Michiel Müller

Third harmonic generation microscopy.

Third harmonic generation microscopy is used to make dynamical images of living systems for the first time. A 100 fs excitation pulse at 1.2 aem results in a 400 nm signal which is generated directly within the specimen. Chara plant rhizoids have been imaged, showing dynamic plant activity, and non-fading image characteristics even with continuous viewing, indicating prolonged viability under these THG-imaging conditions.

3-Dimensional super-resolution by spectrally selective imaging

The mutual spatial positions of individual pentacene molecules embedded in a p-terphenyl host crystal, residing within the same diffraction-limited volume, are determined with far-field optics with an accuracy far below the Rayleigh distance. This is achieved by spectrally selective imaging, an approach based on the combination of confocal microscopy, single-molecule detection and position-sensitive imaging. It is demonstrated that the molecules can be localized in three dimensions with a precision of 40 nm in the lateral and 100 nm in the axial dimension which represents an enhancement by a factor of 20 and 65, respectively, compared to the Rayleigh distance at the used numerical aperture of the microscope.

Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives

The excitation efficiency in two‐photon absorption (TPA) microscopy depends strongly — owing to the square dependence of the TPA fluorescence on the excitation intensity — on the temporal width of the excitation pulse. Because of their inherently large frequency bandwidth, ultrashort optical pulses tend to broaden substantially because of dispersion from propagation through the dispersive elements in the microscope. In this paper, the dispersion characteristics of a wide range of microscope objectives are investigated. It is shown that the induced dispersion can be pre‐compensated in all cases for pulses as short as 15 fs. Because of the excellent agreement between the results from theoretical modelling and the experimental data, predictions of the possibility of dispersion control for microscope objectives in general, as well as for even shorter pulses, can be inferred. Since for TPA imaging the background due to single photon absorption processes and scattering is independent of the pulse width, proper dispersion pre‐compensation — which minimizes the pulse duration at the focal point and hence maximizes the excitation efficiency — provides optimal image contrast in TPA microscopy.

Quantitative label-free imaging of lipid composition and packing of individual cellular lipid droplets using multiplex CARS microscopy

Lipid droplets (LDs) are highly dynamic organelles that perform multiple functions, including the regulated storage and release of cholesterol and fatty acids. Information on the molecular composition of individual LDs within their cellular context is crucial in understanding the diverse biological functions of LDs, as well as their involvement in the development of metabolic disorders such as obesity, type II diabetes, and atherosclerosis. Although ensembles of LDs isolated from cells and tissues were analyzed in great detail, quantitative information on the heterogeneity in lipid composition of individual droplets, and possible variations within single lipid droplets, is lacking. Therefore, we used a label-free quantitative method to image lipids within LDs in 3T3-L1 cells. The method combines submicron spatial resolution in three dimensions, using label-free coherent anti-Stokes Raman scattering microscopy, with quantitative analysis based on the maximum entropy method. Our method allows quantitative imaging of the chemistry (level of acyl unsaturation) and physical state (acyl chain order) of individual LDs. Our results reveal variations in lipid composition and physical state between LDs contained in the same cell, and even within a single LD.

REAL TIME TWO-PHOTON ABSORPTION MICROSCOPY USING MULTI POINT EXCITATION

In this communication we present the development of a real time two‐photon absorption microscope, based on parallel excitation with many foci. This pattern of foci is created by a two‐dimensional microlens array. The fluorescence is detected by direct, non descanned detection on a CCD camera. Due to the parallel nature of both excitation and detection it is possible to speed up image acquisition significantly. This makes the instrument especially suitable for studying living specimens and/or real time processes. The optical design of the instrument is discussed and an imaging example is given. We specifically address the relation between the axial sectioning capability and the distance between the illumination foci at the sample.

Direct extraction of Raman line-shapes from congested CARS spectra.

We show that Raman line-shapes can be extracted directly from congested coherent anti-Stokes Raman scattering (CARS) spectra, by using a numerical method to retrieve the phase-information hidden in measured CARS spectra. The proposed method utilizes the maximum entropy (ME) model to fit the CARS spectra and to further extract the imaginary part of the Raman susceptibility providing the Raman line-shape similar to the spontaneous Raman scattering spectrum. It circumvents the challenges arising with experimentally determining the real and imaginary parts of the susceptibility independently. Another important advantage of this method is that no a priori information regarding the vibrational resonances is required in the analysis. This permits, for the first time, the quantitative analysis of CARS spectra and microscopy images without any knowledge of e.g. sample composition or Raman response.

Chemical specificity in three-dimensional imaging with multiplex coherent anti-Stokes Raman scattering microscopy

We demonstrate the three-dimensional (3D) imaging capabilities and chemical specificity of multiplex coherent anti-Stokes Raman scattering microscopy. The simultaneous acquisition of a significant part of the vibrational spectrum at each specimen position permits straightforward differentiation among chemical species. 3D imaging is illustrated with a lipid multilamellar vesicle, and lateral and axial resolutions are determined.

Measurement of femtosecond pulses in the focal point of a high-numerical-aperture lens by two-photon absorption.

We demonstrate a method for the measurement of femtosecond optical pulses in the focal point of a high-NA lens, using interferometric autocorrelation through two-photon absorption. A chirp-free input pulse of 47 fs is found to broaden by ≈50% after focusing by a well-compensated objective. With proper prechirp compensation, the actual pulse width in the focus of such a lens system can be restored to (almost) its initial value. The unique value of the presented two-photon autocorrelation technique is its capability of measuring the actual pulse width at the focal point of a high-NA lens, an aspect that is of direct importance to two-photon imaging approaches, for example.

Far-field fluorescence microscopy beyond the diffraction limit

A technique has been developed to obtain three-dimensional structural information on a length scale well below the Rayleigh length with conventional far-field optics. By spectrally selecting a single molecule with high-resolution laser spectroscopy and using a CCD camera to register the spatial distribution of the emitted photons in three dimensions, one can determine the position of a molecule with unprecedented accuracy. One can resolve details in the specimen with sub-diffraction-limited resolution in three dimensions by applying this procedure to as many molecules as are present in the same diffraction-limited volume and obtaining their mutual positions. The feasibility of this technique is demonstrated for the system of pentacene in p-terphenyl at cryogenic temperatures for which molecules were localized with an accuracy of better than 40 nm in the lateral and 100 nm in the axial directions.

Collinear type II second-harmonic-generation frequency-resolved optical gating for use with high-numerical-aperture objectives

Ultrashort-pulse lasers are now commonly used for multiphoton microscopy, and optimizing the performance of such systems requires careful characterization of the pulses at the tight focus of the microscope objective. We solve this problem by use of a collinear geometry in frequency-resolved optical gating that uses type II second-harmonic generation and that allows the full N.A. of the microscope objective to be used. We then demonstrate the technique by measuring the intensity and the phase of a 22-fs pulse focused by a 20x, 0.4-N.A. air objective.