We are pleased to provide downloads, or links, to a number of papers on confocal microscopy which were produced using Vertilon's PhotoniQ DAQs.
Shashanka P. Ashili, Laimonas Kelbauskas, Jeff Houkal, Dean Smith, Yanqing Tian, Cody Youngbull, Haixin Zhu, Yasser H. Anis, Michael Hupp, Kristen B. Lee, Ashok V. Kumar, Juan Vela, Andrew Shabilla, Roger H. Johnson, Mark R. Holl, Deirdre R. Meldrum
SPIE Proceedings, Volume 7929, BioMEMS and Medical Microsystems 14 February 2011
Copyright ©2011 Society of Photo-Optical Instrumentation Engineers (SPIE)
We have developed a fully automated platform for multiparameter characterization of physiological response of individual and small numbers of interacting cells. The platform allows for minimally invasive monitoring of cell phenotypes while administering a variety of physiological insults and stimuli by means of precisely controlled microfluidic subsystems. It features the capability to integrate a variety of sensitive intra- and extra-cellular fluorescent probes for monitoring minute intra- and extra-cellular physiological changes. The platform allows for performance of other, post- measurement analyses of individual cells such as transcriptomics. Our method is based on the measurement of extracellular metabolite concentrations in hermetically sealed ~200-pL microchambers, each containing a single cell or a small number of cells. The major components of the system are a) a confocal laser scan head to excite and detect with single photon sensitivity the emitted photons from sensors; b) a microfluidic cassette to confine and incubate individual cells, providing for dynamic application of external stimuli, and c) an integration module consisting of software and hardware for automated cassette manipulation, environmental control and data collection. The custom-built confocal scan head allows for fluorescence intensity detection with high sensitivity and spatial confinement of the excitation light to individual pixels of the sensor area, thus minimizing any phototoxic effects. The platform is designed to permit incorporation of multiple optical sensors for simultaneous detection of various metabolites of interest. The modular detector structure allows for several imaging modalities, including high resolution intracellular probe imaging and extracellular sensor readout. The integrated system allows for simulation of physiologically relevant microenvironmental stimuli and simultaneous measurement of the elicited phenotypes. We present details of system design, system characterization and metabolic response analysis of individual eukaryotic cells.
Multifocal Multiphoton Microscopy: New Detection Methods and Biological Applications
Jörg Martini, Faculty of Physics, Bielefeld University, November 2006
In this work multifocal multiphoton microscopy (MMM) has, on the one hand, been applied as a technique for the investigation of certain biological samples. On the other hand, new detection techniques have been developed and tested in order to advance the capabilities in MMM. This progress in technical possibilities has been driven by the particular difficulties in imaging the samples of interest, i.e. cartilage, tissue engineering products for cartilage implants and tobacco protoplasts transfected with a Arabidopsis thaliana transcription factor. Some of these techniques became a standard measurement protocol for successful imaging of the particular sample of interest, while others were found to be better suited for types of samples that they were not intended for. As this work documents a contribution to the collaboration between partners both in the field of biology, who are interested gaining knowledge on their samples, as well as in the field of microscopy, who are interested in advancing the general techniques, the results serve both groups.
Calibration and Validation of Confocal Spectral Imaging Systems
Jeremy M. Lerner, Robert M. Zucker, U.S. Environmental Protection Agency, Reproductive Toxicology Division
National Health and Environmental Effects Research Laboratory, Cytometry Part A 62A:8–34 (2004)
Copyright ©2004 Wiley-Liss, Inc.
Confocal spectral imaging (CSI) microscopic systems currently on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, some CSI systems have been found to present inconsistent spectral profiles of reference spectra within a particular system and between related and unrelated instruments. This variability confirms that there is a need for a standardized, objective calibration and validation protocol. Our protocol uses an inexpensive multi-ion discharge lamp (MIDL) that contains Hg+, Ar+, and inorganic fluorophores that emit distinct, stable, spectral features in place of a sample. We derived reference spectra from the MIDL data to accurately predict the spectral resolution, ratio of wavelength to wavelength, contrast, and aliasing parameters of any CSI system. We were also able to predict and confirm the influence of pinhole diameter on spectral profiles.
Scattering Suppression and Confocal Detection in Multifocal Multiphoton Microscopy
Jörg Martini, Volker Andresen, Dario Anselmetti
Journal of Biomedical Optics 12(3), 034010 (May/June 2007)
Copyright ©2007 Society of Photo-Optical Instrumentation Engineers
We have developed a new descanned parallel (32-fold) pinhole and photomultiplier detection array for multifocal multiphoton microscopy that effectively reduces the blurring effect originating from scattered fluorescence photons in strongly scattering biological media. With this method, we achieve a fourfold improvement in photon statistics for detecting ballistic photons and an increase in spatial resolution by 21% in the lateral and 35% in the axial direction compared to single-beam non-descanned multiphoton microscopy. The new detection concept has been applied to plant leaves and pollen grains to verify the improvements in imaging quality.
Laser Scanning Confocal Microscopy
Nathan S. Claxton, Thomas J. Fellers, Michael W. Davidson
Department of Optical Microscopy and Digital Imaging, National High Magnetic Field Laboratory, The Florida State University, Tallahassee, Florida
Laser scanning confocal microscopy has become an invaluable tool for a wide range of investigations in the biological and medical sciences for imaging thin optical sections in living and fixed specimens ranging in thickness up to 100 micrometers. Modern instruments are equipped with 3-5 laser systems controlled by high-speed acousto-optic tunable filters (AOTFs), which allow very precise regulation of wavelength and excitation intensity. Coupled with photomultipliers that have high quantum efficiency in the near-ultraviolet, visible and near-infrared spectral regions, these microscopes are capable of examining fluorescence emission ranging from 400 to 750 nanometers. Instruments equipped with spectral imaging detection systems further refine the technique by enabling the examination and resolution of fluorophores with overlapping spectra as well as providing the ability to compensate for autofluorescence. Recent advances in fluorophore design have led to improved synthetic and naturally occurring molecular probes, including fluorescent proteins and quantum dots, which exhibit a high level of photostability and target specificity.
Spectral Imaging Fluorescence Microscopy
Tokuko Haraguchi, Takeshi Shimi, Takako Koujin, Noriyo Hashiguchi, Yasushi Hiraoka
Genes to Cells (2002)
Copyright ©2002 Blackwell Science Limited
The spectral resolution of fluorescence microscope images in living cells is achieved by using a confocal laser scanning microscope equipped with grating optics. This capability of temporal and spectral resolution is especially useful for detecting spectral changes of a fluorescent dye; for example, those associated with fluorescence resonance energy transfer (FRET). Using the spectral imaging fluorescence microscope system, it is also possible to resolve emitted signals from fluorescent dyes that have spectra largely overlapping with each other, such as fluorescein isothiocyanate (FITC) and green fluorescent protein (GFP).
