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The tested performance is six orders of magnitude with 600ps pulse width and sub-fW sensitivity. Combined with 405nm laser illumination and motored monochromator, Laser Induced Fluorescence Photon Spectrometry (LIPS) has been developed with a scan range from 200~900nm at maximum of 500nm/sec and 1nm FWHM. Based on the Planck equation E=hν, this photon counting spectrum provides a fundamental advance in spectral analysis by digital processing. Advantages include its ultimate sensitivity, theoretical linearity, as well as quantitative and logarithmic analysis without use of arbitrary units. Laser excitation is also useful for evaluation of photobleaching or oxidation in materials by higher energy illumination. Traditional typical photocurrent detection limit is about 1pW which includes millions of photons, however using our system it is possible to evaluate the photon spectrum and determine background noise and auto fluorescence(AFL) in optics in any cytometry or imaging system component. In addition, the photon-stream digital signal opens up a new approach for picosecond time-domain analysis. Photon spectroscopy is a powerful method for analysis of fluorescence and optical properties in biology.
An endoscope for simultaneous macroscopic navigation and microscopic inspection of luminal sidewalls
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The physical limitations of biological optical microscopy are well established. However, considerably less attention is paid to the fact that the biological nature of the objects studied imposes additional constraints on microscopic imaging of cells and tissues. Biological systems are, by definition, dynamic. Therefore, any experimental procedure should address the biological and chemical changes during measurement in the studied system. The imaging itself may induce some of such changes, whereas others variations occur independently of microscopic observations.
The goal of this short course is to present the factors that limit the accuracy, resolution, and reproducibility of microscopic imaging of biological objects. The discussion will focus on two methods of 3D optical imaging: confocal microscopy and two-photon microscopy. The course will recapitulate the fundamental physical limitations of optical imaging, and reevaluate their meaning in the context of practical biological microscopy. The following subjects will be discussed: influence of photon statistics and instrumental noise on accuracy and resolution, photophysical and biochemical stability of fluorescence labels, photodamage and phototoxicity, autofluorescence, and intrinsic optical properties of biological specimens.
The course will also address the important issues of calibration and standardization. The performance of microscopic imaging of biological samples is usually evaluated in qualitative and subjective manner. There is no versatile, widely adopted standard for evaluation of optical microscopes used for biological studies, or for the quality of biological images collected. One of the aims of this short-course is to identify a set of statistical procedures for evaluation of microscope performance in the context of cell studies.
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