Photodynamic inactivation of Escherichia coli by porphyrin cytochrome c

Jiang, Guoyu; Lei, Wanhua; Hou, Yuanjun; Wang, Xuesong

doi:10.1039/c2nj40615b

Cited by 8 publications

(5 citation statements)

References 38 publications

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“…Lipofuscin and other lipopigments can emit 450 to 700 nm (3,5). Porphyrins, including heme and its derivatives, can emit in the range of 600-700 nm (6).…”

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confidence: 99%

“…Although in flow cytometry, the term 'autofluorescence' is often referred to as background fluorescence excited by either ultraviolet irradiation or visible, violet or blue light and is characterized by emission in the green part of the spectrum, autofluorescence can also be observed at longer wavelengths. Some sources of background fluorescence in mammalian cells are summarized in Figure 1 (2)(3)(4)(5)(6).…”

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confidence: 99%

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The effect of trypan blue treatment on autofluorescence of fixed cells

2017

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Controlling background fluorescence remains an important challenge in flow cytometry, as autofluorescence can interfere with the detection of chromophores. Furthermore, experimental procedures can also affect cellular fluorescence in certain regions of the emission spectrum. In this work, the effects of fixation, permeabilization, and heating on cellular autofluorescence are analyzed in various spectral regions, along with the influence of trypan blue as a quenching dye for these treatments. The impact of these procedures on the staining of SK-BR-3 cells with a dim green fluorophore, a miniSOG (mini Singlet Oxygen Generator) flavoprotein in the form of the recombinant protein DARPin-miniSOG, is also evaluated. The data presented here indicate that fixation of certain types of cells leads to noticeable increase of the autofluorescence. Our results also suggest that trypan blue should be used as an autofluorescence quencher only with bright green emitters since it interferes with the fluorescent signal in a longer-wavelength region of the spectrum and as a result causes reduction of the signal from dim green fluorescent agents. © 2017 International Society for Advancement of Cytometry.

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“…Lipofuscin and other lipopigments can emit 450 to 700 nm (3,5). Porphyrins, including heme and its derivatives, can emit in the range of 600-700 nm (6).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

The effect of trypan blue treatment on autofluorescence of fixed cells

2017

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“…The emission in orange and red regions excited at 561 nm or 635 nm can instead be associated with porphyrins [46]. The latter have a main absorption peak around 400 nm (Soret band) plus minor bands between 500 and 700 nm (Q bands) and emit in the orange-red region (between 600 and 700 nm) [53,54]. Since this fluorescence emission is usually lower than that excited at 488 nm, single-color fluorescence measurements are often performed using far-red-emitting dyes.…”

Section: Discussionmentioning

confidence: 99%

Optimized two-color single-molecule tracking of fast-diffusing membrane receptors

Spagnolo

Moscardini

Amodeo

et al. 2023

Preprint

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Single particle tracking (SPT) combined with total internal reflection fluorescence (TIRF) microscopy is an outstanding approach to decipher crucial molecular mechanisms on the cell membrane at the nanometric scale. In multicolor configurations it can even be the ideal tool to investigate interactions, but this is hindered by a number of experimental challenges. We systematically and quantitatively analyze the impact of all the necessary sub-elements of any SPT-TIRF setup on signal-to-noise ratio, especially in dynamic studies using minimally-invasive dyes for biomolecule labeling. We show that the dominant limiting factor is the autofluorescence originating from the commonly-used optical glass. We identify and test a different material and show a significant improvement of signal-to-noise ratio in a multichannel TIRF configuration employing the new glass covers. We also address the problem of photobleaching of fluorescent probes by presenting effective approaches suited to a multicolor implementation that requires simultaneous stabilization of multiple dyes. We apply the developed protocol to the analysis of p75 receptors labeled by two fluorophores on the membrane of living cells. Our strategy yields reliable, simultaneous two-color SPT, even for fast-diffusing receptors, enabling this study under conditions not accessible with standard experimental configurations. We argue that the present protocol can pave the way for multicolor super-resolved localization and tracking of single molecules by TIRF microscopy, much expanding the potential of SPT.

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“…[57] The latter have a main absorption peak ≈400 nm (Soret band) plus minor bands between 500 and 700 nm (Q bands) and emit in the orange-red region (between 600 and 700 nm). [56,58] Since this fluorescence emission is usually lower than the one excited at 488 nm, singlecolor fluorescence measurements are often performed using farred-emitting dyes. [59] However, we demonstrated that by selecting suitable TIRFm penetration depths for SPT on the cell membrane, the contribution of cells to the background can be less than 2% under 488 nm excitation (only 1% higher compared to the single-channel 635-case) when using the standard D263M glass; using the proposed N-PK51 cover glass, where the background without cell is ≈40% than for D263M, the contribution of cells to the background is expected to be not higher than 5% upon excitation at 488 nm (and even lower for excitation at 561 nm).…”

Section: Discussionmentioning

confidence: 99%

Optimized Two‐Color Single‐Molecule Tracking of Fast‐Diffusing Membrane Receptors

Schirripa Spagnolo,

Moscardini,

Amodeo

et al. 2023

Advanced Optical Materials

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Single particle tracking (SPT) combined with total internal reflection fluorescence microscopy (TIRFm) is an outstanding approach to decipher mechanisms on the cell membrane at the nanoscale. Multicolor configurations, needed to investigate interactions, are still hindered by several challenges. This work systematically and quantitatively analyzes the impact of necessary elements of SPT‐TIRFm setups on the signal‐to‐noise ratio (SNR), which must be optimized especially in dynamic studies needing minimally invasive dyes for biomolecule labeling. Autofluorescence originating from commonly used optical glass results in the dominant limiting factor in TIRFm, and a cover glass material is tested yielding significant SNR improvements in multichannel TIRFm. Moreover, methodologies are optimized for reducing fluorophore photobleaching in multicolor implementations requiring simultaneous stabilization of multiple dyes. The developed strategies are applied to the fast p75NTR receptors labeled by two fluorophores on the membrane of living cells, achieving reliable, simultaneous two‐color SPT, contrary to configurations using standard cover glasses. This work highlights the importance of optical materials suitable for microscopy and with reduced autofluorescence for increasing sensitivity toward ultimate spatiotemporal resolutions. In particular, the present protocols can pave the way for multicolor super‐resolved localization and tracking of single molecules by TIRFm, greatly expanding the potential of SPT.

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Photodynamic inactivation of Escherichia coli by porphyrin cytochrome c

Cited by 8 publications

References 38 publications

The effect of trypan blue treatment on autofluorescence of fixed cells

The effect of trypan blue treatment on autofluorescence of fixed cells

Optimized two-color single-molecule tracking of fast-diffusing membrane receptors

Optimized Two‐Color Single‐Molecule Tracking of Fast‐Diffusing Membrane Receptors

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