Objectivelens and eyepiece lens magnification

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Collimated beam shaper and collimated diffuser is your solution for applications requiring a wide working distance range or an extended depth of focus.

NOTE: If a 1/3” inch camera (6mm diagonal) is used on the 0.5X microscope adaptor you can apply the ratio of 6/8 for the reduction in the captured field.

The story of GFP begins in the oceans with the jellyfish Aequorea victoria, which has the unusual property that its outer edges glow green when it is agitated. In the early 1960s, Osamu Shimomura collected raw material from thousands of these jellyfish and extracted a small amount of a blue luminescent protein, which was subsequently named aequorin. During this process he also found another substance that glowed green when exposed to ultraviolet light — this was the protein that later became known as GFP. Shimomura and colleagues went on to show that the green glow produced by the jellyfish arises from an energy-transfer process in which the aequorin donor excites the GFP acceptor, which then emits green light.

MICROSCOPE meaning: 1. a device that uses lenses to make very small objects look larger, so that they can be…. Learn more.

What is objectivelens inmicroscope

The very aptly named green fluorescent protein — or GFP as it is almost universally known — is a barrel-shaped protein made up of 238 amino acids. Threaded through the long axis of the β-sheet barrel is an α-helix that contains a chromophore that is responsible for the emission of green light when GFP is exposed to either blue or ultaviolet light. This particular property, coupled with the fact that GFP is well tolerated by many different organisms, has led to its use as a fluorescent tag for monitoring biological processes at the cellular level.

Whatdoesthestage doon a microscope

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The magnification of the image depends on the combination of the eyepiece and the objective used. This combination also affects the field of view. This example shows how these factors inter-relate.

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A variety of microscope objectives are available. All objectives use lenses to focus light. Light is broken down into various wavelengths (colors) as it travels through a lens. The various wavelengths have different focal points. That means that red, green and blue appears to focus at different points. This is called chromatic aberration. Spherical aberrations are focal mismatches caused by the shape of the lens. Quality lenses are designed correct for chromatic and spherical aberration to bring the primary colors to a common focal point. These terms may help you determine the best objective for your application:

The further development of GFP was based on a greater understanding of the molecular structure of the protein and specifically the chromophore responsible for its colourful name. Roger Tsien and co-workers explained how three amino acids in the peptide backbone of GFP — namely serine, tyrosine and glycine in positions 65, 66 and 67, respectively — react in the presence of oxygen to form the fluorescent chromophore p-hydroxybenzylideneimidazolinone. With this more detailed description of GFP, Tsien went on to develop other GFP derivatives with different spectral characteristics and increased stability. Not only could the brightness of the fluorescence be enhanced, but also the colour of emission could be tuned. Today all the colours of the rainbow can be found in a range of GFP and GFP-like proteins.

Opticalmicroscope

Whatdoestheeyepiece lens doon a microscope

In the early 1990s, Martin Chalfie and co-workers demonstrated that GFP could be expressed in organisms other than Aequorea victoria — such as Escherichia coli and Caenorhabditis elegans — and this was the breakthrough that paved the way for the practical implementation of GFP as a fluorescent tag for studying biological processes. It was generally thought that a number of steps requiring other proteins would be needed to produce the chromophore in GFP, but these experiments proved this to be wrong. Significantly, this result meant that GFP could be used as a universal tag, because no other auxiliary agents were needed to induce fluorescence. By engineering the genetic machinery of C. elegans so that it would produce GFP when a protein with a specific activity was expressed inside a cell, Chalfie was able to see cellular processes in a whole new light — albeit a green one!

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TIP: On the trinocular version of the PZMIII or PZMIV stereo microscope with the standard configuration (1.0X objective, 10X eyepieces) and with the optimal camera adaptor (0.5X on a ½” CCD camera) the video capture field of view is up to 40% less than the visual field. By using a 0.5X objective with 20X eyepieces the video capture area doubles, and the resulting video capture more closely matches the visual field of view.

Apochromatic objective–This is the most expensive objective. It is chromatically adjusted for four colors (deep blue, blue, green and red) and spherically corrected for deep blue, blue and sometimes green. This is the best choice for color viewing. These have a higher numerical aperture (N.A.) than achromats or fluorites.

What is thepurpose ofthe objectivelens inalightmicroscope

Solution: Instead of the standard configuration, setup the microscope with a 0.5X objective to increase the working distance to 187 mm. The result of using this lower power objective is that the magnification range decreases by one half and at the same time the field of view double. To restore the microscope system to the original condition (magnification and field of view), replace the 10X eyepieces with 20X eyepieces. The use of these two options restores the visual field of view and magnification range back to the original condition with the added benefit of a larger working distance.

Plan objective–These objectives produces a flat image across the field of view. The three objectives discussed above all produce a curved image. A plan-achromat, plan-fluorite or plan-apochromat are corrected.

Whatdoesthe objectivelens doon a microscope

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Problem: The PZMIII or PZMIV stereo zoom microscope normally comes with a 1.0X objective and a 10X pair of eyepieces. The magnification is 6X to 50X, however the concept of magnification is difficult to visualize. Let's discuss what can be seen at the two zoom extremes. Imagine the visual circle to be a range of 34–4.2 mm. This microscope has a working distance of 100mm. Researchers working with small animals will have difficulty working in this tight space.

What is thestageon a microscope

Infinity Correction–When measuring from the back end of the objective to the primary focal plane, many microscopes are limited to a specific distance (160mm). More expensive microscope use a different series of lenses, prisms and mirrors to allow for an "infinite" distance between those two points. This is called infinity correction.

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Fluorite or semi-apochromat objectives–These lenses are chromatically corrected for red and blue, and the green focus is also close. They are spherically corrected for blue and green. This objective is better suited for color viewing or recording than achromatic objectives.

Harmonic separators used to reflect one wavelength and to transmit the others. Reflectance is higher than 99.5% for the wavelength of interest.

Achromatic objectives–This objective brings red and blue light to a common focus, and is corrected for spherical aberrations for green. It is excellent for black and white viewing. If an objective is not labeled, it is achromatic.

The 2008 Nobel Prize for Chemistry has been awarded to Osamu Shimomura, Martin Chalfie and Roger Tsien for the discovery and development of the so-called green fluorescent protein

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The first image shows the eyepiece view when using a 1.0X objective with a 10X eyepiece. It has a 34mm field of view. The second image shows the video field of view of about 16–4.7mm (COLCAM-NTSC camera with a 0.5X coupler). The third image shows the video view that approximates the eyepiece view. It uses a 0.5X objective with a 20X eyepiece.

Apart from the obvious biomedical implications, GFP sensors have also been developed that can detect chemical species such as metals ions and small molecules. So not only has GFP enabled scientists to see biological processes in a whole new light, but many other chemical opportunities await. The future for GFP is a bright one.