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- Brismar, Hjalmar, et al.
(författare)
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Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, texas red, and cyanine 3.18 fluorophores : influences of some environmental factors recorded with a confocal laser scanning microscope.
- 1995
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 43:7
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Tidskriftsartikel (refereegranskat)abstract
- We report on the spectra and fluorescence lifetimes of four commonly used fluorophores: lissamine rhodamine (LRSC); tetramethyl rhodamine isothiocyanate (TRITC); Texas Red; and cyanine 3.18 (Cy-3). Fluorescence lifetime recordings revealed that these spectrally overlapping fluorophores can be individually detected by their lifetimes, indicating that at least four fluorophores can be individually identified in discrete tissue domains by confocal microscopy. A further advantage of lifetime recordings is that fluorophores that emit light within the same wavelength band can be used and chromatic aberrations are therefore circumvented, thereby improving the spatial accuracy in imaging of multiple fluorophores. Low and high pH, respectively, tended to influence fluorophore emission spectra and fluorescence lifetime. IgG conjugation of the fluorophores tended to shift the spectra towards longer wavelengths and to change the fluorescence lifetimes. The IgG-conjugated form of the fluorophores may, when applied to tissue specimens, change the emission spectrum and lifetime. In addition, different tissue embedding procedures may influence fluorescence lifetime. These observations emphasize the importance of spectral and lifetime characterization of fluorescent probes within the chemical context in which they will be used experimentally. Changes in spectra and fluorescence lifetimes may be a useful tool to gain information about the chemical environment of the fluorophores.
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| 5. |
- Trepte, O, et al.
(författare)
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Studies of Porphyrin-containing Specimens Using An Optical Spectrometer Connected To A Confocal Scanning Laser Microscope
- 1994
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Ingår i: Journal of Microscopy. - 0022-2720. ; 176, s. 238-244
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Tidskriftsartikel (refereegranskat)abstract
- A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user-defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, lambda/Delta lambda, ranges from 350 at lambda = 400 nm to 100 at gamma = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three-dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photodynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated. For our study Wistar/Furth rats were injected either with Photofrin or with ALA 3-5 h before they were killed. The organs were removed directly after, and snap-frozen in carbon dioxide ice with isopentane. No further staining or fixation procedures were adopted.
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