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 (L2778 fluorescent red, L1030 fluorescent yellow-green, L9654 fluorescent orange, Sigma-Aldrich; Dragon Green, Flash Red, Suncoast Yellow, Bangs Laboratories, Inc.) were diluted 1000-fold in distilled water, then further diluted another 100-fold in 1% low-melt agarose.

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 (L2778 fluorescent red, L1030 fluorescent yellow-green, L9654 fluorescent orange, Sigma-Aldrich; Dragon Green, Flash Red, Suncoast Yellow, Bangs Laboratories, Inc.) were diluted 1000-fold in distilled water, then further diluted another 100-fold in 1% low-melt agarose.

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 (L2778 fluorescent red, L1030 fluorescent yellow-green, L9654 fluorescent orange, Sigma-Aldrich; Dragon Green, Flash Red, Suncoast Yellow, Bangs Laboratories, Inc.) were diluted 1000-fold in distilled water, then further diluted another 100-fold in 1% low-melt agarose.

Techniques: Recombinant, Software