Histology & Its Method of Study: Light Microscopy Flashcards
(22 cards)
Bright-field microscopy
- most common method used by students and pathologists
- uses ordinary light
- colors imparted by tissue staining
What are the optical components of a bright-field microscope?
- condenser: focuses light on object
- objective: lens enlarging & projecting image of object toward observer
- eyepiece: (ocular lens) further magnifies image and projects unto viewer’s retina or a charge coupled device (CCD) highly sensitive to low light levels with a camera and monitor (DOES NOT IMPROVE RESOLUTION)
How do you obtain total magnification?
objective X eyepiece/ocular
define resolving power
the smallest distance between two structures at which they can be seen as separate objects
- critical factor in obtaining crisp, detailed image with a light microscope
- determines image quality, clarity
Which optical component does resolving power mainly depend on?
the objective lens and its quality
Virtual microscopy
- permits tissue study sans-stained slide or microscope
- cost efficient, easy, tech savvy
Fluorescence microscopy
- uses UV light to for molecules to be visible
- allows localization of fluorescent probes (much more specific than routine stains)
- acridine orange acid binds DNA & RNA
- DAPI & Hoechst blue bind DNA
- important in immunohistochemistry
Phase-contrast microscopy
- uses differences in refractive index of various natural cell & tissue components to produce an image without staining or fixation
- allows observation of living cells
- uses lens system that produces visible images from transparent objects
What principle is phase-contrast microscopy based on?
“Light changes its speed when passes through cellular and extracellular structures with different refractive indices.”
What is a modification of phase-contrast microscopy?
differential interference microscopy with Nomarski optics
- produces image of living cells with 3D aspect
Confocal microscopy
- involves scanning the specimen at successive focal planes with a focused light beam, often from a laser
- produces a 3D reconstruction from the images
- high resolution, precision
- digital 3D
- sharp, small point high-intensity, laser
Polarizing microscopy
allows recognition of stained or unstained of highly organized and complex subunits
- light passes through polarizing filter and exits vibrating only one way
- produces repetitive, periodic macromolecular structures
What is birefringence in polarizing microscopy?
the ability to rotate the direction of vibration of polarized light
What are the two microscopes used in Electron Microscopy?
- Transmission electron microscope (TEM)
- Scanning electron microscope (SEM)
How are contrast and resolution improved in TEM?
heavy metal ions are added to the fixative to bind to macromolecules and increase their electron density and visibility
What techniques allow TEM to study cells without fixation or embedding?
cyrofracture and freeze etching use frozen specimens in liquid nitrogen with vaporized metal atoms like platinum and split the lipid bilayer to expose the protein’s size, shape, and distribution
What happens in SEM?
surface of specimen is first dried and spray-coated with a very thin layer of heavy metal like gold to reflect electrons in a beam of scanning the specimen
what are the main differences between TEM and SEM?
- TEM requires thin samples (less than 100 nm thick) while SEM can accommodate bulkier samples since it only requires the surface to be exposed to the electron beam
- TEM offers higher resolution while SEM gives less
- TEM uses transmitted electrons to form image while SEM uses secondary, backscattered ones, & X-rays emitted from the surface to form an image and provide compositional information
-SEM is easier to interpret because of 3D view
Cell & Tissue Culture
Cells can be grown in vitro (outside of body) and in the organism (in vivo) cells bathed in blood plasma fluid to survive and grow and can be examined in the living state by phase-contrast light microscopy.
- used to analyze infectious viruses like and chromosomal analyses, cervical cells, HeLa cells first line of cells in 1951
primary cell structures
complex solutions of salts, amino acids, vitamins with added growth factors
cell line
forms when cells remain in vitro for long periods and become immortalized
transformation
the process in which certain changes (some related to oncogenes) promote cell immortality similar to a normal cell becoming a cancer cell
