Greg Norris

A promising new wavelength region for three-photon fluorescence microscopy of live cells

We report three‐photon laser scanning microscopy (3PLSM) using a bi‐directional pumped optical parametric oscillator (OPO) with signal wavelength output at λ= 1500 nm. This novel laser was used to overcome the high optical loss in the infrared spectral region observed in laser scanning microscopes and objective lenses that renders them otherwise difficult to use for imaging. To test our system, we performed 3PLSM auto‐fluorescence imaging of live plant cells at λ= 1500 nm, specifically Spirogyra, and compared performance with two‐photon excitation (2PLSM) imaging using a femtosecond pulsed Ti:Sapphire laser at λ= 780 nm. Analysis of cell viability based on cytoplasmic organelle streaming and structural changes of cells revealed that at similar peak powers, 2PLSM caused gross cell damage after 5 min but 3PLSM showed little or no interference with cell function after 15 min. The λ= 1500 nm OPO is thus shown to be a practical laser source for live cell imaging.

Characterisation of periodically poled materials using nonlinear microscopy

Periodically poled crystalline materials are extremely attractive for processes such as second harmonic generation and optical parametric generation due to their very high conversion efficiency. For optimal performance, fabrication of poled regions with sub-micron tolerance is required. In this paper we introduce multi-photon laser scanning luminescence microscopy as a powerful minimally-invasive measurement technique which provides information about internal device structure with high spatial resolution that cannot be easily obtained with existing methods. A comparative study of confocal and multi-photon imaging of periodically poled crystalline materials is also performed.

A simple but precise method for quantitative measurement of the quality of the laser focus in a scanning optical microscope

We report a method for characterizing the focussing laser beam exiting the objective in a laser scanning microscope. This method provides the size of the optical focus, the divergence of the beam, the ellipticity and the astigmatism. We use a microscopic‐scale knife edge in the form of a simple transmission electron microscopy grid attached to a glass microscope slide, and a light‐collecting optical fibre and photodiode underneath the specimen. By scanning the laser spot from a reflective to a transmitting part of the grid, a beam profile in the form of an error function can be obtained and by repeating this with the knife edge at different axial positions relative to the beam waist, the divergence and astigmatism of the postobjective laser beam can be obtained. The measured divergence can be used to quantify how much of the full numerical aperture of the lens is used in practice. We present data of the beam radius, beam divergence, ellipticity and astigmatism obtained with low (0.15, 0.7) and high (1.3) numerical aperture lenses and lasers commonly used in confocal and multiphoton laser scanning microscopy. Our knife‐edge method has several advantages over alternative knife‐edge methods used in microscopy including that the knife edge is easy to prepare, that the beam can be characterized also directly under a cover slip, as necessary to reduce spherical aberrations for objectives designed to be used with a cover slip, and it is suitable for use with commercial laser scanning microscopes where access to the laser beam can be limited.

Energy shedding during nonlinear self-focusing of optical beams

Self-focusing of intense laser beams and pulses of light in real nonlinear media is in general accompanied by material losses that require corrections to the conservative Nonlinear Schrödinger equations describing their propagation. Here we examine loss mechanisms that exist even in lossless media and are caused by shedding of energy away from the self-trapping beam making it to relax to an exact solution of lower energy. Using the conservative NLS equations with absorbing boundary conditions we show that energy shedding not only occurs during the initial reshaping process but also during oscillatory propagation induced by saturation of the nonlinear effect. For pulsed input we also show that, depending on the sign and magnitude of dispersion, pulse splitting, energy shedding, collapse or stable self-focusing may result.

A compact instrument for adjusting laser beams to be accurately coincident and coaxial and its use in biomedical imaging using wave-mixed laser sources

Biomedical imaging applications that involve nonlinear optical processes such as sum-frequency generation (SFG) and four-wave mixing require that the pulses are synchronized in time and the beams are coaxial to better than 400 μrad. For this reason, folding mirrors are normally used to extend the beam path over a few meters so that detectors can be put into the beams to check their overlap at the start of a long path and also at the end of it. We have made a portable instrument with a footprint of only 22 cm × 11 cm × 16 cm that uses a short focal length lens and a telephoto combination for viewing the near-field and far-field simultaneously. Our instrument is simple to build and use, and we show its application in coherent anti-Stokes Raman scattering microscopy and SFG-based two-photon fluorescence microscopy.

Increased signals from short-wavelength-excited fluorescent molecules using sub-Ti:Sapphire wavelengths

We report the use of an all‐solid‐state ultrashort pulsed source specifically for two‐photon microscopy at wavelengths shorter than those of the conventional Ti:Sapphire laser. Our approach involves sum–frequency mixing of the output from an optical parametric oscillator (λ= 1400–1640 nm) synchronously pumped by a Yb‐doped fibre laser (λ= 1064 nm), with the residual pump radiation. This generated an fs‐pulsed output tunable in the red spectral region (λ= 620–636 nm, ∼150 mW, 405 fs, 80 MHz, M2∼ 1.3). We demonstrate the performance of our ultrashort pulsed system using fluorescently labelled and autofluorescent tissue, and compare with conventional Ti:Sapphire excitation. We observe a more than 3‐fold increase in fluorescence signal intensity using our visible laser source in comparison with the Ti:Sapphire laser for two‐photon excitation at equal illumination peak powers of 1.16 kW or less.

Relaxed damage threshold intensity conditions and nonlinear increase in the conversion efficiency of an optical parametric oscillator using a bi-directional pump geometry

A novel bi-directional pump geometry that nonlinearly increases the nonlinear optical conversion efficiency of a synchronously pumped optical parametric oscillator (OPO) is reported. This bi-directional pumping method synchronizes the circulating signal pulse with two counter-propagating pump pulses within a linear OPO resonator. Through this pump scheme, an increase in nonlinear optical conversion efficiency of 22% was achieved at the signal wavelength, corresponding to a 95% overall increase in average power. Given an almost unchanged measured pulse duration of 260 fs under optimal performance conditions, this related to a signal wavelength peak power output of 18.8 kW, compared with 10 kW using the traditional single-pass geometry. In this study, a total effective peak intensity pump-field of 7.11 GW/cm(2) (corresponding to 3.55 GW/cm(2) from each pump beam) was applied to a 3 mm long periodically poled lithium niobate crystal, which had a damage threshold intensity of 4 GW/cm(2), without impairing crystal integrity. We therefore prove the application of this novel pump geometry provides opportunities for power-scaling of synchronously pumped OPO systems together with enhanced nonlinear conversion efficiency through relaxed damage threshold intensity conditions.

Energy shedding during nonlinear self-focusing of laser pulses

With the development of lasers, self-trapping and self-focusing of intense light due to the intensity-dependent change in the refractive index of certain media was predicted. For a medium with a sufficiently large, negative Kerr coefficient (n2), self-focusing of the incident light takes place when the power exceeds a critical value. For cw and short pulse regimes (~100fs) the phenomenon is well modelled by the nonlinear Schrödinger equation (NLS). We presnt results of numerical simulations of NLS using a diffraction-limited input pulse with a Gaussian spatial profile and address the energy shedding that takes place when entering a nonlinear medium and during propagation.

Promising new wavelengths for multi-photon microscopy: thinking outside the Ti:Sapphire box

Multi-photon excitation (MPE) imaging is dominated by the Ti:Sapphire laser as the source for excitation. However, it is limited when considering 3PE of common fluorophores and efficient 2PE of UV dyes which require wavelengths beyond the range of the Ti:Sapphire. Two ultra-short pulsed sources are presented as alternatives: a novel optical parametric oscillator (OPO) geometry (1400–1600nm) and the sum-frequency mixing of an OPO and Yb-doped fibre laser, providing a tunable output (626-635nm). For long wavelengths, we report three-photon laser scanning microscopy (3PLSM) using a bi-directional pumped optical parametric oscillator (OPO) with signal wavelength output at 1500 nm. This novel laser was used to overcome the high optical loss in the infrared spectral region observed in laser scanning microscopes and objective lenses that renders them otherwise difficult to use for imaging. To test our system, we performed 3PLSM auto-fluorescence imaging of live plant cells at 1500 nm, specifically Spirogyra, and compared performance with two-photon excitation (2PLSM) imaging using a femtosecond pulsed Ti:Sapphire laser at 780 nm. Analysis of cell viability based on cytoplasmic organelle streaming and structural changes of cells revealed that at similar peak powers, 2PLSM caused gross cell damage after 5 minutes but 3PLSM showed little or no interference with cell function after 15 minutes. The 1500 nm OPO was thus shown to be a practical laser source for live cell imaging. For short wavelengths, we report the use of an all-solid-state ultra-short pulsed source specifically for two-photon microscopy at wavelengths shorter than those of the conventional Ti:Sapphire laser. Our approach involved sumfrequency mixing of the output from the long-wavelength OPO described above with residual pump radiation to generate fs-pulsed output in the red spectral region. We demonstrated the performance of our ultra-short pulsed system using fluorescently labelled and autofluorescent tissue, and compared with conventional Ti:Sapphire excitation. We observed a more than 3-fold increase in fluorescence signal intensity using our visible laser source in comparison with the Ti:Sapphire laser for two-photon excitation at equal illumination powers of 22 mW or less.

Methanol immersion reduces spherical aberration of water dipping lenses at long wavelengths used in multi-photon laser scanning microscopy

Dipping objectives were tested for multi-photon laser scanning microscopy, since their large working distances are advantageous for thick specimens and the absence of a coverslip facilitates examination of living material. Images of fluorescent bead specimens, particularly at wavelengths greater than 850 nm showed defects consistent with spherical aberration. Substituting methanol for water as the immersion medium surrounding the beads corrected these defects and produced an increase in fluorescence signal intensity. The same immersion method was applied to two representative biological samples of fixed tissue: mouse brain labeled with FITC for tubulin and mouse gut in which the Peyer’s patches were labeled with Texas Red bilosomes. Tissue morphology was well preserved by methanol immersion of both tissues; the two-photon-excited fluorescence signal was six times higher than in water and the depth of penetration of useful imaging was doubled. No modification of the microscope was needed except the provision of a ring to retain a sufficient depth of methanol for imaging.