Journal: Cell Reports Methods

Article Title: A single-shot hyperspectral phasor camera for fast, multi-color fluorescence microscopy

doi: 10.1016/j.crmeth.2023.100441

Figure Lengend Snippet: SHy-Cam-SPIM tiled volumetric in vivo imaging (A) MIP image acquired from the trunk area of a 4 dpf transgenic zebrafish embryo; five labels were unmixed by LU from SHy-Cam image data (autofluorescence and four fluorescent proteins). The volumetric image consists of 21 tiles, each with 62 optical sections, covering a field of view of 602 × 256 × 120 μ m . Scale bar for (A): 50μm. (B–F) The individual unmixed fluorescence signals displayed for the subregion (dashed box) in (A): (B) cyan: autofluorescence, (C) green: membrane Tg(krt4:GFP), (D) magenta: vasculature Tg(kdrl:mCherry), (E) yellow: neutrophil Tg(lyz:TagRFP), and (F) purple: ubiquitous cell nucleus ubi:H2B-iRFP670.

Article Snippet: 1- μ m , 1% solid fluorescent bead samples Flash Red , Bangs Laboratories, Inc. , N/A.

Techniques: In Vivo Imaging, Transgenic Assay, Fluorescence, Membrane

Journal: Cell Reports Methods

Article Title: A single-shot hyperspectral phasor camera for fast, multi-color fluorescence microscopy

doi: 10.1016/j.crmeth.2023.100441

Figure Lengend Snippet: SHy-Cam spectral unmixing on standard and fixed samples (A) Normalized emission spectra of Rhodamine 6G and Rhodamine B in water. (B) Phasor plot demonstrating distinct clusters of pure Rhodamine 6G (R6G) and Rhodamine B (RB) and of five solution mixtures with different relative concentrations (R6G:RB). (C) Zoomed-in view of phasor plot clusters in (B) visualized using the SEER phasor map encoding (red circle indicates the SEER map center for the morphed angle rendering of the spectral information). (D) SEER pseudo-color bars representing each phasor cluster, with a continuous color map (bottom panel) showing the distinction of relative concentrations of the solutions. The emission profiles (400–700 nm) and peak emission wavelengths of each mixture are shown to the right of the corresponding pseudo-color bars; as RB relative concentration increases, the peak and shape of emission profile resembles pure RB. (E) Fluorescence emission profiles of six different fluorescent beads. (F) Spectral phasor calculated from the SHy-Cam image of a suspension of the beads mixture. Each cluster on the phasor plane represents beads from the same batch that present very similar fluorescence emission profiles (E). Six circular ROIs applied to the distinct phasor clusters are back mapped from phasor plane to a 3D image plane, highlighting the corresponding image pixels with the same ROI color coding to achieve unmixing. (G) The unmixed image stack of the suspension. Beads were color coded with the same pseudo-colors of the corresponding circular ROIs on the phasor plane. (H–J) Images of a triple-labeled zebrafish embryo collected with conventional and SHy-Cam microscopy. Green: membrane Tg(krt4:GFP), yellow: neutrophil Tg(lyz:TagRFP), purple: ubiquitous cell nucleus ubi:H2B-iRFP670. (H) Image acquired with Zeiss LSM 880 with sequential laser excitation for each fluorescent label, rendered as a maximum intensity projection (MIP) of a coronal plane of a 4 dpf fixed zebrafish embryo (inset, K). (I) The same region acquired with Zeiss LSM 880 using three-laser simultaneous excitation in 32-channel hyperspectral mode, rendered using linear unmixing (LU) for fluorescence separaton. (J) Spectral LU result from SHy-Cam image acquired from the same embryo, reasonably close to the confocal microscope images in (H) and (I). Scale bar for (H), (I), and (J): 10μm.

Article Snippet: 1- μ m , 1% solid fluorescent bead samples Flash Red , Bangs Laboratories, Inc. , N/A.

Techniques: Concentration Assay, Fluorescence, Suspension, Labeling, Microscopy, Membrane

Journal: Cell Reports Methods

Article Title: A single-shot hyperspectral phasor camera for fast, multi-color fluorescence microscopy

doi: 10.1016/j.crmeth.2023.100441

Figure Lengend Snippet:

Article Snippet: 1- μ m , 1% solid fluorescent bead samples Flash Red , Bangs Laboratories, Inc. , N/A.

Techniques: Recombinant, Software

Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense

doi: 10.1371/journal.ppat.1009943

Figure Lengend Snippet: (A) Bacterial burden expressed as colony forming units (CFU) after 24h of S. Typhimurium infection in 2-DG-treated WT BMDMs compared to untreated (UT) controls (n = 5). (B) Intracellular S. Typhimurium load in WT and IR Δmyel BMDMs after 24h of infection (n = 3). (C) S. Typhimurium in 4-OHT-treated WT BMDMs compared to untreated (UT) controls after 24h of infection (n = 3). (D) L. monocytogenes (E) S. aureus burden in 2-DG-treated WT BMDMs compared to untreated (UT) controls (n = 3). (F) S. aureus burden in WT and IR Δmyel BMDMs (n = 3) 24h post-infection. (G) Bacterial load in livers of WT and IR Δmyel mice after 3 days post S . Typhimurium infection or (H) 4-OHT-treated mice. Data represent 2 experiments with 5 mice each. Data are shown as mean ± S.E.M. and statistical significance calculated using student t-test and represented as * = p<0.05; ** = p<0.01; *** = p<0.001. (I) MFI of OVA323-339-MHC II complexes on the surface of WT BMDMs pre-treated with 2-DG and LPS (n = 3). (J) MFI of unprocessed Alexa647-labelled OVA in phagosomes isolated from 2-DG-treated BMDMs analyzed by flow cytometry (n = 3). All samples were pre-stimulated with LPS. (K) MFI of unprocessed Alexa647-labelled OVA in bead containing phagosomes isolated from S. Typhimurium-infected BMDMs analyzed by flow cytometry (n = 3).

Article Snippet: To assess the β-galactosidase activity in phagolysosomes, red fluorescent beads (Bangs Laboratories) were coated with 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C 12 FDG, Life Technologies) for 60 min at 37°C in NaHCO 3 pH 9.6 buffer.

Techniques: Infection, Isolation, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense

doi: 10.1371/journal.ppat.1009943

Figure Lengend Snippet: (A) MFI of pH-sensitive pHrodo-E. coli particles in BMDMs untreated (UT) or pre-treated with 2-DG, normalized to Alexa647 MFI (n = 3). (B) MFI of pHrodo-E. coli particles in WT and IR Δmyel BMDMs normalized to Alexa647 MFI (n = 3). (C) MFI of pHrodo-E. coli particles in S. Typhimurium-infected WT BMDMs (2h p.i.) normalized to Alexa647 MFI (n = 3). (D) MFI of pHrodo-E. coli particles in 4-OHT-treated BMDMs infected with S. Typhimurium for 2h normalized to Alexa647 MFI (n = 3). (E-F) Expression of v-ATPase subunits (V0, V1) in isolated bead-containing phagosomes from untreated and 2-DG-treated BMDMs. The image shown is representative of 3 individual experiments. Immunoblot band intensities were quantified using ImageJ and the V1/ V0 ratio was determined and plotted (n = 3). (G-H) Expression of v-ATPase subunits (V0, V1) in isolated bead phagosomes from WT and IR Δmyel BMDMs. Western blot was quantified and V1/V0 ratios are shown. Expression of v-ATPase subunits (V0, V1) in isolated S. Typhimurium phagosomes and cytoplasm. V1/V0 ratios were quantified and plotted. (I-J) V0 subunit A was immunoblotted from isolated S . Typhimurium phagosomes and probed for V1 subunit B. (K-L) Proximity Ligation Assay (PLA) analysis of V0-aldolase A interaction in 2-DG treated BMDMs. The image shown is representative of 3 individual experiments. (M) Immunoprecipitation of V0 subunit from S . Typhimurium infected phagosomes at indicated times probed for V1, V0 and actin. Rabbit IgG was used as a control for immunoprecipitation. (N) Phagosomes isolated from 2DG-treated and untreated macrophages were subjected to Native-PAGE and immunoblotted for v-ATPase subunits V0 and V1.

Article Snippet: To assess the β-galactosidase activity in phagolysosomes, red fluorescent beads (Bangs Laboratories) were coated with 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C 12 FDG, Life Technologies) for 60 min at 37°C in NaHCO 3 pH 9.6 buffer.

Techniques: Infection, Expressing, Isolation, Western Blot, Proximity Ligation Assay, Immunoprecipitation, Control, Clear Native PAGE

Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense

doi: 10.1371/journal.ppat.1009943

Figure Lengend Snippet: Schematic representation of S. Typhimurium-mediated evasion of phagosome degradation in macrophages by preventing glycolysis-regulated assembly of the v-ATPase.

Article Snippet: To assess the β-galactosidase activity in phagolysosomes, red fluorescent beads (Bangs Laboratories) were coated with 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside (C 12 FDG, Life Technologies) for 60 min at 37°C in NaHCO 3 pH 9.6 buffer.

Techniques: