Pekka Hänninen

Two-photon excitation 4Pi confocal microscope: enhanced axial resolution microscope for biological research

The applicability of two photon excitation 4Pi confocal fluorescence microscopy to biological imaging is demonstrated. We show that 4Pi confocal microscopy in combination with a simple deconvolution algorithm allows axial localization and quantification with 0.14 μm resolution in a biological sample. The 4Pi‐confocal microscope extends the applicability of far field fluorescence microscopy to high resolution three‐dimensional imaging and quantification of subcellular structures.

A new microvolume technique for bioaffinity assays using two-photon excitation

Bioaffinity binding assays such as the immunoassay are widely used in life science research. In an immunoassay, specific antibodies are used to bind target molecules in the sample, and quantification of the binding reaction reveals the amount of the target molecules. Here we present a method to measure bioaffinity assays using the two-photon excitation of fluorescence. In this method, microparticles are used as solid phase in binding the target molecules. The degree of binding is then quantified from individual microparticles by use of two photon excitation of fluorescence. We demonstrated the effectiveness of the method using the human α-fetoprotein (AFP) immunoassay, which is used to detect fetal disorders. The sensitivity and dynamic range we obtained with this assay indicate that this method can provide a cost-effective and simple way to measure various biomolecules in solution for research and clinical applications.

Ultrasonic enrichment of microspheres for ultrasensitive biomedical analysis in confocal laser-scanning fluorescence detection

An ultrasonic particle concentrator based on a standing-wave hemispherical resonator is combined with confocal laser-scanning fluorescence detection. The goal is to perform ultrasensitive biomedical analysis by concentration of biologically active microspheres. The standing-wave resonator consists of a 4 MHz focusing ultrasonic transducer combined with the optically transparent plastic bottom of a disposable 96-well microplate platform. The ultrasonic particle concentrator collects suspended microspheres into dense, single-layer aggregates at well-defined positions in the sample vessel of the microplate, and the fluorescence from the aggregates is detected by the confocal laser-scanning system. The biochemical properties of the system are investigated using a microsphere-based human thyroid stimulating hormone assay.

CONTINUOUS WAVE EXCITATION TWO-PHOTON FLUORESCENCE MICROSCOPY

Two‐photon excitation fluorescence imaging is feasible with continuous wave lasers. Images of biological specimens are obtained by employing photon counting in conjunction with an increased recording time. The approach allows two‐photon three‐dimensional imaging of fluorescently labelled specimens with inexpensive lasers.

Annular aperture two-photon excitation microscopy

Abstract We investigate the effect of annular apertures in two-photon excitation confocal and non-confocal microscopy. In a theoretical study, we show that the use of annular apertures in two-photon excitation microscopy leads to resolution increase without diffraction fringes. We determine experimentally the axial resolution of annular aperture confocal and non-confocal two-photon excitation microscopes and compare it with that of the standard two-photon excitation contrasts. Images of fluorescence beads obtained with a transmission annular aperture two-photon excitation microscope demonstrate the applicability of annular apertures for enlarging the focal depth in high resolution two-photon excitation imaging.

Two-photon excitation fluorometric measurement of homogeneous microparticle immunoassay for C-reactive protein.

Recent developments in infrared laser technology have enabled the design of a compact instrumentation for two-photon excitation microparticle fluorometry (TPX). The microparticles can be used in immunoassays as the antibody-coated solid phase to capture an antigen and then detect it with a fluorescently labeled tracer antibody. Unlike most other methods, TPX technology allows low-volume, homogeneous immunoassays with real-time measurements of assay particles in the presence of a moderate excess of fluorescent tracer. In this study, the TPX assay system was used for the reagent characterization and the measurement of C-reactive protein (CRP) in diluted plasma samples, targeting the assay range useful in infectious disease diagnosis. The pentameric structure of the CRP permitted the optimization of an assay with the lowest detectable concentration of 1 microg/L (7.5 pM) by using a single monoclonal antibody both for capture and as the tracer. With a 1:200 predilution of samples, the measurement range of the assay was 1-150 mg/L, but an additional 1:10 dilution was required for higher concentrations. The TPX method showed a good correlation with the reference result obtained in a routine hospital laboratory, demonstrating the feasibility of the technology for immunodiagnostic applications.

Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy

We investigate a novel concept to efficiently generate multiphoton induced fluorescence from organic molecules. The concept is based on frustrating the energy transfer between a fluores- cent donor and one or more acceptors in conjugated molecules. The nonlinearity is not based on higher order molecular susceptibilities but entirely on their linear properties. Therefore, in contrast to nonresonant multiphoton absorption, this method does not require high local intensities. Like- wise, the production of visible fluorescence does not require an infrared excitation wavelength. Hence, when applied to scanning microscopy this property is predicted to increase spatial resolu- tion. Instead of the10 GW/cm 2 required in non-resonant multiphoton excitation, focal intensities of10 MW/cm 2 are expected to produce an equally strong nonlinear signal. The predicted resolu- tion is up to 30% greater than that of an ideal confocal microscope operating at the same fluores- cence wavelength. The resolution improvement over non-resonant two-photon absorption micro- scopes is about two-fold in all directions.

Two-photon excitation microfluorometer for multiplexed single-step bioaffinity assays

A new type of instrumentation for single-step bioaffinity assays and microvolume fluorometry is presented. The concept is based on the use of two-photon excitation by a low-cost near-infrared laser and individual observation of bioactive fluorescent microparticles. The applicability of the instrument is demonstrated by a microparticle based multiplexed bioaffinity assay where several fluorescent markers are simultaneously excited. This instrument can be applied in the growing fields of drug discovery, in life science research, and in routine laboratory diagnostics.

Refractive-index-induced aberrations in two-photon confocal fluorescence microscopy

The effect of refractive index mismatch on the image quality in two‐photon confocal fluorescence microscopy is investigated by experiment and numerical calculations. The results show a strong decrease in the image brightness using high‐aperture objectives when the image plane is moved deeper into the sample. When exciting at 740 nm and recording the fluorescence around 460 nm in a glycerol‐mounted sample using a lens of a numerical aperture of 1·4 (oil immersion), a 25% decrease in the intensity is observed at a depth of 9 μm. In an aqueous sample, the same decrease is observed at a depth of 3 μm. By reducing the numerical aperture to 1·0, the intensity decrease can be avoided at the expense of the overall resolution and signal intensity. The experiments are compared with the predictions of a theory that takes into account the vectorial character of light and the refraction of the wavefronts according to Fermats principle. Advice is given concerning how the effects can be taken into account in practice.

Ultrasensitive protein concentration measurement based on particle adsorption and fluorescence quenching.

A new easy-to-use method for quantification of proteins in solution has been developed. It is based on adsorption competition of the sample protein and fluorescently labeled bovine serum albumin (BSA) onto gold particles. The protein concentration is determined by observing the magnitude of fluorescence altered by quenching the fluorescence on the gold particles in a homogeneous assay format. Under optimal low pH conditions, the assay allowed the determination of picogram quantities (7.0 microg/L) of proteins with an average variation of 4.5% in a 10 min assay. The assay sensitivity was more than 10-fold improved from those of the commonly used most sensitive commercial methods. In addition, the particle sensor provides a simple and rapid assay format without requirements for hazardous test compounds and elevated temperature. Eleven different proteins were tested with the constructed sensor exhibiting a protein-to-protein variability less than 15% allowing protein concentration measurements without the need for recalibration of different proteins.