ABSTRACT
Multidimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterized by its arrival time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multidimensional TCSPC makes a fluorescence lifetime technique with multiwavelength capability, near-ideal counting efficiency, and the capability to resolve multiexponential decay functions. We show that the same technique and the same hardware can be used for precision fluorescence decay analysis and fluorescence correlation spectroscopy (FCS) in selected spots of a sample.
Subject(s)
Microscopy, Fluorescence, Multiphoton/methods , Cell Line , Cell Nucleus/chemistry , Fluorescence Resonance Energy Transfer , Green Fluorescent Proteins/analysis , Humans , Intracellular Signaling Peptides and Proteins , Microscopy, Confocal/methods , Photons , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Recombinant Fusion Proteins/analysis , Skin/ultrastructureABSTRACT
Techniques based on laser scanning microscopes for nanoprocessing of periodic structures on silicon with ultra-short laser pulses have been developed. Ripples of 800-900 nm spacing were obtained after laser irradiation at a wavelength of 1040 nm, a repetition rate of 10 kHz and a fluence of 2 J/cm2 in air. Smaller features of 70-100nm spacing were achieved in oil at a wavelength of 800 nm, a repetition rate of 90 MHz and a fluence of 200-300 mJ/cm2 by using a high numerical focusing objective.
ABSTRACT
In this paper we present recent single molecule detection experiment using a solid immersion lens (SIL) for fluorescent correlation spectroscopy measurements. We compared the performance of the SIL in combination with an air objective (40x, numerical aperture (NA)=1.15) with a water immersion objective (40x, NA=0.6) in a confocal microscope system (ConfoCorr 1). Important parameters for single molecule experiments such as collection efficiency and excitation field confinement were investigated. Although the two set-ups have similar numerical aperture the measurements demonstrated higher field confinement and better collection efficiency for the SIL system in comparison to the conventional confocal set-up. Adding spherical aberrations shifts the sample volume up to 4 microm away from the plane surface of the SIL and conserves a diffraction limited focal volume. In this case the FCS autocorrelation demonstrates a free 3D diffusion of dye molecules in a highly confined light field.
