A new UV-visible confocal laser scanning microspectrofluorometer designed for spectral cellular imaging

Favard, Cyril; Valisa, P.; Egret‐Charlier, Marguerite; Sharonov, S.; Herben, C.; Manfait, Michel; Silva, Edouard Da; Vigny, Paul

doi:10.1002/(sici)1520-6343(1999)5:2<101::aid-bspy4>3.0.co;2-w

Cited by 16 publications

(7 citation statements)

References 48 publications

(27 reference statements)

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“…Spectral measurements Fluorescence and absorption spectra were measured using a Hitachi MPF‐3 fluorometer (Tokyo, Japan) and a Cary‐209 spectrophotometer (Varian, Sunnyvale, CA). CSI measurements were performed with a V‐45 installation (DILOR SA, Lille, France) (20). The measurements were carried out with a 100× oil‐immersion objective (Nikon, Kawasaki, Japan) at ca 0.6 μm lateral resolution and ca 3 μm axial resolution.…”

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confidence: 99%

Near-infrared Photosensitizer Based on a Cycloimide Derivative of Chlorin p6: 13,15-N-(3′-Hydroxypropyl)Cycloimide Chlorin p6¶

Feofanov

Grichine

Karmakova

et al. 2007

Photochemistry and Photobiology

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The 13,15‐N‐(3′‐hydroxypropylcycloimide) chlorin p6 (CIC), which absorbs at 711 nm, possesses considerable photoinduced cell‐killing activity. It is 43‐, 61‐ and 110‐fold more active than chlorin p6, 3‐formyl‐3‐devinyl chlorin p6 and Photogem, respectively, and has no cytotoxicity without irradiation as estimated on A549 human adenocarcinoma cells. To attain the highest intracellular penetration and activity the monomeric form of CIC should be stabilized. This stabilization in an aqueous environment can be achieved using 0.002–0.005% of Cremophor EL emulsion (polyoxyethylene derivative of hydrogenated castor oil). The intracellular accumulation of CIC occurs in cytoplasm in a monomeric form bound to cellular membranes. This form of the dye is characterized by a high quantum yield of singlet oxygen generation (0.66 ± 0.02). Besides diffuse staining of intracellular membranous structures, CIC accumulates 3‐ to 4‐fold more intensely in mitochondria and Golgi apparatus, thus indicating these organelles to be the initial targets of its photodynamic action. The incubation time providing 50% accumulation level of CIC in cells is 30 ± 5 min. The time for 50% release of CIC from the cells is 60 ± 10 min. A 10‐fold decrease in CIC intracellular penetration at 22°C proves that temperature‐sensitive mechanisms of transport, rather than diffusion, are responsible for the dye uptake. The average cytoplasmic concentration of CIC was seven times the extracellular concentration in the 0.2–1.6 μM range, used for the photodynamic activity measurements. The concentration of CIC and the light dose that correspond to ca 50% level of phototoxicity induce predominantly an apoptotic‐type of cell death, whereas the conditions providing 100% level of phototoxicity induced necrosis. The results obtained indicate that cycloimide derivatives of chlorin p6 may serve as a base for the development of an efficient near‐IR photosensitizer.

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Near-infrared Photosensitizer Based on a Cycloimide Derivative of Chlorin p6: 13,15-N-(3′-Hydroxypropyl)Cycloimide Chlorin p6¶

Feofanov

Grichine

Karmakova

et al. 2007

Photochemistry and Photobiology

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“…Often the full size of the CCD chip is used. 4,6,7,8 Here, the direction on the chip perpendicular to the dispersion direction contains spatial information of the sample. This allows for faster recording of the spectra from a complete line in the sample; however, longer readout times are required to read out the chip.…”

Section: Resultsmentioning

confidence: 99%

High Sensitivity Spectrograph for Use in Fluorescence Microscopy

et al. 2001

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In this paper a versatile, high sensitivity spectrograph is presented for use in uorescence m icroscopy. The high sensitivity is achieved by using a prism for the dispersion in combination with a state-ofthe-art back illuminated charge-coupled device (CCD) camera. The spectrograph, including the CCD camera, has a detection ef ciency of 0.77 6 0.05 at 633 nm. Full emission spectra with a 1-5 nm spectral resolution can be recorded at a maximum rate of 800 spectra per second. Two applications are shown, in which the spectrograph is ber-coupled to a commercia l confocal laser scanning microscope. In the rst example, Fö rster resonance energy transfer imaging experiments were carried out on double-labeled actin laments in the in vitro motility assay. A 160 3 160 point im age was recorded in 1.5 m in at 3 ms dwell time per image point. In the second application, a time-resolved study of single quantum dots is presented at 5.2 ms time resolution.

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“…Confocal spectral measurements were performed using a V-45 installation (DILOR SA, France) for scanning confocal microspectroscopy and imaging (29). The measurements were carried out with a 40ϫ oil-immersion objective (Nikon).…”

Section: Methodsmentioning

confidence: 99%

Oligonucleotide-conjugated Thiazole Orange Probes as “Light-up” Probes for Messenger Ribonucleic Acid Molecules in Living Cells¶

Privat

Melvin

Asseline

et al. 2007

Photochemistry and Photobiology

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Light-up'' probes, icosa-␣-thymidylate-thiazole orange conjugates, for the in situ time-resolved detection of messenger ribonucleic acid (mRNA) in living cells are evaluated. Upon annealing with polyA in aqueous solutions, the icosa-␣-thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes into adherent fibroblasts resulted in high yields of hybridization and fluorescent signals. Incubation of cells in the presence of these probes resulted in facile internalization of the probe and similar painting of the messenger RNA in the nuclear and cytosolic regions.

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A new UV-visible confocal laser scanning microspectrofluorometer designed for spectral cellular imaging

Cited by 16 publications

References 48 publications

Near-infrared Photosensitizer Based on a Cycloimide Derivative of Chlorin p6: 13,15-N-(3′-Hydroxypropyl)Cycloimide Chlorin p6¶

Near-infrared Photosensitizer Based on a Cycloimide Derivative of Chlorin p6: 13,15-N-(3′-Hydroxypropyl)Cycloimide Chlorin p6¶

High Sensitivity Spectrograph for Use in Fluorescence Microscopy

Oligonucleotide-conjugated Thiazole Orange Probes as “Light-up” Probes for Messenger Ribonucleic Acid Molecules in Living Cells¶

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