Journal: Scientific Reports

Article Title: α-Gal on the protein surface affects uptake and degradation in immature monocyte derived dendritic cells

doi: 10.1038/s41598-018-30887-8

Figure Lengend Snippet: Uptake of BSA-α-Gal (left) and BSA (right) after 1 h (top) and 4 h (bottom) of iMDDC incubation at 37 °C analyzed by confocal laser scanning microscopy. Green = BSA-α-Gal or BSA, red = HLA-DR and blue = DAPI stained nuclei.

Article Snippet: Confocal Laser Scanning Microscopy (CLSM) was performed using the LSM 510 Meta system (Carl Zeiss, Jena, Germany) individually modified to allow imaging with improved detection efficiency using avalanche photodiodes .

Techniques: Incubation, Confocal Laser Scanning Microscopy, Staining

Journal:

Article Title: H 2 O 2 -induced mitochondrial fragmentation in C 2 C 12 myocytes

doi: 10.1016/j.freeradbiomed.2010.08.024

Figure Lengend Snippet: C2C12 cells were treated with 250 μM H2O2 or 0 μM H2O2 (control) for 6 hours, then stained with MitoTracker Deep Red 633 for 30 minutes and visualized by CLSM immediately, or after a 24- or 48-hour incubation. For each image taken, the total number of cells and the number of cells displaying a reticular mitochondrial network were counted. A, representative images for each of the conditions. B, the percentage (mean ± SEM) of total imaged cells displaying a reticular mitochondrial network under each condition. An average of 235 cells was scored per condition. *Significantly different from all other bars, P < 0.001. Six-hour treatment with 250 μM H2O2 significantly decreased the percentage of cells having a reticular mitochondrial network, but the cells recovered within 24 hours.

Article Snippet: Live cell imaging using confocal laser scanning microscopy (CLSM) Single images, typically corresponding to optical sections <1.8 μm thick, were taken of live MitoTracker-stained cells using a Zeiss LSM 510 META confocal scan head mounted on an upright Axioplan 2 microscope with Achroplan IR 63×/0.90 NA or Achroplan 40×/0.80 NA water dipping objectives.

Techniques: Control, Staining, Incubation

Journal:

Article Title: H 2 O 2 -induced mitochondrial fragmentation in C 2 C 12 myocytes

doi: 10.1016/j.freeradbiomed.2010.08.024

Figure Lengend Snippet: A, C2C12 cells were treated with 0, 62.5, 125, 250, and 500 μM H2O2 for 3 hours, stained with MitoTracker Deep Red, visualized by CLSM, and categorized by network morphology. The percent of cells in the “fragmented” category is shown for each condition. Mock-treated (0 μM H2O2) myocytes showed a predominantly reticular network morphology, which became increasingly fragmented as H2O2 dose was increased. An average of 52 cells was scored per condition. Data are presented as percents of the total number of cells scored in each treatment group. Data from 2 independent experiments are shown. B, myocytes were treated with 0, 125, 250, 500, 1000, or 2000 μM H2O2 for 6 hours and assayed for cell viability with WST-8 immediately (triangles), or washed and returned to H2O2-free medium, and assayed 24 hours later (squares). Data are presented as means ± SD of 5-6 replicates. Notably, the H2O2 concentration used in subsequent experiments (250 μM) did not significantly lower cell viability. Student's t-test was used to test for statistically significant differences between each condition and the respective 0 μM H2O2 control. NS, not significantly different from mock-treated (0 μM H2O2); *P < 0.05; **P = 0.001; ***P < 10-7. C, cells were incubated in medium II initially containing 250 μM H2O2 for 5 minutes or 1 to 6 hours, then stained with MitoTracker for 30 minutes and visualized by CLSM. The percent of cells in the “fragmented” category is shown for each time point. The occurrence of fragmented mitochondria appears to peak at 5.5 hours. An average of 51 cells was scored per time point. Data from 2 independent experiments are shown.

Article Snippet: Live cell imaging using confocal laser scanning microscopy (CLSM) Single images, typically corresponding to optical sections <1.8 μm thick, were taken of live MitoTracker-stained cells using a Zeiss LSM 510 META confocal scan head mounted on an upright Axioplan 2 microscope with Achroplan IR 63×/0.90 NA or Achroplan 40×/0.80 NA water dipping objectives.

Techniques: Staining, Concentration Assay, Control, Incubation

Journal: bioRxiv

Article Title: Revealing the Ultrastructure of Live Candida albicans using Stimulated Emission Depletion Microscopy

doi: 10.1101/2024.11.25.625149

Figure Lengend Snippet: (A) A comparison of the emission profile acquired using confocal laser scanning microscopy (CLSM) and STED microscopy, along with a Perrin-Jablonski diagram describing the emission depletion during the fluorescence process. A depletion beam is aligned with the excitation laser during STED imaging to deplete the emission of fluorophores in the depletion region, resulting in a smaller overall emission profile and enhanced spatial resolution. Disruption of the internal conversion process causes relaxing electrons to immediately return from the excited state (S1) to the ground state (S0), releasing a red-shifted photon which is blocked by the microscope fluorescence emission filters, while fluorescence from S1 to S0 is collected from the confined excitation volume and used to form an image. (B) A methods workflow showing the growth, staining and preparation methods for live-cell STED imaging of C. albicans .

Article Snippet: Imaging was performed using an inverted microscope equipped with confocal laser scanning microscopy (CLSM) and STED capability (STEDYCON, Abberior Instruments).

Techniques: Comparison, Confocal Laser Scanning Microscopy, Microscopy, Fluorescence, Imaging, Disruption, Staining

Journal: bioRxiv

Article Title: Revealing the Ultrastructure of Live Candida albicans using Stimulated Emission Depletion Microscopy

doi: 10.1101/2024.11.25.625149

Figure Lengend Snippet: (A) C. albicans labelled with Nile Red, imaged with a CLSM. A digitally magnified ROI is shown with a green box, confirming intracellular staining. (B) C. albicans labelled with actin LIVE 460L, imaged with a CLSM. (C) C. albicans labelled with DNA LIVE 590, imaged with a CLSM. (D), (E), and (F) show the STED counterparts of (A), (B), and (C). A digitally magnified ROI is shown in (D), showing the level of detail improvement possible with STED microscopy compared to CLSM.

Article Snippet: Imaging was performed using an inverted microscope equipped with confocal laser scanning microscopy (CLSM) and STED capability (STEDYCON, Abberior Instruments).

Techniques: Staining, Microscopy

Journal: bioRxiv

Article Title: Revealing the Ultrastructure of Live Candida albicans using Stimulated Emission Depletion Microscopy

doi: 10.1101/2024.11.25.625149

Figure Lengend Snippet: CLSM and STED microscopy of live C. albicans stained with Nile Red. (A) Representative confocal microscopy image. (B) Representative STED microscopy image of the same field of view as shown in (A). Cyan and yellow boxes highlight regions of interest (ROIs). Digitally magnified ROIs of CLSM data are shown in (C) and (E), while digitally magnified ROIs of STED data are shown in (D) and (F). Cyan arrows in (D) show mitochondria with the cristae visible, yellow arrow in (F) shows a sub-micron diameter vacuole interacting with mitochondria, and the magenta arrow in (F) highlights an example of a lipid-rich vesicle. Features indicated by arrows are not clearly visible in the corresponding CLSM micrographs (C) and (E). Scale bars for (A) and (B) = 10 µm, scale bars for (C) to (E) = 1 µm.

Article Snippet: Imaging was performed using an inverted microscope equipped with confocal laser scanning microscopy (CLSM) and STED capability (STEDYCON, Abberior Instruments).

Techniques: Microscopy, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Revealing the Ultrastructure of Live Candida albicans using Stimulated Emission Depletion Microscopy

doi: 10.1101/2024.11.25.625149

Figure Lengend Snippet: (A) CLSM image of C. albicans labelled with Nile Red. A green line shows a ROI. (B) Line intensity profile of the ROI shown in (A). (C) Image decorrelation analysis of (A) gives a resolution of 417 nm for this CLSM micrograph. (D) STED microscopy image of C. albicans labelled with Nile Red, showing the same ROI as (A) with a magenta line. (E) Line intensity profile of the ROI shown in (D) for the STED data. (F) Image decorrelation analysis of (D) gives a resolution of 136 nm for these STED data. Scale bars for (A) and (D) = 1 µm.

Article Snippet: Imaging was performed using an inverted microscope equipped with confocal laser scanning microscopy (CLSM) and STED capability (STEDYCON, Abberior Instruments).

Techniques: Microscopy