The ability to accurately measure the mobility of particles at low concentrations in small volumes is very
useful for a broad range of applications. The coupling of micro- and nano-fluidic devices and confocal microscopy
offers an efficient and rapid technique for multiplexed single molecule detection and analysis. Microfluidic channels
at micron and sub-micron scales were designed and fabricated on fused silica wafers. Fluorescence correlation
spectroscopy and fluorescence lifetime were applied to measure and analyze the mobility of fluorescent species in
micro-droplets, micro-channels, and nano-channels. The experimental results show
We describe the fabrication of sub-100-nanometer-sized channels in a fused silica lab-on-a-chip device and experiments
that demonstrate detection of single fluorescently labeled proteins in buffer solution within the device with high signal
and low background. The fluorescent biomolecules are transported along the length of the nanochannels by
electrophoresis and/or electro-osmosis until they pass into a two-focus laser irradiation zone. Pulse-interleaved excitation
and time-resolved single-photon detection with maximum-likelihood analysis enables the location of the biomolecule to
be determined. Diffusional transport of the molecules is found to be slowed within the nanochannel, and this facilitates
fluidic trapping and/or prolonged measurements on individual biomolecules. Our goal is to actively control the fluidic
transport to achieve rapid delivery of each new biomolecule to the sensing zone, following the completion of
measurements, or the photobleaching of the prior molecule. We have used computer simulations that include
photophysical effects such as triplet crossing and photobleaching of the labels to design control algorithms, which are
being implemented in a custom field-programmable-gate-array circuit for the active fluidic control.
A freely diffusing single fluorescent molecule may be scrutinized for an extended duration within a confocal microscope
by actively trapping it within the femtoliter probe region. We present results from computational models and ongoing
experiments that research the use of multi-focal pulse-interleaved excitation with time-gated single photon counting and
maximum-likelihood estimation of the position for active control of the electrophoretic and/or electro-osmotic motion
that re-centers the molecule and compensates for diffusion. The molecule is held within a region with approximately
constant irradiance until it photobleaches and/or is replaced by the next molecule. The same photons used for
determining the position within the trap are also available for performing spectroscopic measurements, for applications
such as the study of conformational changes of single proteins. Generalization of the trap to multi-wavelength excitation
and to spectrally-resolved emission is being developed. Also, the effectiveness of the maximum-likelihood position
estimates and semi-empirical algorithms for trap control is discussed.
Multidimensional fluorescence microscopy is finding service in forefront biological studies that require separation of
images from different fluorophores. For example, commercial microscopes are available with multi-band analog
detectors and user-friendly software for "linear unmixing" of species with overlapping emission spectra. To extend such
techniques to ultrasensitive and single-molecule applications, we have developed a custom-built microscope, which
incorporates two tunable-wavelength picosecond dye lasers for pulse-interleaved laser excitation, angle-tuned reflection
of the laser beams from narrow-band Raman notch filters to introduce epi-illumination and provide strong rejection of
scattered laser wavelengths, diffraction-limited confocal imaging with 3-dimensional piezo-scanning, an adjustable
prism spectrometer for high-throughput resolution of collected fluorescence into 4 spectral bands, and a 4-channel high-quantum
efficiency avalanche diode for sub-nanosecond-resolved single-photon detection. Custom software enables
multi-band fluorescence correlation spectroscopy and identification of photon bursts for single-molecule detection. For
unmixing of spectrally-overlapping signatures for ultrasensitive molecular imaging applications, we find that maximum-likelihood
analysis can out-perform least-squares-based linear unmixing in the regime of low photon numbers per
spectral/temporal channel. Also, the likelihood surface provides the confidence of the parameter estimates and the
covariance of the species contributions. Monte Carlo simulations show that bias in the results of the analysis, which
stems from the constraint that photon numbers should be positive, becomes more pronounced at low signal levels, for
both maximum-likelihood and least-squares based unmixing.
Fluorescence correlation spectroscopy (FCS) could provide a more useful tool for intracellular studies and biological sample characterization if measurement times could be reduced. While an increase in laser power can enable an autocorrelation function (ACF) with adequate signal-to-noise to be acquired within a shorter measurement time, excitation saturation then leads to distortion of the ACF and systematic errors in the measurement results. An empirical method for achieving reduced systematic errors by employing a fitting function with an additional adjustable parameter has been previously introduced for two-photon FCS. Here we provide a unified physical explanation of excitation saturation effects for the three cases of continuous-wave, pulsed one-photon excitation, and two-photon excitation FCS. When the time between laser pulses is longer than the fluorescence lifetime, the signal rate at which excitation saturation occurs is lower for pulsed excitation than for cw excitation, and due to the disparate timescales of the photophysical processes following excitation, it is lower still for two-photon excitation. We use a single-molecule description of FCS to obtain improved analytical ACF fitting functions for the three cases. The fitting functions more accurately account for saturation effects than those previously employed without the need for an additional empirical parameter. Use of these fitting functions removes systematic errors and enables measurements to be acquired more quickly by use of higher laser powers. Increase of background, triplet photophysics, and the cases of scanning FCS and fluorescence cross-correlation spectroscopy are also discussed. Experimental results acquired with a custom built apparatus are presented.
Fluorescence correlation spectroscopy (FCS) is a sensitive research tool for studying molecular dynamics at the single molecule level. Photophysical dynamics often dramatically influence FCS measurements, as we show here in characterizing the role of excitation saturation in two-photon fluorescence correlation measurements. We introduce a physical model that characterizes the influence of excitation saturation on the two-photon fluorescence observation volume, and derive an analytical expression for the correlation function that includes the influence of saturation. With this model, we can accurately describe both the temporal decay and the amplitude of measured fluorescence correlation functions over a wide range of illumination powers.
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