Martin Schrader

Three-photon excitation in fluorescence microscopy

We show experiments proving the feasibility of scanning fluorescence microscopy by three-photon excitation. Three-photon excitation fluorescence axial images are shown of polystyrene beads stained with the fluorophore 2,5-bis(4-biphenyl)oxazole (BBO). Three-photon excitation is performed at an excitation wavelength of 900 nm and with pulses of 130 fs duration provided by a mode-locked titanium sapphire laser. Fluorescence is collected between 350 and 450 nm. The fluorescence image signal features a third-order dependence on the excitation power, also providing intrinsic 3-D imaging. The resolution of a three-photon excitation microscope is increased over that of a comparable two-photon excitation microscope.

Far-field fluorescence microscopy with three-dimensional resolution in the 100-nm range

We report three‐dimensional (3D) microscopy with nearly isotropic resolution in the λ/5 − λ/10 range. Our approach combines 4Pi‐confocal two‐photon fluorescence microscopy with image restoration. The 3D resolution is demonstrated with densely clustered beads as well as with F‐actin fibers in mouse fibroblast cells. A comparison with unrestored two‐photon confocal images reveals a total reduction of the uncertainty volume up to a factor of 15.

THREE-DIMENSIONAL SUPER-RESOLUTION WITH A 4PI-CONFOCAL MICROSCOPE USING IMAGE RESTORATION

The combination of two-photon excitation 4Pi-confocal fluorescence microscopy with image restoration leads to a fundamental improvement of three-dimensional resolution in the imaging of transparent, fluorescent specimens. The improvement is exemplified by randomly dispersed fluorescent beads and with actin filaments in a mouse fibroblast cell. For an illumination wavelength of 810 nm, we obtained lateral and axial full-width at half-maxima of point-like objects of 120–140 nm, and 70–100 nm, respectively. Fluorescent beads that are 150 nm apart are imaged with an intensity dip of ∼25%. This amounts to a ∼sixfold improvement of the axial resolution over standard two-photon confocal microscopy. In the cell, the 3D-images reveal details otherwise not resolvable with focused light.

4Pi-confocal imaging in fixed biological specimens.

By combining the wavefronts produced by two high-aperture lenses, two-photon 4Pi-confocal microscopy allows three-dimensional imaging of transparent biological specimens with axial resolution in the 100-140-nm range. We reveal the imaging properties of a two-photon 4Pi-confocal microscope as applied to a fixed cell. We demonstrate that a fast, linear point deconvolution suffices to achieve axially superresolved 3D images in the cytoskeleton. Furthermore, we describe stringent algorithms for alignment and control of the two lenses. We also show how to compensate for the effects of a potential refractive index mismatch of the mounting medium with respect to the immersion system.

4Pi-confocal images with axial superresolution

We present two‐photon excitation 4Pi‐confocal images of clustered fluorescence beads demonstrating three‐dimensional far‐field light microscopy with unprecedented resolution. For an excitation wavelength of 760 nm, the lateral and axial resolution amounts to 200 and 145 nm, respectively. The four‐fold improved axial resolution is achieved by engineering the point‐spread function through a suitable combination of aperture enlargement, two‐photon excitation, confocalization and three‐point deconvolution. In contrast to their confocal counterparts, 4Pi‐confocal images do not exhibit the typical axial elongation. The axial resolution in the 4Pi‐confocal images corresponds to about one‐fifth of the wavelength and surpasses the lateral resolution by 25%.

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.

Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy

Monomolecular films of polymerized dimethyl‐bis[pentacosadiinoic‐oxyethyl] ammonium bromide (EDIPAB) provide one‐ and two‐photon excited fluorescence that is sufficiently high to quantify the axial resolution of 3‐D fluorescence microscopes. When scanned along the optical axis, the fluorescence of these layers is bright enough to allow online observation of the axial response of these microscopes, thus facilitating alignment and fluorescence throughput control. The layers can be used for directly measuring and monitoring the axial response of 4Pi‐confocal microscopes, as well as for their initial alignment and phase adjustment. The proposed technique has the potential to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Coverslips with EDIPAB‐layers can be used as substrates for the cultivation of cells.

Refractive index mismatch induced intensity and phase variations in fluorescence confocal, multiphoton and 4Pi-microscopy

We examine the focal shift and the phase when focusing into a refractive index mismatched sample. We show that, while the phase at the location of the intensity maximum is not a linear function of the focusing depth, it can be linearly approximated in intervals that depend on the aperture, on the refractive index and the wavelength. The first axial interval is the maximum object thickness up to which confocal or multiphoton axial images only differ by a wavelength-independent factor from the true axial dimension. The first axial interval also defines the maximum thickness up to which 4Pi-microscopy can be employed. The imaging properties of 4Pi-microscopy of samples that are mounted in mismatched media are investigated. We show how to compensate mismatch induced phase shifts to obtain superresolved 4Pi-confocal images of mismatched objects.

Potential of confocal microscopes to resolve in the 50-100 nm range

We determine the resolution of high‐performance confocal microscopes by measuring the three‐dimensional point–spread function (3D‐PSF) of an optimized confocal setup. The 3D‐PSF is standardized by recording the scattered light of pointlike objects. For a wavelength of 543 nm and a specified numerical aperture of 1.4 (oil), we find an axial and lateral focal full width at half‐maximum (FWHM) of 460±20 and 145±10 nm, respectively. A high signal‐to‐noise ratio is obtained by using recording times comparable to those of near‐field scanning optical microscopy. We further reduce the effective PSF extent by means of a three‐dimensional deconvolution technique exploiting the information gained from the measurement of the focus. We show that it is possible to obtain an axial and lateral FWHM of the far‐field effective PSF after deconvolution of 80 and 40 nm, respectively.

Avalanche photodiode detection with object scanning and image restoration provides 2–4 fold resolution increase in two‐photon fluorescence microscopy

High-quantum-efficiency photodetection, millisecond pixel dwell time stage scanning and image restoration by maximum-likelihood estimation are synergetically combined and shown to improve the resolution of two-photon excitation microscopy 2–4 fold in all directions. Measurements of the two-photon excitation point-spread function (PSF) of a 1.4 aperture oil immersion lens are carried out by imaging fluorescence beads with a diameter of one seventh of the excitation wavelength (830 nm) and subsequent deconvolution with the bead object function. The proposed method of resolution increase is applied to beads as well as to rhodamine labelled actin fibres in mouse fibroblast cells. As the resolution improvement is not based on the non-linear effect of two-photon excitation, the results imply a comparable resolution increase in single-photon excitation confocal microscopy. In the fibroblasts, we established a three-fold improvement in axial resolution, namely from 840 nm before, to 280 nm after restoration (full-width at half-maximum). Actin fibres with axial distances of 850 nm, otherwise difficult to discern, are fully separated. In the lateral direction, images of fluorescence beads of about 110 nm diameter are restored to the real dimensions of the beads with an accuracy of better than one pixel (41 nm).