Multiphoton imaging has developed into an important technique for in-vivo research in life sciences. With the laser System DermaInspect (JenLab, Germany) laser radiation from a Ti:Sapphire laser is used to generate multiphotonabsorption deep in the human skin in vivo. The resulting autofluorescence radiation arises from endogenous fluorophores such as NAD(P)H, flavines, collagen, elastin, porphyrins und melanin. Second harmonic generation (SHG) was used to detect collagen structures in the dermal layer. Femtosecond laser multiphoton imaging offers the possibility of high resolution optical tomography of human skin as well as fluorescence lifetime imaging (FLIM) with picosecond time resolution. In this work a photon detector with ultrashort rise time of less than 30ps was applied to FLIM measurements of human skin and hair with different pigmentation. Fluorescence lifetime images of different human hair types will be discussed.
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.
Multiphoton Microscopy with a femtosecond pulsed Ti:sapphire laser in the near infrared (NIR) enables the user not only to image cells and tissues with a subcellular resolution but also to perform highly precise nanosurgery. Intratissue compartments, single cells and even cell organelles like mitochondria, membranes or chromosomes can be manipulated and optically knocked out. Working at transient TW/cm2 laser intensities, single cells of tumor-sphaeroids were eliminated efficiently inside the sphaeroid without damaging the neighbour cells. Also single organelles of cells inside tissues could be optically knocked out with the nanoscalpel without collateral damage. Tissue structures inside a human tooth have been ablated with sizes below 1 μm. This method may become a useful instrument for nano-manipulating and surgery in several fields of science, including targeted transfection.
Multiphoton imaging has developed into an important technique for in-vivo research in life sciences in the last few years. A near-infrared laser beam is focused into a sample such that multiphoton-absorption can be generated which stimulates a fluorescence signal as well as second harmonic generation (SHG). Recently it has been shown
that it is possible to image the epidermis in vivo with a resolution of about 1 μm. It was possible to produce 3-dimensional autofluorescence maps of the investigated tissue. However, the depth range of this technique is limited through the working distance of the focusing optics mostly to the epidermal part of the skin. Gradient index lenses offer possibilities to expand the imaging depth into the dermal layer. With typical diameters of up to 2mm and lengths between 2 and 6 cm they are capable of transmitting the laser, fluorescence and SHG radiation and to be integrated into an imaging system. First results of the applicability of gradient index lenses for imaging of skin are presented.
Multi-dimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterised by its time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multi-dimensional TCSPC makes a fluorescence lifetime technique with multi-wavelength capability, near-ideal counting efficiency, and the capability to resolve multi-exponential decay functions. We show that the same technique and the same hardware can be used to for precision fluorescence decay analysis, fluorescence correlation spectroscopy (FCS), and fluorescence intensity distribution analysis (FIDA and FILDA) in selected spots of a sample.
Fluorescence correlation spectroscopy (FCS) has evolved to a valuable tool for biomolecular analysis on the single molecule level. Measurements on a single molecule level can only be performed if the measurement volume is small enough to contain on average only very few molecules. Common FCS-systems are therefore based on a confocal
geometry in which a laser spot is focused into a liquid sample. This illumination concept in combination with a pinhole in the detection path leads to an observation volume in the order of one femtoliter. On the other hand, many biomolecular interactions need to be measured on surfaces. To study such interactions or the fluctuating
signal of surface bound molecules itself, as for instance during single molecule enzyme catalysis, evanescent field based excitation seems advantageous as compared to confocal FCS. We discuss different schemes for evanescent field FCS and present an efficient excitation-detection scheme in an objective-based TIR-FCS configuration.
We present parallel single molecule detection (SMD) and fluorescence correlation spectroscopy (FCS) experiments with a fully integrated complementary metal oxide semiconductor (CMOS) single-photon 2×2 detector array. Multifocal excitation is achieved with a diffractive optical element (DOE). Special emphasis is placed on parallelization of the total system. The performance of the novel single-photon CMOS detector is investigated and compared to a state-of-the-art single-photon detecting module [having an actively quenched avalanche photodiode (APD)] by measurements on free diffusing molecules at different concentrations. Despite the order of magnitude lower detection efficiency of the CMOS detector compared to the state-of-the-art single-photon detecting module, we achieve single molecule sensitivity and reliably determine molecule concentrations. In addition, the CMOS detector performance for the determination of the fraction of slowly diffusing molecules in a primer solution (two-component analysis) is demonstrated. The potential of this new technique for high-throughput confocal-detection-based systems is discussed.
We present multipoint Fluorescence Correlation Spectroscopy (FCS) experiments with a fully integrated Complementary Metal Oxide Semiconductor (CMOS) single photon 2x2 detector array. Multifocal excitation was achieved with a diffractive optical element (DOE). Special emphasis was put on parallelization of the total system. In particular the performance of the single-photon CMOS detector was investigated and compared to a state-of-the art single-photon detecting module (actively quenched avalanche photo diode) by measurements on free diffusing molecules at different concentrations. The potential of our new technique for high throughput FCS based systems is discussed.
In this paper we present recent spectroscopic studies using a Solid Immersion Lens for Fluorescent Correlation Spectroscopy measurements. We compare the performance of the Solid Immersion Lens confocal microscope built-up in our group to the performance of a conventional confocal microscope used for FCS. The novelty of the new SIL-FCS microscope is a system containing a conventional objective (NA = 0.6) combined with a Solid Immersion Lens used for single molecule experiment. Important parameters for single molecule experiments such as collection efficiency and excitation field confinement are investigated for different modes of the SIL objective system.
The crucial role played by the source's degree of spatial coherence in wide-field optical coherence tomography is shown experimentally. Spatially coherent illumination, as obtained with a pulsed laser, generates a considerable amount of coherent optical cross-talk. The latter can be suppressed with spatially incoherent illumination as provided by a thermal or a pseudothermal light source. Demonstration is made for a US air force resolution target covered with a scattering solution made of polystyrene microspheres suspended in water. The origin and nature of cross-talk signals are discussed, as well as specific limitations of spatially incoherent sources.
KEYWORDS: CMOS sensors, Microscopy, Digital signal processing, Signal processing, Spatial frequencies, Imaging systems, Microscopes, Signal detection, Objectives, Structured light
Real-time optically sectioned microscopy is demonstrated using an AC-sensitive detection concept realized with smart CMOS image sensor and structured light illumination by a continuously moving periodic pattern. We describe two different detection systems based on CMOS image sensors for the detection and on-chip processing of the sectioned images in real time. A region-of-interest is sampled at high frame rate. The demodulated signal delivered by the detector corresponds to the depth discriminated image of the sample. The measured FWHM of the axial response depends on the spatial frequency of the projected grid illumination and is in the μm-range. The effect of using broadband incoherent illumination is discussed. The performance of these systems is demonstrated by imaging technical as well as biological samples.
We demonstrate phase space tomography for the measurement of the transversal spatial coherence function of light after propagation through a scattering medium. The results of this approach are compared to measurements performed with shearing-interferometry. Implications for parallel Optical Coherence Tomography will be briefly discussed.
Monitoring biological relevant reactions on the single molecule level by the use of fluorescent probes has become one of the most promising approaches for understanding a variety of phenomena in living organisms. By applying techniques of fluorescence spectroscopy to labelled molecules a manifold of different parameters becomes accessible i.e. molecular dynamics, energy transfer, DNA fingerprinting, etc... can be monitored at the molecular level.
However, many of these optical methods rely on oversimplified assumptions, for example a three-dimensional Gaussian observation volume, perfect overlap volume for different wavelength, etc. which are not valid approximations under many common measurement conditions. As a result, these measurements will contain significant, systematic artifacts, which limit their performance and information content.
Based on Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Spectroscopy we will present representative examples including a thorough signal analysis with a strong emphasis on the underlying optical principles and limitations. An outlook to biochip applications, parallel FCS and parallel Lifetime measurements will be given with cross links to optical concepts and technologies used in industrial inspection.
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