Second harmonic detection of spatially filtered two-photon excited fluorescence

Abstract

Spatlal fllterlng of two-photon exclted fluorescence comblned wlth wcond harmonlc detection has duinonstrated a redduc#on ln the rlgnal blank resuHlng In Improved detectkn Iknlts. The second harmonlc detectkn fluorometer k basad on the recovery of the second harmonlc produced as a resun of nonhar abrorptbn of two photons from a dnwddaUy moddated laser. Thk Instrument k capable of acqulrlng corrected twoghoton exdtatlon spectra rapidly w h h shnultaneousty provkllng detectlon llmltr as low as 200 pM (S/N = 3)fwstwmgakorknruch as^^^ wlth an 82 MHr traln of 12-p u h havlng an average power of 180 mW. Varlour Instrumental contrlbutlons to the ndw spectrum are examined. A tltanlum:sapphke laser was usod io demonstrate the compatlblllty of very rhort laser pulses wlth second harmonic detectlon. A detectbn llmn of 7.55 nM was 0btaiCl.d for the l a m dye coumarln-480 (WN = 9) wlth an 82 MHz traln of 1 -p u h havlng an average power of 850 mW.

Simultaneous Two‐Photon Activation of Type‐I Photodynamic Therapy Agents

“…The resulting modulated and filtered signal was detected using a photomultiplier tube (Hamamatsu, model R1527) operated at a potential of 1000 V, and the signal component at the second harmonic of the modulation frequency was monitored using a lock-in amplifier (Stanford Research Systems, model SR850). This approach allowed two-photon-excited fluorescence to be isolated effectively from linear interferences, such as residual scattered laser light (47,48). The average power of the NIR laser light incident on the sample was monitored using a volume-absorbing power meter (Scientech, model AD30); average powers <200 mW were used for all spectroscopic measurements.…”

“…Methods for quantibing two-photon power response. The crosssection for simultaneous TPE is typically about a million-fold smaller than that for an equivalent single-photon excitation process (42,48). As a consequence of this relatively low efficiency, the ratio of the combined scattered excitation radiation and single-photoncxcited fluorescence to two-photon-excited fluorescence emission can be very high.…”

“…Optical filtering may be used to discriminate twophoton emission spectrally from this background; unfortunately, these interferences can be exceedingly difficult to eliminate using such spectral means alone. A convenient method for eliminating linear interferences utilizes second harmonic detection to monitor response from the sample at twice the modulation frequency that is related to only the two-photon-excited fluorescence (47,48). Although the second-harmonic fluorescence signal is only approximately 12% of the total two-photon fluorescence produced, the rejection of linear interferences afforded by this detection scheme more than compensates for the loss in absolute signal level, resulting t n an overall increase in the signal-to-noise ratio.…”

Single Molecule Detection by Laser Two-Photon Excited Fluorescence in a Capillary Flowing Cell

“…Detection limits as low as 120 pM were obtained by two-photon excitation. [7][8][9] A single-molecule detection by two-photon excited fluorescence was carried out on a glass plate using a Ti-sapphire laser (100 fs) and confocal geometry, where molecules diffusing into and out of the twophoton excitation volume produced fluorescence bursts. 10 A detection limit of 3 pM has been reported using a microchannel plate, and only one molecule was expected to stay under observation at any given moment.…”

Two-Photon-Excited Phase-Resolved Fluorescence Spectroscopy

“…Multiphoton excited uorescence (M PEF) has been used to perform measurem ents in strongly absorbing m edia, [8][9][10] tissue, 11 and whole blood. 12 M PEF has been used to measure uorescent species in dilute solutions [13][14][15][16][17][18][19][20][21] down to the single m olecule level. 19,20 M PEF-based imaging has also been improved signi cantly by using time resolu-tion.…”