Journal: PLoS ONE

Article Title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

doi: 10.1371/journal.pone.0162419

Figure Lengend Snippet: Qdots molar extinction coefficient ε (cm -1 M -1 determined at 405nm) and dilutions used to achieve a similar level of fluorescence (adapted from [ 11 ]).

Article Snippet: Having initially settled on a single excitation system with 425nm (PE2-CoolLED) excitation which was found to be optimum for Qdots work, we investigated the possibility of performing multicolour fluorescence combining Qdots with other fluorophores.

Techniques: Fluorescence

Journal: PLoS ONE

Article Title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

doi: 10.1371/journal.pone.0162419

Figure Lengend Snippet: FFPE section stained with Qdot 655 was imaged successively using mercury bulb, LED405 & Metal Halide source. The images are intensity images at 655nm. Red arrows: specific 655 fluorescence; green arrows: autofluorescence. The image taken with the first exposure to LED405nm illumination. (LED405 (1) ) shows the specific staining with little autofluorescence when the section is illuminated with LED 405nm. Metal halide exposure led to high level autofluorescence and fast photobleaching of the specific fluorescence, confirmed with a second set of images with LED 405nm (LED405 (2) ). Similar results were obtained with all other Qdots.

Article Snippet: Having initially settled on a single excitation system with 425nm (PE2-CoolLED) excitation which was found to be optimum for Qdots work, we investigated the possibility of performing multicolour fluorescence combining Qdots with other fluorophores.

Techniques: Staining, Fluorescence

Journal: PLoS ONE

Article Title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

doi: 10.1371/journal.pone.0162419

Figure Lengend Snippet: Comparison of fluorescence intensities when illuminating Qdots with mercury light, LED 405nm or LED 425nm.

Article Snippet: Having initially settled on a single excitation system with 425nm (PE2-CoolLED) excitation which was found to be optimum for Qdots work, we investigated the possibility of performing multicolour fluorescence combining Qdots with other fluorophores.

Techniques: Comparison, Fluorescence

Journal: PLoS ONE

Article Title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

doi: 10.1371/journal.pone.0162419

Figure Lengend Snippet: Top panel: An example of the autofluorescence intensity images under illumination with 405+460nm is shown for the indicated emission wave lengths which correspond to the peak of emission of the studied Qdots. ROI shows a typical thresholded region of interest selected for quantification of the intensity of autofluorescence. Graphs : The spectra of autofluorescence were found to be fairly similar between 500 and 720nm. However the level of autofluorescence between 530 to 650nm increases with the excitation wave length with PE4000 (A&C). SOLA2, PE2 425 & PE4000 405+460nm lead to similar autofluorescence spectra (D).

Article Snippet: Having initially settled on a single excitation system with 425nm (PE2-CoolLED) excitation which was found to be optimum for Qdots work, we investigated the possibility of performing multicolour fluorescence combining Qdots with other fluorophores.

Techniques:

Journal: PLoS ONE

Article Title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

doi: 10.1371/journal.pone.0162419

Figure Lengend Snippet: Top panel: An example of the autofluorescence intensity images under illumination with 405+460nm is shown for the indicated emission wave lengths which correspond to the peak of emission of the studied Qdots. ROI shows a typical thresholded region of interest selected for quantification of the intensity of autofluorescence. Graphs : The spectra of autofluorescence were found to be fairly similar between 500 and 720nm. However the level of autofluorescence between 530 to 650nm increases with the excitation wave length with PE4000 (A&C). SOLA2, PE2 425 & PE4000 405+460nm lead to similar autofluorescence spectra (D).

Article Snippet: Slides labelled with each Qdots were imaged using mercury bulb or CoolLED PE2 with either a 405 or a 425nm LAM (LED Array Module).

Techniques:

Journal: PLoS ONE

Article Title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

doi: 10.1371/journal.pone.0162419

Figure Lengend Snippet: Autoexposure times to achieve 70% of the dynamic range.

Article Snippet: Slides labelled with each Qdots were imaged using mercury bulb or CoolLED PE2 with either a 405 or a 425nm LAM (LED Array Module).

Techniques:

Journal: PLoS ONE

Article Title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

doi: 10.1371/journal.pone.0162419

Figure Lengend Snippet: Autofluorescence and specific fluorescence emission.

Article Snippet: Slides labelled with each Qdots were imaged using mercury bulb or CoolLED PE2 with either a 405 or a 425nm LAM (LED Array Module).

Techniques: Fluorescence

Journal: PLoS ONE

Article Title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

doi: 10.1371/journal.pone.0162419

Figure Lengend Snippet: The intensity of fluorescence at the peak of emission for each Qdot was recorded in macrophages labelled with antiCD68 and a secondary antibody conjugated with the indicated Qdot. The intensity of autofluorescence at the same wavelength was obtained from an unstained slide. The figure shows the specificity of fluorescence for each Qdot, illuminated with PE4000 at 405nm (405); 435nm (435); 460nm (460), simultaneous illumination with 405 and 460nm (405+460), 435+460 (435+460), PE2 and Sola2. The crude data are given in .

Article Snippet: Slides labelled with each Qdots were imaged using mercury bulb or CoolLED PE2 with either a 405 or a 425nm LAM (LED Array Module).

Techniques: Fluorescence

Journal: PLoS ONE

Article Title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

doi: 10.1371/journal.pone.0162419

Figure Lengend Snippet: Autofluorescence and specific fluorescence at peak of emission for Cy3 & Cy5 excited by white light LED (SOLA), PE2 (535nm & 615nm) or PE4000 (555 & 635nm).

Article Snippet: Slides labelled with each Qdots were imaged using mercury bulb or CoolLED PE2 with either a 405 or a 425nm LAM (LED Array Module).

Techniques: Fluorescence

Journal: PLoS ONE

Article Title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

doi: 10.1371/journal.pone.0162419

Figure Lengend Snippet: Comparison of unmixed specific fluorescence Cy3 (A) & Cy5 (B) when excited by white light LED (SOLA), PE2 (535nm & 615nm) or PE4000 (555 & 635nm). The same field of view illuminated with the indicated LED system was captured through the appropriate filter (for Cy3 & for Cy5) between 500 and 720nm. The fluorescence intensity (scaled counts/s) at emission peak wavelengths was obtained after spectral unmixing. The excitation peaks of Cy3 and Cy5 are 554 & 649 nm respectively. Despite the LAMs used with PE2 not being the optimum available, the results were better than with the white light system.

Article Snippet: Slides labelled with each Qdots were imaged using mercury bulb or CoolLED PE2 with either a 405 or a 425nm LAM (LED Array Module).

Techniques: Fluorescence

Journal: PLoS ONE

Article Title: Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections

doi: 10.1371/journal.pone.0162419

Figure Lengend Snippet: Bleed through test.

Article Snippet: Slides labelled with each Qdots were imaged using mercury bulb or CoolLED PE2 with either a 405 or a 425nm LAM (LED Array Module).

Techniques: