Week 6 Flashcards
What are the instruments in Microtomy that need temperature control
Tissue processor - 2-4 C above paraffin MP
Embedding center - 2-4 C above paraffin MP
Dry air over - - 2-4 C above paraffin MP
Floatation bath - 5-10 below the MP of the paraffin
What does a light or compound microscope have
Two magnifying lenses
-objective attacked to rotating nosepiece
-ocular (eye pieces)
Set Kohler, scan at 10, zoom at 40
how to calculate total magnification
multiply magnification of the objective lens
and the ocular lens.
x10 ocular and x45 objective would give a total
magnification of x450
What is resolution or resolving power
-gives fine details
-ability to differentiate between objects
The smallest distance where two objects are seen as two and not blurred into one
what is the path from eyes to lamp
eyes- eyepiece-objective- specimen-condenser-iris diaphragm-field diaphragm-lamp
what is chromatic aberration
white after entering a lens is split into colors of the visible spectrum and refracted at different angles with different focus point.
A lens that has not been corrected will show with colored border
The refractive index decreases with increasing wavelength .
what type of microscope corrective objectives are used
Achromatic objective - correct for red and blue
Parfocal objective -all objectives are focused in the same plane so magnification can be changed without needing to refocus but you see may need to adjust oculars for parfocality
What is micrometery
-uses a micrometer scale in one eyepiece and stage micrometer
-used for measuring or counting on mm scale
how to keep microscope in maintenance
-keep covered
-clean lenses with lens paper
-remove oil immediately
-dont dismantle objectives
-keep light off when not in use
-remove slides off stage once done
-focus up not down especially on 40x
what is a polarizing microscope
-used to ID crystals (talc, silica, urates) and amyloid stained with Congo red
-target amyloid on tissue is confirmed with this microscope by seeing the apple green birefringence/double refraction/anisotropism (under a light microscope it will appear salmon)
– Birefringence - transmission of light unequally in
different directions.
– Anisotropism – having unlike properties in different
directions
how does the polarizing microscope work
-light from this microscope vibrates on one plane
-two filters : Polarizer between light and specimen
Analyzer between specimen and eye
-The analyzer is rotated so optical paths are perpendicular with polarizer and the field will go dark
-birefringent will appear bright on dark background
When is phase contrast microscopy used
-for unstained samples with living cells allowing transparent objects to be seen clearly
What is dark field microscopy
-used for fine structures -unstained micro orgs or radioactive labelling with silver
-rare in histopath lab
-NO TRANSMITTED LIGHT only scattered light
-stray light is directed on objects and they will be self luminous on a dark background and they will also look bigger
What is fluorescent microscopy
-one wavelength of light is absorbed and remitted as light of longer wavelength
-object is hit with short wavelength rays in UV range and light is emitted is violet or blue in visible range
-uses a halogen or mercury lamp with exciter lens and barrier lens
What is fluorescent microscopy used for
Autofluorescence - when substances like collagen fibers fluoresce naturally
-used for AB -AG reaction IHC
-* Acid fast bacilli (tagged with auramine – rhodamine) and amyloid (tagged with thioflavin T) can be identified using this technique when they get tagged by fluorescent dye.
What is electron microscopy
-high resolution
-electron gun instead of light source
-electron gun, electron beam and specimen are maintained in a vacuum
-Electron beam is directed at sample and focused by changing the strength of the EMF
-sample image is seen by projection onto a fluorescent screen
What is transmission EM
- A thin section either transmits electrons (clear areas) or deflects electrons (dark areas).
- A 2-D image is produced.
- For the diagnosis of muscle and kidney diseases, different type of tumors.
What is SCANNING E.M
-Electron beam sweeps the surface of the specimen
and releases secondary electrons.
* Produces a 3-D image.
* Used in research to study the surfaces of the surface membranes of cells.
What are different types of microtome
**mostly used in pathology labs
Ultramicrotome
– Used for cutting very thin sections 0.5 µm.
- rotary microtome**
– Most commonly used for paraffin sectioning and found in most cryostats. - Sliding microtome**
– Use for sectioning celloidin and large paraffin blocks. - Clinical freezing microtome**
– Replaced by cryostat but easier to obtain free floating sections for special stains.
What are the parts of a flotation bath
- Set at 5-10 C < MP of wax
- Remove bubbles.
- can use adhesives on slides (albumin, silane, glue, poly-L-lysine, gelatin, agar).
- Charged slides – electrostatic attachment.
- Clean the water surface regularly.
- Use chemicals to lower the surface tension – help flatten out the tissues.
Why are dryers and ovens used in microtomy
-to help dewax the slide before staining and introducing water to prep for staining.
-if there is incomplete drying the tissues can wash off during staining and cause incomplete dewaxation
-must be maintained at 60C for routine and 37 for enzyme, ISH, IHC stains
-if there samples get too hot there will be nuclear bubbling, parched earth and cracking
What are robotic stainers like
-flexible because you can program it
-allows EXACT staining time
-can do many types of stains at once
-reduce chemical exposure
What are linear stainers
-move slides from one container to next
-good for progressive H&E but less flexible for regressive
-can be continuously loaded
What is a coverslipper
-automated coverslippers use film coverslips or glass coverslips
-reduce chemical exposure
-needs regular cleaning
-ensure there is correct xylene to resin ratio for glass coverslip
What are slide and cassette printers
-cassette ink resists chemicals through processing and staining
-can track location, status, reduces human error
What is the gravimetric factor
-method that compensates for variances in dye content of dry dyes that are used in the preparation of staining solutions
present dye/new dye
