Download
1 / 150
1.63k likes | 2.04k Views
MICROSCOPY. Microscopy is the technical field of using microscopes to view samples or objects. There are three well-known branches of microscopy, optical, electron and scanning probe microscopy.
E N D
Microscopy is the technical field of using microscopes to view samples or objects. There are three well-known branches of microscopy, optical, electron and scanning probe microscopy.
Optical and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic radiation/electron beam interacting with the subject of study, and the subsequent collection of this scattered radiation in order to build up an image.
The optical microscope uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest and simplest of the microscopes.
New designs of digital microscopes are now available which use a CCD camera to examine a sample and the image is shown directly on a computer screen without the need for expensive optics such as eye-pieces.
Chromatic aberration • Chromatic aberration is caused by a lens having a different refractive index for different wavelengths of light (the dispersion of the lens).
Since the focal length “ f ” of a lens is dependent on the refractive index “ n ”, different wavelengths of light will be focused on different positions.
Numerical aperture • Numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light.
Where ’ n ‘ is the index of refraction of the medium in which the lens is working and θ is the half-angle of the maximum cone of light that can enter or exit the lens.
Refractive Index • 1.0 for air, 1.33 for pure water, and up to 1.56 for oils
NA is important because it indicates the resolving power of a lens. The size of the finest detail that can be resolved is proportional to λ / NA, where λ is the wavelength of the light.
WAVELENGTH • Wavelength is the distance between repeating units of a propagating wave of a given frequency. It is commonly designated by the Greekletterlambda (λ).
A lens with a larger numerical aperture will be able to visualize finer details than a lens with a smaller numerical aperture. Lenses with larger numerical apertures also collect more light and will generally provide a brighter image.
Optical resolution • Optical resolution describes the ability of an imaging system to resolve detail in the object that is being imaged. The ability of a lens to resolve detail is usually determined by the quality of the lens but is ultimately limited by diffraction
The resolution of a microscope is defined as the minimum separation needed between two objects under examination in order for the microscope to discern them as separate objects.
This minimum distance is labeled δ. If two objects are separated by a distance shorter than δ, they will appear as a single object in the microscope.
DEPTH OF FIELD • Depth of field (DOF) is the portion of a scene that appears sharp in the image.
The DOF is determined by the subject distance (that is, the distance to the plane that is perfectly in focus), the lens focal length, and the lens f-number (relative aperture).
Magnification • Magnification is the process of enlarging something only in appearance, not in physical size. Magnification is also a number describing by which factor an object was magnified.
When this number is less than one it refers to a reduction in size, sometimes called Minification.
Real image • A real image is a representation of an actual object (source) formed by rays of light passing through the image.
If a screen is placed in the plane of a real image, the image will generally become visible. Real images can be produced by concave mirrors and converging lenses.
Virtual image • A virtual image is an image in which the outgoing rays from a point on the object never actually intersect at a point. A simple example is a flat mirror where the image of oneself is perceived at twice the distance from yourself to the mirror.
That is, if you are half a meter in front of the mirror, your image will appear at a distance of half a meter inside or behind the mirror.
Oil Immersion Objective • oil immersion is a technique used to increase the resolution of a microscope.
This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens.
The refractive indices of the oil and of the glass in the first lens element are nearly the same, which means that the refraction of light will be small upon entering the lens In addition to improving resolution.
The use of oil is also advantageous in that it reduces the reflective losses as light enters the lens. • Cedar wood Oil is used in Oil immersion.
Stereo microscope • The stereo or dissecting microscope is designed differently , and serves a different purpose.
It uses two separate optical paths with two objectives and two eyepieces to provide slightly different viewing angles to the left and right eyes.
In this way it produces a three-dimensional visualization of the sample being examined
The stereo microscope is often used to study the surfaces of solid specimens or to carry out close work such as sorting, dissection, microsurgery, watch-making, small circuit board manufacture or inspection, etc.
Unlike compound microscopes, illumination in a stereo microscope most often uses reflected (episcopic) illumination rather than transmitted (diascopic) illumination, that is, light reflected from the surface of an object rather than light transmitted through an object.
Digital Microscope • Low power microscopy is also possible with digital microscopes, with a camera attached directly to the USBport of a computer, so that the images are shown directly on the monitor.
Often called "USB" microscopes, they offer high magnifications (up to about 200×) without the need to use eyepieces, and at very low cost.
Digital microscope • A digital microscope uses optics and a charge-coupled device (CCD) camera to output a digital image to a monitor. A digital microscope differs from an optical microscope in that there is no provision to observe the sample directly through an eyepiece.
Since the optical image is projected directly on the CCD camera, the entire system is designed for the monitor image
Resolution of the image is dependent on the CCD used in the camera. Using a typical 2 Megapixel CCD, an image with 1600 x 1200 pixels is generated. The resolution of the image is dependent on the field of view of the lens used with the camera.
The approximate pixel resolution can be determined by dividing the horizontal field of view (FOV) by 1600. Most common instruments have a relatively low resolution of 1.3 Megapixels, but higher resolution cameras are available.
The images can be recorded and stored in the normal way on the computer. The camera is usually fitted with a light source, although extra sources (such as a fibre-optic light) can be used to highlight features of interest in the object. They also offer a large depth of field, a great advantage at high magnifications.
Electron microscope • The electron microscope uses a particle beam of electrons to illuminate a specimen and create a highly-magnified image.
Electron microscopes have much greater resolving power than light microscopes and can obtain much higher magnifications of up to 2 million times, while the best light microscopes are limited to magnifications of 2000 times.
Transmission Electron Microscope (TEM) • The original form of electron microscope, the transmission electron microscope (TEM) uses a high voltage electron beam to create an image.
