Two-photon excitation imaging based on a compact scanning head

Diaspro, Alberto; Corosu, M.; Ramoino, Paola; Robello, Mauro

doi:10.1109/51.790987

Cited by 13 publications

(8 citation statements)

References 3 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At 5 and 45 min AE uptake was analyzed with fluorescence two‐photon excitation microscopy (TPE) in viable cells. Optical sections were acquired with a TPE architecture described in detail elsewhere 24…”

Section: Methodsmentioning

confidence: 99%

Involvement of p53 in specific anti‐neuroectodermal tumor activity of aloe‐emodin

Pecere

Sarinella

Salata

et al. 2003

Intl Journal of Cancer

View full text Add to dashboard Cite

Previously, we have identified aloe-emodin (AE) as a new type of anticancer agent, with activity that is based on apoptotic cell death promoted by a neuroectodermal tumorspecific drug uptake. We attempt to clarify the intracellular target of AE and the apoptosis-signaling pathway activated by AE in neuroblastoma cell lines. Two-photon excitation microscopy and spectroscopic titrations documented that AE is highly concentrated in susceptible cells and binds to DNA. One of the most important mediators of apoptotic response to genotoxic stimuli, such as anticancer agents, is the p53 tumor suppressor gene. To evaluate the role played by p53 in AE-induced apoptosis a p53 mutant cell line, which lacks transcriptional activity of p53 targeted genes, was tested. AE displayed a reduced growth inhibitory and proapoptotic activity in p53 mutant cells (SK-N-BE(2c)) with respect to the p53 wild-type line (SJ-N-KP). This effect was not caused by a reduced drug uptake in the mutant neuroblastoma cell line but was related to a different apoptotic cell phenotype. Whereas SJ-N-KP cells were susceptible to a p53 transcription-dependent pathway of apoptosis, SK-N-BE(2c) cells underwent apoptosis with up-regulation of p53 expression but not of p53-target genes. After AE treatment p53 translocates to the mitochondria inter-membrane space in both neuroblastoma cell lines. Due to its high accumulation in neuroectodermal tumor cells AE could also kill tumor cells harboring p53 mutant genes. This property would further contribute to AE specific anti-tumor activity and might be exploitable in the clinic.

show abstract

Section: Methodsmentioning

confidence: 99%

Involvement of p53 in specific anti‐neuroectodermal tumor activity of aloe‐emodin

Pecere

Sarinella

Salata

et al. 2003

Intl Journal of Cancer

View full text Add to dashboard Cite

show abstract

“…Filipin‐labelled images (Fig. 1) were acquired by a two‐photon excitation modified PCM 2000 scanning head (Wilson & Sheppard, 1984; Diaspro et al ., 1999b). Cells excited with an ultrafast pulsed laser at 720 nm, 5 mW, were imaged using a 450 nm emission filter and a Nikon Plan apochromatic oil immersion objective 100×/1.3 NA.…”

Section: Methodsmentioning

confidence: 99%

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy

Ramoino

Fronte

Fato

et al. 2002

Journal of Microscopy

Self Cite

View full text Add to dashboard Cite

SummaryIn Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl-BODIPY ® FL C 12 (BODIPY-CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red-shifted as the probe concentrates. In cells incubated with 25 µ m BODIPY-CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10-15 min of continuous labelling, then it decreases until no food vacuoles are found in 30-min fed cells. BODIPY-CE accumulates in red-labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green-labelled vesicles at the apical pole. The inhibition of clathrin-mediated endocytosis does not interfere in P. primaurelia with BODIPY-CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol-binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH 4 Cl reduces the lipid storage. Low temperature (4 ° C) does not prevent the internalization of BODIPY-CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY-CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY-CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.

show abstract

“…The fluorescence signal, collected by an objective and spectrally resolved by either emission filters (HQ535/30=535±15 nm, HQ440/50=440±25 nm and SP670=short pass at 670 nm, Chroma Inc., Brattelboro, VT) or a Jobin–Ivon monochromator, is fed to a multi‐mode fiber that brings the light to a photomultiplier (R928, Hamamatsu, Milano, Italy) in the PCM2000 controller 23. The point spread function widths of the setup measured at λ =740 nm are 220±40 nm in the radial direction and 790±50 nm in the axial direction (statistical accuracy means standard deviation herein) 24…”

Section: Methodsmentioning

confidence: 99%

Selective Fluorescence Recovery after Bleaching of Single E²GFP Proteins Induced by Two‐Photon Excitation

et al. 2005

Self Cite

View full text Add to dashboard Cite

We report the two-photon excitation and emission or a recently developed green fluorescent protein (GFP) mutant, E(2)GFP. Two main excitation bands are found at 780 and 870 nm. Blinking and irreversible and reversible bleaching were observed. Fluorescence blinking occurs in the millisecond range and has been ascribed to conversions between the neutral, anionic and dark zwitterionic states. Bleaching is observed after approximately 10 to 400 ms depending on the excitation power, and it is probably due to a conversion to a dark state. The striking feature of this GFP mutant is that the fluorescence can be recovered with very high efficiency only upon irradiation at 720 +/- 10 nm. This GFP mutant therefore seems promising as an almost permanent chromophore for two-photon excitation (TPE) microscopy or for applications in single-molecule memory arrays.

show abstract

Two-photon excitation imaging based on a compact scanning head

Cited by 13 publications

References 3 publications

Involvement of p53 in specific anti‐neuroectodermal tumor activity of aloe‐emodin

Involvement of p53 in specific anti‐neuroectodermal tumor activity of aloe‐emodin

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy

Selective Fluorescence Recovery after Bleaching of Single E²GFP Proteins Induced by Two‐Photon Excitation

Contact Info

Product

Resources

About

Two-photon excitation imaging based on a compact scanning head

Cited by 13 publications

References 3 publications

Involvement of p53 in specific anti‐neuroectodermal tumor activity of aloe‐emodin

Involvement of p53 in specific anti‐neuroectodermal tumor activity of aloe‐emodin

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy

Selective Fluorescence Recovery after Bleaching of Single E2GFP Proteins Induced by Two‐Photon Excitation

Contact Info

Product

Resources

About

Selective Fluorescence Recovery after Bleaching of Single E²GFP Proteins Induced by Two‐Photon Excitation