Erkki Soini

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.

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.

Lanthanide chelates as new fluorochrome labels for cytochemistry.

Anti-rabbit IgG labeled with a new fluorescent europium chelate was used to localize rabbit IgG to human smooth muscle myosin in a histological section. The antibody labeled with the europium chelate could be viewed with a conventional fluorescence microscope with a steady-state light source. This result encourages the development of a time-resolved fluorescence microscope, because a significant improvement in the signal-to-noise ratio can be anticipated.

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.

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.

A new design of the flow cuvette and optical set‐up for the scanning flow cytometer

We introduce a new design for the optical cuvette and a new optical lay-out for the Scanning Flow Cytometer (SFC) that permits measurement of the angular dependency of the scattered light from individual moving particles. The improved optical scheme of the SFC allows measurement of the angular scattering pattern of individual particles at polar angles from 10 degrees to 120 degrees with integration at azimuthal angles from 0 degrees to 360 degrees and with angular resolution of better than 0.5 degrees. The performance of the SFC is demonstrated using certified polystyrene particles as reference material The aim of this work is to develop a flow cytometer, which, by recording the entire light scattering pattern of individual biological particles, would provide more information about the particle structure than the ordinary wide angle, forward and side scattering concepts.

Absolute real-time measurement of particle size distribution with the flying light-scattering indicatrix method

The flying light-scattering indicatrix (FLSI, angular dependency of the intensity of light scattered by a moving individual particle) method, based on a scanning flow cytometer (SFC) that permits measurement of individual particle characteristics from light-scattering data, has been used for the determination of size distribution of the following particles: polystyrene latex, milk fat, and spores (Penicillium levitum, Aspergillus pseudoglaucus). The optical system of the SPC and empirical equations provided absolute sizing at the rate of 50 particles/s. Size distributions obtained with the FLSI method and a best-fit procedure using Mie scattering theory have been compared.