Histology Wk 2 Flashcards
In bright field microscopy - what are the 3 components
Condenser - focusing light on the object to be studied
Objective - lens enlarging and projecting the image of object toward the observer
Eyepiece- further magnifying this image and projecting it onto the viewer’s retina or a charged coupled device highly sensitive to low light levels with a camera and monitor
What is the resolving power
Small distance between two structures at which they can be seen as separate objects
The maximal resolving power is approx. 0.2 micrometers
Single ribosome or cytoplasmic filament cannot be distinguished
What is virtual microscopy used for
Study of bright field microscopic preparations, involves the conversion of stained tissue preparation to high resolution digital images and permits the study of tissues using a computer or other digital device
What is fluorescence microscopy
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
What do fluorescent compounds have an affinity for
Specific cell macromolecules maybe used as fluorescent stains.
Acridine orange - which binds both dna and rna
DAPI and Hoeschst - stain specifically bind DNA and are used to stain cell nuclei, emitting a characteristic blue fluorescence under UV.
What is phase contrast microscopy
Uses a lens system that produces visible images from transparent objects and importantly can be used with living, cultured cells
What is phase contrast microscopy based on
Based on the principle that light changes its speed when passing through cellular and extra cellular structures with different refractive indices
This causes structures to appear lighter or darker in relation to each other
What is the modification of phase contrast microscopy
Differential interference contrast microscopy with Nomarski optics which produces an image of living cells with a more apparent 3D aspect
What is confocal microscopy and why is it good
Achieve high resolution and sharp focus by using
- a small point of high intensity light, often from a laser
- a plate with a pinhole aperture in front of the image detector
This greatly improves the resolution of the object in focus and allows the localization of specimen components with much greater precision than the bright field microscope
What is polarizing microscopy
It allows the recognition of stained or untainted structures made of highly organised subunits
When normal light passes through a polarizing filter, it exits vibrations in only one direction. If the second filter is placed in the microscope above the first one, with its main axis perpendicular to the first filter, no light passes through.
What is birefringence
The ability to rotate the direction of vibration of polarised light
It is a feature of crystalline substances or substances containing highly oriented molecules such as cellulose, collagen, micro tubules and actin filaments
What is transmission electron microscopy
Is an imaging system that permits resolution around 3 nm
What does the TEM high resolution suggest
Allows isolated particles magnified as much as 400,000 times to be viewed in detail.
Very thin resin embedded tissue sections are typically studied at magnifications up to 120,000 times 2D
What is done to TEM to improve contrast and resolution
Heavy metal ions are often added to the fixative or dehydrating solutions used for tissue preparation.
These include osmium tetroxide, lead citrate and uranyl compounds which bind cellular macromolecules increasing their electron density and visibility
What is used to study cells without fixation or embedding
Cryofracture and freeze etching - very small tissue specimens are rapidly frozen in liquid nitrogen and then cut or fractured with a knife
