Enhanced immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells analyzed by phase-sensitive flow cytometry

Steinkamp, John A.; Lehnert, Nancy M.; Keij, Jan F.; Lehnert, Bruce E.

doi:10.1002/(sici)1097-0320(19991201)37:4<275::aid-cyto4>3.0.co;2-u

Cited by 17 publications

(17 citation statements)

References 32 publications

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“…The samples are later labeled with fluorescent antibodies to yield a dramatically improved signal-to-noise ratio. Finally, as the backgound autofluorescence has a short lifetime (Steinkamp et al, 1999), it is possible to use time-gated imaging to remove autofluorescence from the image (see Time-Gated Imaging).…”

Section: Cells Labels and Experimental Design Fixation And Backgroumentioning

confidence: 99%

Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology

Petty

2007

Microscopy Res & Technique

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Cutting-edge biophysical technologies including total internal reflection fluorescence microscopy, single molecule fluorescence, single channel opening events, fluorescence resonance energy transfer, high-speed exposures, two-photon imaging, fluorescence lifetime imaging, and other tools are becoming increasingly important in immunology as they link molecular events to cellular physiology, a key goal of modern immunology. The primary concern in all forms of microscopy is the generation of contrast; for fluorescence microscopy contrast can be thought of as the difference in intensity between the cell and background, the signal-to-noise ratio. High information-content images can be formed by enhancing the signal, suppressing the noise, or both. As improved tools, such as ICCD and EMCCD cameras, become available for fluorescence imaging in molecular and cellular immunology, it is important to optimize other aspects of the imaging system. Numerous practical strategies to enhance fluorescence microscopy experiments are reviewed. The use of instrumentation such as light traps, cameras, objectives, improved fluorescent labels, and image filtration routines applicable to low light level experiments are discussed. New methodologies providing resolution well beyond that given by the Rayleigh criterion are outlined. Ongoing and future developments in fluorescence microscopy instrumentation and technique are reviewed. This review is intended to address situations where the signal is weak, which is important for emerging techniques stressing super-resolution or live cell dynamics, but is less important for conventional applications such as indirect immunofluorescence. This review provides a broad integrative discussion of fluorescence microscopy with selected applications in immunology. Microsc. Res. Tech. 70:687-709, 2007. V

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Section: Cells Labels and Experimental Design Fixation And Backgroumentioning

confidence: 99%

Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology

Petty

2007

Microscopy Res & Technique

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“…33,34 Another source of nonspecific fluorescence signal might lie in the fixation procedure itself. 35,36 The presence of autofluorescence may reduce the effective dynamic range of the light detector available for registration of specific fluorescence signal. Consequently, the presence of autofluorescence can be a precision-limiting factor in the measurement of concentration ͑amount͒ of a fluorophore of interest.…”

Section: Autofluorescence and Image Qualitymentioning

confidence: 99%

Loss of image quality in photobleaching during microscopic imaging of fluorescent probes bound to chromatin

et al. 2005

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Prolonged excitation of fluorescent probes leads eventually to loss of their capacity to emit light. A decrease in the number of detected photons reduces subsequently the resolving power of a fluorescence microscope. Adverse effects of fluorescence intensity loss on the quality of microscopic images of biological specimens have been recognized, but not determined quantitatively. We propose three human-independent methods of quality determination. These techniques require no reference images and are based on calculation of the actual resolution distance, information entropy, and signal-to-noise ratio (SNR). We apply the three measures to study the effect of photobleaching in cell nuclei stained with propidium iodide (PI) and chromomycin A3 (CA3) and imaged with fluorescence confocal microscopy. We conclude that the relative loss of image quality is smaller than the corresponding decrease in fluorescence intensity. Furthermore, the extent of quality loss is related to the optical properties of the imaging system and the noise characteristics of the detector. We discuss the importance of these findings for optimal registration and compression of biological images.

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“…Time-gated Luminescence (TGL) techniques employing long-lived (1-2000 µs) luminescent probes (typically europium and terbium chelates) with time-gated detection can provide effective suppression of background autofluorescence to greatly enhance signal detection [1][2][3][4] .…”

Section: Introductionmentioning

confidence: 99%

UV LED excited time-gated luminescence flow cytometry: concepts and experimental evaluation

Jin

Connally

Piper

2006

Smart Medical and Biomedical Sensor Technology IV

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This paper presents experimental and theoretical studies of time-gated discrimination of long-lived luminescence (lifetime: 1~2000 µs) labelled target-organisms against non-target autofluorescence background (lifetime: <100 ns) in flow cytometry. A theoretical model of such a TGL flow cytometer is developed which takes account of flow speed, illumination and detection apertures, fluorescence label lifetime, and pulsed illumination and gated detection timing sequences. Ultraviolet LED and channel photomultiplier were found to be practical as pulsed excitation sources and gated detector for TGL flow cytometry. The prototype cytometer was constructed and optimized to operate at 6 k Hz repetition rate of TGL cycles consisting of 100 µs LED excitation and ~60 µs gated detection. The spatial counting efficiency was evaluated by enumerating 5.5 µm diameter europium microspheres resulting in a counting accuracy approaching 100%.

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Enhanced immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells analyzed by phase-sensitive flow cytometry

Cited by 17 publications

References 32 publications

Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology

Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology

Loss of image quality in photobleaching during microscopic imaging of fluorescent probes bound to chromatin

UV LED excited time-gated luminescence flow cytometry: concepts and experimental evaluation

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