Flash lamp-excited time-resolved fluorescence microscope suppresses autofluorescence in water concentrates to deliver an 11-fold increase in signal-to-noise ratio

Journal of Biomedical Optics
Russell ConnallyJames Piper

Abstract

The ubiquity of naturally fluorescing components (autofluorophores) encountered in most biological samples hinders the detection and identification of labeled targets through fluorescence-based techniques. Time-resolved fluorescence (TRF) is a technique by which the effects of autofluorescence are reduced by using specific fluorescent labels with long fluorescence lifetimes (compared with autofluorophores) in conjunction with time-gated detection. A time-resolved fluorescence microscope (TRFM) is described that is based on a standard epifluorescence microscope modified by the addition of a pulsed excitation source and an image-intensified time-gateable CCD camera. The choice of pulsed excitation source for TRFM has a large impact on the price and performance of the instrument. A flash lamp with rapid discharge characteristics was selected for our instrument because of the high spectral energy in the UV region and short pulse length. However, the flash output decayed with an approximate lifetime of 18 micros and the TRFM required a long-lived lanthanide chelate label to ensure that probe fluorescence was visible after decay of the flash plasma. We synthesized a recently reported fluorescent chelate (BHHCT) and conjugated it to a...Continue Reading

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Citations

Sep 17, 2005·Journal of Fluorescence·Jingli Yuan, Guilan Wang
Jan 7, 2009·Journal of Biomedical Optics·Venkat K Ramshesh, John J Lemasters
Jun 11, 2016·Scientific Reports·Nima SayyadiAnwar Sunna
Aug 5, 2006·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Russell ConnallyJames Piper
Sep 18, 2007·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Dayong JinJames Piper
Jul 4, 2008·Annals of the New York Academy of Sciences·Russell E Connally, James A Piper
Mar 22, 2019·Chemical Communications : Chem Comm·Youichi TsuchiyaChihaya Adachi
Feb 3, 2011·Nutrition Research and Practice·Nam-E KangAe Wha Ha
Mar 31, 2011·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Dayong Jin

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