Optical sectioning by two-pinhole confocal fluorescence microscopy

A two-pinhole axially superresolving confocal fluorescence imaging system is presented. Based on the concept of subtractive incoherent imaging, the system described here is equipped with a zero-focus complex-transmittance pupil filter in one of the collector paths. The optical sectioning capacity of the system is 25% superior to that of a free-pupil one-pinhole instrument.     

Improving the lateral resolution in confocal fluorescence microscopy using laterally interfering excitation beams

The confocal fluorescence microscope is the instrument of choice for biologists. However, compared to other instruments, its resolution is still limited. We propose a simple technique, based on laterally interfering beams, to improve the resolution. One technique consists in using a halve phase plate to modify the illumination, combined with a laterally offset detection. A 90 nm lateral resolution is obtained for properly prepared specimens using readily available dyes. Another approach is to use several excitation beams, slightly shifted and properly dephased, to decrease the lateral extension of the PSF. With this approach, a lateral resolution of 75 nm is predicted with the advantage of a regular confocal detection. Finally, we show how using these techniques in combination with a two-color two-photon excitation could permit to further improve the resolution to 60 nm.     

Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy

We report a confocal total-internal-reflection fluorescence (TIRF) microscope that generates a detection volume for analyte molecules of less than 5 al (5 x 10(-18) l) at a water-glass interface. Compared with conventional confocal microscopy, this represents a reduction of almost 2 orders of magnitude, which is important in isolating individual molecules at high analyte concentrations, where many biologically relevant processes occur. Diffraction-limited supercritical focusing and fluorescence collection is accomplished by a parabolic mirror objective. The system delivers TIRF images with excellent spatial resolution and detects single molecules with a high signal-to-background ratio.     

Far-field fluorescence microscopy with repetitive excitation

We introduce the concept of repeatedly exciting an excited state of a photostable fluorescent entity to generate a nonlinear fluorescence signal which is solely based on the linear susceptibility of the molecule. The excitation cycle between the fluorescent state and another state prolongs the average lifetime of , with emphasis on those molecules that are in the center of the focus. The photons emitted by the long-lived molecules in the center are recorded by a temporal filter and constitute fluorescence that depends nonlinearly on the excitation intensity. Theoretical analysis reveals that this concept can provide three-dimensional imaging and improve the spatial resolution in far-field fluorescence microscopy. We show that despite the presence of diffraction the effective focal waist can in principle be narrowed down to the molecular scale at the expense of signal. Received: 3 December 1998     

Three-dimensional resolution enhancement in fluorescence microscopy by harmonic excitation

A method for increasing lateral as well as axial resolution in fluorescence microscopy is presented. A passband with a high cutoff frequency throughout reciprocal space can be achieved by illumination of the object with spatially harmonic excitation patterns generated by the interference of two collimated laser beams. Theoretical calculations show an almost isotropic point-spread function with a FWHM near 100 nm.     

4Pi microscopy with linear fluorescence excitation

Practical 4Pi microscopy has so far exclusively relied on multiphoton excitation of fluorescence, because the nonlinear suppression of contributions from higher-order sidelobes was mandatory for unambiguous axial superresolution. We show that novel lenses of 74 degrees semiaperture angle enable biological 4Pi microscopy with regular one-photon fluorescence excitation, thus increasing the signal and reducing system complexity and cost. An axial resolution of 95 nm, corresponding to a more than fourfold improvement over confocal microscopy, is verified in the imaging of microtubules in mammalian cells.     

Molecular photodynamics involved in multi-photon excitation fluorescence microscopy

We present a simple model for calculating the fluorescence generated by the multi-photon excitation (MPE) of molecules in solution. The model takes into account internal molecular dynamics such as ground-state depletion due to inter-system crossing (ISC), as well as external molecular dynamics associated with diffusion into and out of an excitation volume confined in 3-dimensions. Internal and external molecular dynamics are combined by using a technique of linearization of a modified diffusion equation which takes into account the possibility of concentration depletion due to photobleaching. In addition, we discuss the phenomenon of pulse saturation which effectively limits the molecular excitation rate constant in the case of short pulsed excitation. Our results are specifically applied in the context of fluorescence autocorrelation functions and single-molecule detection. In the latter case, we discuss some consequences of high-order multi-photon photobleaching. Finally, we include three appendices to rigorously define the temporal and spatial profiles of an arbitrary excitation beam, and also to discuss some properties of an exact evaluation of concentration depletion due to photobleaching. Received: 9 March 1998 / Accepted: 20 April 1998     

Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM)

Multidirectional selective plane illumination microscopy (mSPIM) reduces absorption and scattering artifacts and provides an evenly illuminated focal plane. mSPIM solves two common problems in light-sheet-based imaging techniques: The shadowing in the excitation path due to absorption in the specimen is eliminated by pivoting the light sheet; the spread of the light sheet by scattering in the sample is compensated by illuminating the sample consecutively from opposing directions. The resulting two images are computationally fused yielding a superior image. The effective light sheet is thinner, and the axial resolution is increased by square root 2 over single-directional SPIM. The multidirectional illumination proves essential in biological specimens such as millimeter-sized embryos. The performance of mSPIM is demonstrated by the imaging of live zebrafish embryos.     

Improved axial resolution in confocal fluorescence microscopy using annular pupils

The axial image of a thick fluorescent layer is studied in a confocal microscope consisting of either circular or annular pupils. The response for circular pupils is sharper than that for annular pupils if a small pinhole detector is used. But when the size of the pinhole is larger than a certain value, the response in the latter case can become sharper than that in the former. This result implies that for a given detector of finite size, axial resolution in confocal fluorescence microscopy can be improved when an annular lens is used. Our experimental results qualitatively demonstrate the theoretical prediction. The strength of optical sectioning and the axial cross-section of the three-dimensional optical transfer function are also derived from the measured data.     

Two-photon near- and far-field fluorescence microscopy with continuous-wave excitation

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50-200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.     

Visible-super-resolution infrared microscopy using saturated transient fluorescence detected infrared spectroscopy

A scanning visible-super-resolution microscope based on the saturation behaviour of transient fluorescence detected infrared (TFD-IR) spectroscopy is proposed. A Gaussian IR beam, a Gaussian visible beam and a Laguerre-Gaussian (LG) visible beam are used to obtain two separate two-color excitation fluorescence (2CF) images of the sample. The final image is obtained as the difference between the two recorded images. If the peak intensity of the LG beam is high enough to induce saturation in the fluorescence signal, the image can, in principle, have unlimited spatial resolution. A ∼3-fold improvement in transverse resolution over the visible diffraction limit (and far exceeding the IR diffraction limit) is easily achievable in present experimental setups.     

High-resolution confocal fluorescence thermal imaging of tightly pumped microchip Nd:YAG laser ceramics

A modified two-path confocal microscope was used to obtain fluorescence images of a Nd:YAG microchip element in the presence of a tightly focused 808 nm pump beam. Based on the temperature-induced homogeneous line broadening, high-resolution (<1 μm, <1°C) thermal images of the pump volume and surroundings were obtained from the spatial variation of Nd³⁺ linewidth. Based on this direct, pure optical and non-contact method, thermal gradients as low as 0.02°C/μm were detected at focal volume, this being in agreement with theoretical predictions. The thermal imaging technique here presented opens the possibility of accurate compensation of thermal effects for the development of stable and efficient microchip lasers.     

Three-dimensional superresolution in two-color excitation fluorescence microscopy using theta illumination method

In two-color excitation fluorescence (2CE) theta microscopy, the point spread function (PSF) of the system is the product of the two PSFs of both excitation beams and due to the ellipsoidal shape of the PSFs, the axial width of the PSF of one excitation beam is strongly reduced after multiplication with the lateral section of another PSF in nonconfocal theta microscopy. Taking the advantages of this setup, we propose using two leaky filters inserted in the two illumination arms, respectively, to compress its own lateral width of each PSF; it means, the lateral and axial superresolution (three-dimensional superresolution) of the whole system in 2CE theta microscope with two orthogonal illumination beams are realized.     

Polarized confocal theta microscopy

We propose a comprehensive treatment of theta microscopy based on dipole emission, which better describes fluorescence emission than the isotropic emission model, as fluorescence emission is often polarized. Formulas describing the point spread function for polarized confocal fluorescence theta microscopy are given. Examples are given and some advantages of polarized theta fluorescence microscopy are presented. To cite this article: O. Haeberlé et al., C. R. Physique 3 (2002) 1445–1450.     

Spectrally encoded confocal microscopy

An endoscope-compatible, submicrometer-resolution scanning confocal microscopy imaging system is presented. This approach, spectrally encoded confocal microscopy (SECM), uses a quasi-monochromatic light source and a transmission diffraction grating to detect the reflectivity simultaneously at multiple points along a transverse line within the sample. Since this method does not require fast spatial scanning within the probe, the equipment can be miniaturized and incorporated into a catheter or endoscope. Confocal images of an electron microscope grid were acquired with SECM to demonstrate the feasibility of this technique.     

Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy

Optical Review - Hybrid digital holographic microscopy that combines fluorescence microscopy and digital holographic microscopy into a single system for biological applications is proposed. In the...     

Fibre-optic double-pass confocal microscopy

This paper reports on the construction and characterisation of a novel double-pass confocal scanning microscope that uses a four-port fibre coupler. The new imaging system is self-aligned, compact, vibration-free, and purely coherent. Compared with fibre-optic transmission- and reflection-mode confocal systems, the fibre-optic double-pass confocal imaging system exhibits higher resolution.