Advances in laser sources for confocal and multiphoton microscopy

The illumination source for all high-resolution, optical sectioning, scanning microscopes is crucially important to the overall performance of the system. We examine advances that have been made in laser sources for both confocal and multiphoton microscopy where the emphasis has been on the development of potentially low-cost, easy to use sources. Growing interest in temporally and spatially resolved techniques has directed laser research towards addressing these challenges. We present the most recent developments in sources for confocal and multiphoton microscopy along with the considerations that should be made when a new source is being considered.     

Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy

We report on the introduction of active optical elements into confocal and multiphoton microscopes in order to reduce the sample-induced aberration. Using a flexible membrane mirror as the active element, the beam entering the rear of the microscope objective is altered to produce the smallest point spread function once it is brought to a focus inside the sample. The conventional approach to adaptive optics, commonly used in astronomy, is to utilise a wavefront sensor to determine the required mirror shape. We have developed a technique that uses optimisation algorithms to improve the returned signal without the use of a wavefront sensor. We have investigated a number of possible optimisation methods, covering hill climbing, genetic algorithms, and more random search methods. The system has demonstrated a significant enhancement in the axial resolution of a confocal microscope when imaging at depth within a sample. We discuss the trade-offs of the various approaches adopted, comparing speed with resolution enhancement.     

Enhancing collection efficiency in large field of view multiphoton microscopy

Many multiphoton imaging applications would benefit from a larger field of view; however, large field of views (>mm) require low magnification objectives which have low light collection efficiencies. We demonstrate a light collection system mounted on a low magnification objective that increases fluorescence collection by as much as 20-fold in scattering tissues. This peripheral detector results in an effective numerical aperture of collection >0.8 with a 3-4 mm field of view.     

3D microscopy of transparent objects using third-harmonic generation

It is demonstrated that third-harmonic generation (THG) near interfaces in the refractive index or the third-order nonlinear susceptibility (χ⁽³⁾) permits three-dimensional imaging of transparent objects. The nonlinear dependence of THG on the excitation power provides inherent optical sectioning. At the same time, the nonresonant nature of THG, in combination with the near-IR excitation wavelengths used (1–2 μm), render this technique potentially (biologically) nondamaging and nonbleaching. A specific property of THG imaging is its sensitivity to — and potential use for imaging of — the relative orientation of interfaces with respect to the axis of propagation of the excitation radiation.     

Highly efficient upconverters for multiphoton fluorescence microscopy

A new generation of efficient two-photon absorbing fluorescent molecules has been developed and used effectively for two-photon laser scanning microscopy. Several examples of the use of these new fluorophores have been presented. In addition, issues relating to the two-photon absorption cross-section, excitation power, sample properties and resolution in two-photon laser scanning microscopy are discussed.     

FLIM and FCS detection in laser‐scanning microscopes: Increased efficiency by GaAsP hybrid detectors

Photon counting detectors currently used in fluorescence lifetime microscopy have a number of deficiencies that result in less‐than‐ideal signal‐to‐noise ratio of the lifetimes obtained: Either the quantum efficiency is unsatisfactory or the active area is too small, and afterpulsing or tails in the temporal response contribute to overall timing inaccuracy. We have therefore developed a new FLIM detector based on a GaAsP hybrid photomultiplier. Compared with conventional PMTs and SPADs, GaAsP hybrid detectors have a number of advantages: The detection quantum efficiency reaches or surpasses the efficiency of fast SPADs, and the active area is on the order of 5 mm², compared with 2.5 10^?3 mm² for a SPAD. The TCSPC response is clean, without the bumps and the diffusion tails typical for PMTs and SPADs. Most important, the hybrid detector is intrinsically free of afterpulsing. FLIM results are therefore free of signal‐dependent background, and FCS curves are free of the known afterpulsing peak. We demonstrate the performance of the new detector for multiphoton NDD FLIM and for FCS. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.