channel. Due to the confocality of the
microscope (1 airy unit), it was possible to image exclusively cells
adhered to the top of the microfluidic device, and hence exposed to
NBs. Images were taken in sequential mode using and 488 and 532 nm
laser with emission windows of 493–749 nm and 557–781
nm, corresponding to the CellTracker Green and TexasRed-dextran, respectively.
These values were determined by the in-built DyeAssistant software
to maximize fluorescence intensity and minimize cross talk.
Fluorescence and brightfield maps of each microfluidic channel were
taken using the TileScan feature, consisting of multiple images (512
× 512 px) which were then combined to create the final image.
The autofocus setting was used in between each image location, determining
the focal plane with the maximum intensity in the green fluorescence
channel across 5 steps within a user-centered 60 μm window.
During imaging, devices were maintained as 37 °C (iBidi heating
system, iBidi, Germany).
imaged using a laser scanning confocal microscope (Leica DMi8/SP8)
to determine the location of live cells and dextran fluorescence.
During the cell culture process, a small proportion of cells did not
successfully adhere to the top of the channel and continued adhered
culture on the bottom of the channel. Due to the confocality of the
microscope (1 airy unit), it was possible to image exclusively cells
adhered to the top of the microfluidic device, and hence exposed to
NBs. Images were taken in sequential mode using and 488 and 532 nm
laser with emission windows of 493–749 nm and 557–781
nm, corresponding to the CellTracker Green and TexasRed-dextran, respectively.
These values were determined by the in-built DyeAssistant software
to maximize fluorescence intensity and minimize cross talk.
Fluorescence and brightfield maps of each microfluidic channel were
taken using the TileScan feature, consisting of multiple images (512
× 512 px) which were then combined to create the final image.
The autofocus setting was used in between each image location, determining
the focal plane with the maximum intensity in the green fluorescence
channel across 5 steps within a user-centered 60 μm window.
During imaging, devices were maintained as 37 °C (iBidi heating
system, iBidi, Germany).
3
Raman Spectroscopy of Cellular Samples
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The Raman system used was an inVia Raman confocal inverted microscope (Renishaw) integrated with a Leica DMi8/SP8 laser scanning confocal microscope system. Raman excitation lasers were a 785 nm diode laser (laser power of 45 mW at the sample, intensity of ∼5.7 MW cm–2) and a 532 nm laser (laser power of 22 mW on the sample, intensity of ∼2.7 MW cm–2) and a 1200 l mm–1 and 1800 l mm–1 grating for each laser, respectively. Light was collected using a near infrared enhanced CCD array detector (1024 × 256 pixels). Prior to every experiment a spectrum of a silicon sample was collected and the microscope was calibrated to the peak position (520.5 cm–1). The cell spectra were obtained using a 100× oil immersion objective (HC PL APO CS2 FWD 0.13 mm NA 1.4) with a slit size of 20 μm. This setting gave an axial resolution (full width half maximum) of 1.7 μm when Z-scanning from a silicon sample and tracking the 520.5 cm–1 peak. For the individual spectra taken with the 785 nm laser, the exposure time was 1 s with 15 accumulations. For the individual spectra taken with the 532 nm laser exposure time was 1 s with 10 accumulations. For the mapping with the 785 nm laser the step size was 0.7–1 μm and exposure time was 1.8–2 s px–1. Confocal fluorescence imaging used excitation at 405 nm and collected emission in the range 432–543 nm with Pinhole size of 1 Airy disc.
4
Cellular Uptake Quantification of Nanomaterials
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Cells were seeded
at a density of 175 000 cells/well in 3 mL glass-bottom Petri
dishes and treated with AS-GSH and AS-GSH-FA at 50 μg Ag/mL
for 6 h. The medium was removed, and cells were washed three times
with PBS. Then, cells were incubated with 4% paraformaldehyde for
20 min, washed with PBS, and stained with DAPI (10 μg/mL). After
15 min of incubation, cells were rewashed three times with PBS to
remove the unbound dye, and 1 mL PBS was left in each well to protect
cells against drying. Cells that were not treated with the test materials
were used as controls. Images were obtained by a confocal microscope
(Leica dmi8/SP8) using filters for DAPI (λex = 325–375
nm and λem = 435–485 nm) and NIR fluorescence
(λex = 510–550 nm and λem = 710 nm long pass).
Cellular uptake was quantitatively analyzed
by measuring the intracellular NIR emission signal of cells after
6 h of incubation with the agents. Cells were seeded at a density
of 17 500 cells/well in a 96-well plate and treated with AS-GSH,
AS-GSH-FA, AS-GSH-FA/Hemi-Br, and free Hemi-Br at 57 μg Ag/mL
or 10 μg Hemi-Br/mL concentrations for 6 h. The cells were washed
with PBS to remove uninternalized particles, and the fluorescence
intensity from each well was measured with a Synergy H1 (Biotek Instruments)
microplate reader equipped with a NIR filter set (ex/em: 528/818 nm).
at a density of 175 000 cells/well in 3 mL glass-bottom Petri
dishes and treated with AS-GSH and AS-GSH-FA at 50 μg Ag/mL
for 6 h. The medium was removed, and cells were washed three times
with PBS. Then, cells were incubated with 4% paraformaldehyde for
20 min, washed with PBS, and stained with DAPI (10 μg/mL). After
15 min of incubation, cells were rewashed three times with PBS to
remove the unbound dye, and 1 mL PBS was left in each well to protect
cells against drying. Cells that were not treated with the test materials
were used as controls. Images were obtained by a confocal microscope
(Leica dmi8/SP8) using filters for DAPI (λex = 325–375
nm and λem = 435–485 nm) and NIR fluorescence
(λex = 510–550 nm and λem = 710 nm long pass).
Cellular uptake was quantitatively analyzed
by measuring the intracellular NIR emission signal of cells after
6 h of incubation with the agents. Cells were seeded at a density
of 17 500 cells/well in a 96-well plate and treated with AS-GSH,
AS-GSH-FA, AS-GSH-FA/Hemi-Br, and free Hemi-Br at 57 μg Ag/mL
or 10 μg Hemi-Br/mL concentrations for 6 h. The cells were washed
with PBS to remove uninternalized particles, and the fluorescence
intensity from each well was measured with a Synergy H1 (Biotek Instruments)
microplate reader equipped with a NIR filter set (ex/em: 528/818 nm).
5
Raman Spectroscopy of Materials
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A Renishaw inVia Raman confocal microscope coupled to a Leica DMi8/SP8 laser scanning confocal microscope system with a 532 nm diode laser as the excitation source (power of 25 mW) was directed at the sample, and an 1800 L/mm grating was used for the measurements. The laser was focused on the sample using an xL50 objective.
6
Microfluidic Trap Dimension Characterization
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A total number of five different microfluidic trap dimensions with radii of 7.5, 12.5, 17.5, 25, and 45 µm are used. To determine the actual microfluidic traps dimensions, the microfluidic traps were imaged with a confocal laser scanning microscope (SP8 DMi8, Leica) equipped with 63× objective (water immersion, NA=1.2, Leica). For this purpose, the fluidic and control layers were initially filled with milliQ water. The water in the fluidic layer was then exchanged with a fluorescent solution containing 25 μM of calcein and 10 mM HEPES at pH 7.0, and the valves were subsequently actuated to close the traps. For each size, 10 microfluidic traps were imaged and z-stack including 50 z positions were recorded with a 488 nm laser with a z-stack height of 356 nm per microfluidic trap. The microfluidic trap radii were determined by fitting a circle to the fluorescent area (Figure 8—figure supplement 2 , and Table 3 ). The height was determined by multiplying the number of z-stacks bearing a fluorescent signal by the z-stack height.
7
Quantifying EGFR and Endosomal Colocalization
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HeLa cells grown on coverslips were fixed and permeabilized in ice-cold methanol for 5 min, indirect fluorescence microscope procedures were processed as described previously [29 (link)]. After blocking with 2% BSA in PBS for 30 min, cells were costained with anti-EGFR (1:200) and anti-EEA1 (1:200) or anti-LAMP1 (1:200) primary antibodies for 1 h at room temperature followed by 1 h incubation with Alexa Fluor 488 conjugated donkey antirabbit and Alexa Fluor 555 conjugated donkey antimouse secondary antibodies (Thermo Fisher Scientific). Coverslips were mounted on glass microscope slides using Dako fluorescence mounting medium (Agilent, Santa Clara, CA, USA), and the images were taken under a Leica SP8 DMi8 inverted confocal laser scanning microscope equipped with 63× glycerol (Numerical Aperture, NA = 1.3) objective. Images were processed and colocalization analysis was performed using Fiji Image J (version 1.53s, National Institute of Health, Bethesda, MD, USA) as described previously. Multichannel images were threshold in each channel; the colocalization was quantified using Fiji Image J JACoPplugin and was represented as colocalization Pearson’s correlation coefficient (r).
8
Confocal Microscopy Imaging of Plant Cells
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For image acquisition, a Leica TCS SP5 (DM6000 CS) or a Leica SP8 (DMi8) confocal laser scanning microscope, equipped with a Leica HCX PL APO CS 63×1.20 water-immersion objective, was used. GFP and BCECF were excited at 488 nm (fluorescence emission: 500–550 nm), YFP and FM4-64 at 514 nm (fluorescence emission YFP: 525–578 nm; fluorescence emission FM4-64: 670–790 nm), and PI at 561 nm (fluorescence emission: 644–753 nm). Roots were mounted in PI solution (0.02 mg/ml) for the counterstaining of cell walls. Z-stacks were recorded with a step size of 420 nm (for 3D reconstructions) or 1.5 µm (for maximum projections of VAC1 bodies).
9
Neurite Outgrowth Analysis in Astrocyte-Myotube Coculture
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Neurite outgrowth was analysed as previously described [25 (link)]. On days 28 and 35 of coculture (1 and 2 weeks after adding astrocytes, respectively), tile scan confocal images were taken at 10X magnification of the NEFH fluorescence in the myotube compartment using an inverted Leica SP8 DMI8 confocal microscopy equipped with a HC PL APO CS2 10x/0.40 dry objective lens. Motor neuron neurites were isolated using ilastik 1.3.3post1 Pixel Classification software [48 (link)], and the number of pixel intersections was calculated per intersection line using an ImageJ 1.52p software linear Scholl analysis script.
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