摘要:

AbstractWe have determined the broadening of 130 fs and 80 fs pulses in the focus of a high numerical aperture microscope. The focal pulse length has been measured by cross‐correlating two counter‐propagating pulses at the focus of a 4Pi confocal microscope. This method allowed us to determine the pulse length directlyin the sample. Focusing through refractive index interfaces leads to pulse broadening depending on the change in refractive index. Our results are relevant to the field of two‐photon fluorescence microscopy and studies of nonlinear phenomena with high spatial resol

ISSN:0966-9051    

DOI:10.1002/1361-6374(199409)2:3<117::AID-BIO1>3.0.CO;2-9

出版商:IOP Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

AbstractMicroscopical imaging methods, based on the confocal‐line approach, offer practical advantages over confocal‐point techniques, and some unique peculiarities. However, the extended illumination geometry, and the consequent spatial filtering limitations at the detection level, cause a semi‐confocal performance affecting the spatial resolution.The present paper describes modifications to the confocal‐line method, resulting from the introduction of an aligned‐confocal‐points illumination and detection approach. Firstly, with the aid of an appropriate shaping technique, the spatial distribution of the illumination beam is modified, in order to concentrate the light in a sequence of spaced spots. Secondly, a spatially matched electronic gating process is applied to the response of a linear image sensor, obtaining the equivalent of individual point detectors being positionally conjugated to the illumination spots. The coverage of the full specimen is afforded with the association of electro‐mechanical scanning devices.The technique maintains the advantages present in confocal‐line instrumentation, while achieving true confocal performance, typical of other point and multi‐point confocal systems. The measurements reported, regarding confocal transmission imaging of thick specimens, demonstrate some of the peculiarities and the improved capabilities of the method, compared to the results obtained in conventional and confocal

ISSN:0966-9051    

DOI:10.1002/1361-6374(199409)2:3<122::AID-BIO2>3.0.CO;2-K

出版商:IOP Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

AbstractIt is shown here that the intensity probe CalciumGreen is suitable for quantitative Ca2+concentration determinations using fluorescence lifetime imaging. The probe is characterized by two distinct fluorescence lifetimes over a large Ca2+concentration range. This indicates that the probe exists in two forms, one free and one bound to Ca2+. This behaviour is exploited for the determination of the absolute free Ca2+concentration from fluorescence lifetime measurements.Fluorescence lifetime confocal imaging affords the determination of an effective lifetime of the probe in buffers and single rat myocytes. The images are used for the quantification of the local concentrations of free Ca2+. We find CalciumGreen to be a useful lifetime probe at physiological Ca2+concentrations. A similar photophysical behaviour has also been found for the intensity probe Fluo‐3. However, it is less suitable for the determination of Ca2+concentrations in cell

ISSN:0966-9051    

DOI:10.1002/1361-6374(199409)2:3<131::AID-BIO3>3.0.CO;2-J

出版商:IOP Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

AbstractThe novel techniques of fluorescence lifetime imaging (FLI) and FLI‐microscopy (FLIM) in the frequency domain enable the mapping of the spatial distribution of fluorescence lifetimes of a specimen. The analysis of FLI(M) data is described. Two computer programs were developed to determine nanosecond fluorescence lifetimes from digitized phase‐resolved images produced by the FLI(M) instrument, for single‐ and double‐component systems, respectively. Image processing routines were incorporated for optimal display of the lifetime analysis. The combination of analysis and image processing yields images of the fluorescence lifetimes and of the associated analytical and statistical parameters. The error analysis enables the estimation of the quality of the FLI(M) data at each pixel of the image. The features of the programs are illustrated by simulated and experimental FLIM data, generated in the latter case with commercially available fluorescence microspheres as references for spatial and temporal res

ISSN:0966-9051    

DOI:10.1002/1361-6374(199409)2:3<139::AID-BIO4>3.0.CO;2-T

出版商:IOP Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

AbstractIn the preparation of fluorescencein situhybridization (FISH) specimens, when multiple DNA probes, each tagged with a different fluorophore, are used in combination, several different cellular structures can be visualized simultaneously. When such specimens are digitized, the different cellular components appear in separate color channels of the multi‐channel digital image. Spectral overlap among the emission spectra of the fluorophores, the sensitivity spectra of the image sensor and the passbands of the optical filter set, however, spread the image of each fluorophore across the color channels. The result can be poor isolation of each fluorophore to a separate color channel. Each probe image is thus contaminated by ‘crosstalk’ from the other channels, to a greater or lesser degree.In an earlier paper we presented a processing method that effectively removes the spreading of fluorophore brightness among the color channels, thereby isolating each fluorophore to a single (monochrome) image. That technique assumes the black level is zero, and the integration time is the same for each channel. This paper extends the technique to account for non‐zero black level and for unequal integration periods for the different fluor

ISSN:0966-9051    

DOI:10.1002/1361-6374(199409)2:3<160::AID-BIO5>3.0.CO;2-D

出版商:IOP Publishing Ltd    

年代:1994

数据来源: WILEY