microscopy; principles and application. types of microscopes Flashcards
what is light microscopy?
light microscopy is conventional bright-field microscopy and more specialized applications like fluorescence, phase-contrast, confocal, and polarizing microscopy are all based on the interaction of light with tissue components and are used to reveal and study tissue features.
what are the types of light microscopy?
- bright field microscopy
- flouresnece microscopy
- phase contracst microscopy
- confocal microscopy
- polarizing microscopy
what does bright field microscopy include?
- With the bright-field microscope, stained tissue is examined with ordinary light passing through the preparation
- the microscope includes an optical system and mechanisms to move and focus the specimen.
what are the optical components of the bright field microscope
- the condenser focusing light on the object to be studied
- the objective lens enlarging and projecting the image of the object toward the observer
- the eyepiece (or ocular lens) further magnifying this image and projecting it onto the viewer’s retina or a charge-coupled device (CCD) highly sensitive to low light levels with a camera and a monitor.
what is the resolving power of the bright field microscope
the resolving power of the microscope is 0.2um, meaning objects smaller or thinner than this cannot be distinguished (e.g. a ribosome, membrane, etc.). Likewise if two objects are closer than this together they cannot be distinguished.
what is virtual microscopy?
- used for the study of bright-field microscopic preparations
- involves the conversion of a stained tissue preparation to high-resolution digital images and permits study of tissues using a computer or other digital device, without an actual stained slide or a microscope.
what is meant by the term flourescence?
When certain cellular substances are irradiated by light of a proper wavelength, they emit light with a longer wavelength— a phenomenon called fluorescence
how does flourence microscopy work?
- tissue sections are usually irradiated with UV light and the emission is in the visible portion of the spectrum.
- The fluorescent substances appear bright on a dark background.
- the instrument has a source of UV or other light and filters that select rays of different wavelengths emitted by the substances to be visualized.
what are fluorescent compounds used for?
Fluorescent compounds with affinity for specific cell macromolecules used as fluorescent stains. e.g Acridine orange, which binds both DNA and RNA
coupling compounds such as fluorescein to molecules that will specifically bind to certain cellular components and thus allow the identification of these structures under the microscope
what is phase contrast microscopy?
- phase-contrast microscopy uses a lens system that produces visible images from transparent objects
- can be used with living cultured cells as unstained cells and tissue sections which are usually transparent ans colourless
how does phase contrast microscopy work?
based on the principle that light changes its speed when passing through cellular and extracellular structures with different refractive indices
These changes are used by the phase-contrast system to cause the structures to appear lighter or darker in relation to each other.
what is differential interference contrast microscopy?
A modification of phase-contrast microscopy, with Nomarski optics, which produces an image of living cells with a more apparent three-dimensional (3D) aspect
what is confocal microscopy?
-a small point of high-intensity light, often from a laser
a plate with a pinhole aperture in front of the image detector. The point light source, the focal point of the lens, and the detector’s pin- point aperture are all optically conjugated or aligned to each other in the focal plane (confocal), and unfocused light does not pass through the pinhole
