We present a multiphoton imaging system mounted on a microsurgery experimental set-up using a Nd:glass femtosecond laser. The system permits to induce laser incisions in human cornea and sclera and to perform nonlinear imaging during the intervention. The laser is a chirped pulse amplification (CPA) system with a regenerative amplifier delivering pulses at a wavelength of 1.06 μm, pulse durations of 400 fs and a maximum energy of 60 μJ at repetition rates up to 10 kHz. The delivery system provides spot sizes down to the micron range. The samples are human corneas retracted from the transplant circuit mounted on a moveable anterior chamber system. Photons generated by non-linear processes in the cornea travel backwards through the beam delivery optics and are captured by a photomultiplier tube behind a dichroic mirror. The signal is filtered by a lock-in amplifier tuned to the laser repetition rate. Scanning the sample permits the acquisition of three-dimensional microscopic images. Above the incision threshold the set-up permits to induce laser cuts in human cornea following complex geometries. Below the threshold the laser pulses create secondary photons by the stimulation of non-linear optical processes in the samples which could be identified as being predominantly second harmonic generation (SHG). The in situ images obtained from the multi-photon module permit to control and optimise the surgical intervention. The combination of multiphoton imaging and corneal surgery necessitates only minimal modifications of the optical system of a femtosecond surgical laser system. A combined system significantly improves parameter control and permits the monitoring of the surgical procedure.

In order to understand the distribution of endogenous fluorescence in the ovary, ovarian biopsies were maintained with a viable tissue imaging system and characterized with multiphoton imaging. It was imperative to maintain a stable in vitro environment so that tissue images could provide accurate correlative data for in vivo spectroscopic measurements. Evaluating tissue viability in real time poses a difficult task given that viability assays are tailored for cell culture. The focus of this study was to design a robust in vitro imaging chamber for assessment of ovarian autofluorescence and simple, reliable viability assays for tissue status monitoring.

Native hyaline cartilage from a human knee joint was directly investigated with laser scanning microscopy via 2-photon autofluorescence excitation with no additional staining or labelling protocols in a nondestructive and sterile manner. Using a femtosecond, near-infrared (NIR) Ti:Sa laser for 2-photon excitation and a dedicated NIR long distance objective, autofluorescence imaging and measurements of the extracellular matrix (ECM) tissue with incorporated chondrocytes were possible with a penetration depth of up to 460 μm inside the sample. Via spectral autofluorescence separation these experiments allowed the discrimination of chondrocytes from the ECM and therefore an estimate of chondrocytic cell density within the cartilage tissue to approximately 0.2-2•10⁷cm³. Furthermore, a comparison of the relative autofluorescence signals between nonarthritic and arthritic cartilage tissue exhibited distinct differences in tissue morphology. As these morphological findings are in keeping with the macroscopic diagnosis, our measurement has the potential of being used in future diagnostic applications.

We investigate the effects of topical application of optical clearing agents (OCA) in two-photon microscopy of ex-vivo human dermis tissue. We demonstrate that the hyperosmotic agents; glycerol, propylene glycol, and glucose in aqueous solution, are effective in improving the penetration depth and enhancing image contrast. We present results of applying the three agents, including the dynamical behaviour of the clarifying effect. Our results show that propylene glycol and glycerol are more effective than glucose in enhancing contrast and, relative to glucose, penetrate three and five times slower, respectively. At suitable concentrations, such agents have the potential to be compatible with living tissue and may possibly enhance in-vivo deep-tissue imaging.

Bacterial interaction with host tissues plays a major role in the cause and persistence of diseases. It has been confirmed by different clinical investigations that Enterococcus faecalis resist root canal treatment and commonly persist in tooth with post treatment infection. The purpose of this study is to apply different microscopic techniques to study the dynamics of the E. faecalis biofilm on root-canal-dentine tissues. Method- Ten intact non-carious human maxillary molars were prepared and incubated with bacterium in nutrient media under anaerobic condition for 16 weeks. Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray microanalysis (EDX), Fluorescents microscopy Light microscopy and Laser Confocal Scanning Microscopy (LCSM) were carried out to characterize the ultrastructure of biofilm. In addition Fourier Transfer Infra Red Spectroscopy (FTIR) and Von-Kossa staining and Fluorescent microscopy were also carried out to confirm the biochemical characteristics of the biofilm structure. Result- The mature biofilm formed on the root-canal wall showed a honey-comb like structure with viable cells bacterial cells inside. The EDX and FTIR analysis showed a significant increase in the levels of Calcium (Ca) and Phosphorus (P) and evidence of biomineralization of the matured biofilm.

Fluorescence is widely used as a spectroscopic tool or for biomedical imaging, in particular for DNA chips. In some cases, detection of very low molecular concentrations and precise localization of biomarkers are limited by the weakness of the fluorescence signal. We present a new method based on sample substrates that improve fluorescence detection sensitivity. These active substrates consist in glass slides covered with metal (gold or silver) and dielectric (alumina) films and can directly be used with common microscope set-up. Fluorescence enhancement affects both excitation and decay rates and is strongly dependant on the distance to the metal surface. Furthermore, fluorescence collection is improved since fluorophore emission lobes are advantageously modified close to a reflective surface. Finally, additional improvements are achieved by structuring the metallic layer. Substrates morphology was mapped by Atomic Force Microscopy (AFM). Substrates optical properties were studied using mono- and bi-photonic fluorescence microscopy with time resolution. An original set-up was implemented for spatial radiation pattern's measurement. Detection improvement was then tested on commercial devices. Several biomedical applications are presented. Enhancement by two orders of magnitude are achieved for DNA chips and signal-to-noise ratio is greatly increased for cells imaging.

A standard method in confocal microscopy to form an extended focus image is to merely add together (integrate) a number of optical sections taken throughout the specimen volume of interest. If we use this method in a conventional microscope the image that results is of rather poor quality. However since the image has been degraded in a known fashion and it is straightforward, by using simple inverse filtering techniques, to restore a high quality extended depth of focus image. Examples will be shown obtained in both the fluorescence and brightfield imaging modes. The method is also suited to high resolution stereo imaging.

A diode-pumped Yb:glass femtosecond laser oscillator with electro-optical cavity-dumping has been applied for nonlinear laser-scanning microscopy and processing of biomaterials. The high-energy pulses delivered by this source in combination with its unique parameters proved very efficient for micro-processing of biomaterials.

Two-photon microscopy is a key method for biological and medical research on cells and tissues mainly due to the submicronic spatial resolution. Unfortunately in its conventional form, this technique leads to long time recording for three-dimensional and fluorescence lifetime imaging because it requires a single point laser scanning. The most suitable way to improve acquisition time is to illuminate the biological sample with several excitation points simultaneously. We thus present a time-resolved multifocal multiphoton microscope. Besides the advantage of preserving biological samples by reducing by a factor 64 the exposition time, this method keeps also the possibility of measuring both intensity and lifetime images of the samples.

Multi-beam confocal sectioned fluorescence lifetime imaging microscopy is demonstrated using a Yokogawa spinning disk. The single-photon excitation source is a supercontinuum generated from a Ti:sapphire seeded photonic crystalline fibre.

Microlenses have been implemented in confocal systems successfully as components of aperture arrays and as arrays of objective lenses. The use of the novel technology of variable focal length (VFL) microlenses in the confocal system has also shown potential. The properties of the VFL microlenses are controlled by the physical and chemical parameters of the microlenses. Arrays of microlenses with varying parameters are fabricated and their characteristics tuned to meet the demands of confocal microscopy.

We present a simple method to measure fluorescence lifetime based on switched excitation coupled with appropriately gated detection. Preliminary results are presented. The technique, which is extendable to multiple lifetime components, is inexpensive to implement.

Research in the Life Sciences increasingly involves the investigation of fast dynamic processes at the cellular and sub-cellular level. It requires tools to image complex systems with high temporal resolution in three-dimensional space. For this task we introduce a fast fluorescence line scanner with image acquisition speeds in excess of 100 frames per second at 512x512 pixels and with a more than 10- fold increased sensitivity compared to point scanning confocal systems. Since the system preserves the capability for optical sectioning of confocal systems it allows to observe processes in three dimensions. We describe the principle of operation, the optical characteristics of the microscope and cover several applications in particular from the field of developmental biology.

Second-harmonic-generation (SHG) microscope is applied to observe collagen fiber structure in porcine dermis and mouse tendon. Difference of collagen fiber structure among different samples is clearly visualized as high contrast SHG images using a sample-scanning SHG microscope based on a transmission configuration. From comparison of the SHG image between a transmission detection mode and a reflection one, we confirm that the reflection-mode SHG imaging is also useful to observe the collagen fiber structure in the porcine dermis. Finally, we applied a laser-scanning confocal SHG microscope to optical-sectioning SHG imaging of the thick porcine dermis every 10-μm depth and confirmed that the collagen fiber structure in the dermis is spatially evolved along the depth direction. The proposed method will be a powerful tool for in vivo measurement of human dermis to monitor skin diseases and cosmetics.

We propose a method for sub-resolution axial localization of particles in fluorescence microscopy, based on maximum-likelihood estimation. Given acquisitions of a defocused fluorescent particle, we can estimate its axial position with nanometer range precision.