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Essay on laser scanning microscopy
Until recently, optical resolution below ~200 nm in x-y and ~500 nm in z has been impossible due to the diffraction limit of light. This has hampered the study of many facets of developmental biology arising over small length scales, such as molecular processes in small structures such as tight junctions synapses, microfilaments, and nuclear pores. Advances in super-resolution microscopy are changing this, enabling optical examina- tion of nanometer-scale phenomena. One strategy for pushing the limits of spatial resolution employs stimulated emission to narrow the focal spot of the microscope. Called stimulated emission depletion (STED) microscopy (Hell and Wichmann, 1994), this technique uses a pair of overlapping concentric laser beams scanned together, with the first beam exciting fluorophores lying within a diffraction-limited spot and the second beam using stimulated emission to narrow this spot by preventing fluorescence at its periphery. STED microscopy can typically achieve 10-fold higher resolution than conventional fluorescence imaging, al- lowing new insights into topics as diverse as tracking synaptic vesicles in neurons, monitoring shape changes in dendritic spines, and measuring lipid dynamics in the plasma membrane (Na¨ gerl et al., 2008; Eggeling et al., 2009). Another approach for breaking the constraints of diffraction is saturated structured illumination microcrospy (SSIM) (Gustafsson, 2005; Heintzmann et al., 2002). It achieves this by illuminating the sample with a sequence of periodic patterns of high spatial frequencies that can reach satu- rating excitation intensities. Fine spatial details in the sample at less than 100 nm resolution are then extracted computationally from the raw images using deconvolution algorithms and Fourier transformations (Schermelleh et al., 2008).
Impressive technological innovations of modern microscopes also extend to the study of whole, living organisms. Conventional confocal microscopes usually allow imaging of no more than 44 mm deep into a tissue due to light scattering. But many important processes relevant for understanding tissue and developmental function occur deeper than this, so scientists are working to push the depth resolution capabilities of microscopes. A powerful approach for achieving increased depth penetration into a specimen is two-photon microscopy (Helmchen and Denk, 2005). It uses near infrared illumination, which goes deeper than visible light, to convert two or more incoming photons into an outgoing photon of distinct color. The spatial confinement of the excitation volume permits imaging deep into a specimen with inherent optical sectioning. To allow imaging of depths in the centimeter range into tissues, two-photon imaging can be combined with microendoscopy, which employs a microendoscope com- prised of a thin but rigid optical probe that inserts into tissue to conduct light to and from deep tissue locations (Flusberg et al., 2005). By scanning a laser focal spot outside the tissue, the probe device projects and demagnifies the scanning pattern to a focal plane inside the tissue. In this way, it becomes possible to ex- plore cell properties in the context of the whole organism, such as in the cavities of internal organs or in the pathways of blood capillaries (Monfared et al., 2006).
Essay on laser scanning microscopy - Bruce White Galleries
The present generation of light microscopes has been modified in nearly all parameters compared to similar micro- scopes of only a decade ago, enabling imaging over unprecedented spatial scales and experimental situations. Due to key improvements, it is now possible to obtain speeds of image acquisition of ~ 120 images/s or even higher, and to have multispectral imaging due to minimization of spectral emission overlap. Microscope systems incorporating these modifications include commercial light scanning confocals, spinning disk confocals, and wide-field microscopes with total internal reflection. Many of these systems have built-in macros for perform- ing kinetic experiments such as FRAP, FRET, or FCS. Advances in automation and image analysis are additionally making it possible to do rapid screening and large-scale anatomical reconstruc- tion using these microscope platforms.
Because the thickness of the light sheet in plane illumination microscopy diverges greatly over the field of view, the tech- nique has until recently been limited to the multicellular, micron-level domain. However, with the use of Bessel beams to create thinner light sheets, it is now possible to extend plane illumination microscopy to the subcellular, nanomet- ric-level domain (Planchon et al., 2011). Creation of the Bessel beam is accomplished by positioning an annular apodization mask in front of the excitation objective. This creates a thin light sheet of less than 0.6 mm that can be scanned rapidly over 60 3 80 mm fields of view. The resulting 3D high-speed live cell imaging (i.e., 10 ms per image plane) is unprecedented and can provide astonishing time-lapse sequences of 3D orga- nization within and between cells. This advance promises to be highly influential in clarifying many aspects of the dynamics and relationships of cell interactions within complex tissues that have eluded other methods such as two-photon and traditional light sheet planar microscopy because of their limited z resolution and slower optical sectioning speeds.
Microscopes: Scanning Electron Microscope Essay - …
Inverted LSM 710, Zeiss, supported with ZEN 2009 software, Germany.
The system has 6 laser lines for excitation and any dye that is excited at one of these following wavelengths can be used: 405; 458; 488; 514; 543 and 633 nm.
Good for fixed samples
Continuous Spectral imaging Online spectral un-mixing
Transmitted light detector (DIC objectives)
Live imaging (CO2, humidity and temperature controlled chamber)
Multi-point experiments (XYZ positions, tiling).
What is Confocal Laser Scanning Microscope (CLSM)
To view the nodal cilia, fluorescent microscopy using a confocal laser microscope, and immunoelectron microscopy using electron microscopy was used without electron staining (Nonaka & Tanaka, 1998).
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