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How to transform your dark field microscopy ppt into a dark field microscopy ppt.

dark field microscopy ppt

When to use dark field illumination?

dark field microscopy ppt is most readily set up at low magnifications (up to 100x), although it can be used with any dry objective lens. Any time you wish to view everything in a liquid sample, debris and all, dark field is best. Even tiny dust particles are obvious. Dark field is especially useful for finding cells in suspension. Dark field makes it easy to obtain the correct focal plane at low magnification for small, low contrast specimens. Use dark field for

Initial examination of suspensions of cells such as yeast, bacteria, small protists, or cell and tissue fractions including cheek epithelial cells, chloroplasts, mitochondria, even blood cells (small diameter of pigmented cells makes it tricky to find them sometimes despite the color).
Initial survey and observation at low powers of pond water samples, hay or soil infusions, purchased protist or metazoan cultures.
Examination of lightly stained prepared slides. ? Initial location of any specimen of very small size for later viewing at higher power.
Determination of motility in cultures

 

dark field microscopy ppt

dark field microscopy ppt TECHNOLOGY:

 

Darkfield microscopy creates contrast in transparent unstained specimens such as living cells. It depends on controlling specimen illumination so that central light which normally passes through and around the specimen is blocked. Rather than light illuminating the sample with a full cone of light (as in brightfield microscopy) the condenser forms a hollow cone with light travelling around the cone rather than through it.

This form of illumination allows only oblique rays of light to strike the specimen on the microscope stage and the image is formed by rays of light scattered by the sample and captured in the objective lens. When there is no sample on the microscope stage the view is completely dark.

Care should be taken in preparing specimens as features above and below the plane of focus can also scatter light and compromise image quality (for example, dust, fingerprints). In general, thin specimens are better because the possibility of diffraction artifacts is reduced.

dark field microscopy ppt

dark field microscopy ppt at High Magnifications

 

For more precise work and blacker backgrounds, you may choose a condenser designed especially for darkfield, i.e. to transmit only oblique rays. There are several varieties: “dry” darkfield condensers with air between the top of the condenser and the underside of the slide–and immersion darkfield condensers which require the use of a drop of immersion oil (some are designed to use water instead) establishing contact between the top of the condenser and the underside of the specimen slide. The immersion darkfield condenser has internal mirrored surfaces and passes rays of great obliquity and free of chromatic aberration, producing the best results and blackest background.

Perhaps the most widely used darkfield condenser is the paraboloid, consisting of a solid piece of glass ground very accurately into the shape of a paraboloid, as illustrated in Figure 5(b). Light incident upon the reflecting surface (between the glass and condenser housing in Figure 5(b)) of a paraboloid condenser will be focused at the focal point of the reflector. Most paraboloid condensers are cut to ensure that the focal point is slightly beyond the top of the condenser so that parallel light rays will be focused at a position that maximizes illumination of the specimen. The light stop at the bottom of the glass condenser serves to block central rays from reaching the specimen. Light rays that are reflected by the condenser are angled higher than the critical angle of reflection and converge at the principal focus of the condenser. The combination of a glass slide, mounting medium, and immersion oil (between the condenser and the microscope slide) complete the optical homogeneity of the paraboloid shape.

As discussed above, the dry darkfield condenser is useful for objectives with numerical apertures below 0.75 (Figure 5(a)), while the paraboloid and cardioid immersion condensers (Figures 1 and 5(b)) can be used with objectives of very high numerical aperture (up to 1.4). Objectives with a numerical aperture above 1.2 will require some reduction of their working aperture since their maximum numerical aperture may exceed the numerical aperture of the condenser, thus allowing direct light to enter the objective. For this reason, many high numerical aperture objectives designed for use with darkfield as well as brightfield illumination are made with a built-in adjustable iris diaphragm that acts as an aperture stop. This reduction in numerical aperture also limits the resolving power of the objective as well as the intensity of light in the image. Specialized objectives designed exclusively for darkfield work are produced with a maximum numerical aperture close to the lower limit of the numerical aperture of the darkfield condenser. They do not have internal iris diaphragms, however the lens mount diameters are adjusted so at least one internal lens has the optimum diameter to perform as an aperture stop.

Table 2 lists several properties of the most common reflecting high numerical aperture darkfield condensers. This table should be used as a guide when selecting condenser/objective combinations for use with high numerical aperture darkfield applications.

dark field microscopy ppt

What is CytoViva® Enhanced dark field microscopy ppt Optics

 

CytoViva’s enhanced darkfield microscope optics improve signal-to-noise up to ten times (10x) over standard darkfield optics1.  This enables nanomaterials as small as 10nm-20nm to be imaged right from your laboratory benchtop2.

CytoViva’s patented (US patents No. 7,542,203, 7,564,623) enhanced darkfield illumination system, which replaces the standard microscope condenser, works by coupling the source illumination directly to the condenser optics. In this optical path, collimating lenses and mirrors align and fix the geometry of the light to match the geometry of the condenser annulus. This creates a very narrow, oblique angle of source illumination that can be precisely focused into the sample but bypasses the objective. The result is very intense  scatter from nanoscale samples against a very dark background.  Source illumination compatible with this system can be halogen, xenon or even laser, depending on the application.

Enhanced Darkfield Illumination Optics

CytoViva’s enhanced darkfield optics enable scientists to optically observe a wide range of nanoscale materials quickly and easily in solution, live cells, tissue and materials based matrices. In addition, non-fluorescent live cells and pathogens can be easily observed at a level of detail not possible with traditional optical imaging techniques such as phase contrast or differential interference contrast.

Finally, when combined with CytoViva’s Hyperspectral Imaging capability this high signal-to-noise microscopy method enables researchers to spectrally characterize and map nanoscale samples in a wide range of environments.

To see just how easy CytoViva is to use, simply watch this brief video overview of the installation and alignment process.
Please email sales@CytoViva.com to request your private web demonstration.

dark field microscopy ppt

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