Combination of ellipsometry, laser scanning microscopy and Z -scan fluorescence correlation spectroscopy elucidating interaction of cryptdin-4 with supported phospholipid bilayers

2007 ◽  
Vol 14 (4) ◽  
pp. 503-509 ◽  
Author(s):  
Adam Miszta ◽  
Radek Macháň ◽  
Aleš Benda ◽  
Andre J. Ouellette ◽  
Wim Th. Hermens ◽  
...  
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Laser Scanning ◽  
Correlation Spectroscopy ◽  
Phospholipid Bilayers ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Fluorescence Correlation

scholarly journals Fluorescence Correlation Spectroscopy Combined with Multiphoton Laser Scanning Microscopy—A Practical Guideline

2021 ◽  
Vol 11 (5) ◽  
pp. 2122
Author(s):  
Jeemol James ◽  
Jonas Enger ◽  
Marica B. Ericson
Keyword(s):  
Biomedical Research ◽  
Fluorescence Correlation Spectroscopy ◽  
Laser Power ◽  
Laser Scanning ◽  
Correlation Spectroscopy ◽  
Operating Conditions ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Fluorescence Correlation ◽  
Practical Guideline

Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue—being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research.

scholarly journals Physicochemical Properties of Chromosomes in Live Cells Characterized by Label-Free Imaging and Fluorescence Correlation Spectroscopy

2019 ◽  
Author(s):  
Tae-Keun Kim ◽  
Byong-Wook Lee ◽  
Fumihiko Fujii ◽  
Kee-Hang Lee ◽  
YongKeun Park ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Fluorescence Correlation Spectroscopy ◽  
Three Dimensional ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Optical Diffraction ◽  
Live Cells ◽  
Label Free ◽  
Mitotic Chromosomes ◽  
Fluorescence Correlation

AbstractThe cell nucleus is a three-dimensional, dynamic organelle that is organized into many subnuclear bodies, such as chromatin and nucleoli. The structure and function of these bodies is maintained by diffusion and interactions between related factors as well as dynamic and structural changes. Recent studies using fluorescent microscopic techniques suggest that protein factors can access and are freely mobile in mitotic chromosomes, despite their densely packed structure. However, the physicochemical properties of the chromosome itself during cell division are not yet fully understood. Physical parameters, such as the refractive index (RI), volume of the mitotic chromosome, and diffusion coefficients of fluorescent probes inside the chromosome were quantified using an approach combining label-free optical diffraction tomography with complementary confocal laser scanning microscopy and fluorescence correlation spectroscopy. Variance in these parameters correlated among various osmotic conditions, suggesting that changes in RI are consistent with those in the diffusion coefficient for mitotic chromosomes and cytosol. Serial RI tomography images of chromosomes in live cells during mitosis were compared with three-dimensional confocal micrographs to demonstrate that compaction and decompaction of chromosomes induced by osmotic change were characterized by linked changes in chromosome RI, volume, and the mobility of fluorescent proteins.

scholarly journals QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

2011 ◽  
Vol 25 (3) ◽  
pp. 111 ◽  
Author(s):  
Merete Krog Raarup ◽  
Jens Randel Nyengaard
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
3D Structure ◽  
Correlation Spectroscopy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Two Photon ◽  
Photon Excitation

This paper discusses recent advances in confocal laser scanning microscopy (CLSM) for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm) tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer), FLIM (Fluorescence Lifetime Imaging Microscopy), FCS (Fluorescence Correlation Spectroscopy) and FRAP (Fluorescence Recovery After Photobleaching) are introduced and their applicability for quantitative imaging of biomolecular (co-)localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

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