Module 1: Methods of Cytology and Histology Flashcards
Collects and focuses a cone of light that illuminates the tissue slide on the stage
Condenser
Most commonly used staining dyes?
Combi of Eosin & Hematoxylin
Fixative of choice for Light Microscopy?
Buffered solution of isotonic formaldehyde (formalin)
Cellular parts that Eosin stains? What color is produced?
a. Stains other cytoplasmic stx b. Collagen Pink
What cellular antigens can IHC target?
a. Cytoplasmic b. Nuclear d. Cell membrane e. Lipid F. Proteins
What is the maximum resolving power in light microscopy?
0.2 um (1000-1500 times)
Cellular parts that Hematoxylin stains? What color is produced?
a. DNA in cell nucleus b. RNA-rich portion of the cytoplasm c. Matrix of cartilage Dark blue/purple
What are the complex solution for cell& tissue culture
a. Salts, amino acid, vitamins b. Serum & specific growth factor
Why the need section/ slice tissue samples?
Inorder to obtain thin translucent sections for the light to pass through and be visualized
What is currently used in tissue processing?
Automated tissue processor
What is Histology?
Study of TISSUE arrangements of the body to constitute organs
Most common procedure done in histologic research
Preparation of tissue slices. (Can be examined visually with transmitted light)
What does antigens produced by viruses diagnose?
Specific virus infections
This process uses microtome to slice & obtain tissue sample based on size for a particular visualization method
Sectioning
General Methods used in studying histology?
a. Microscope b. Molecular method
A phenomenon where substances irradiated by light of a proper wavelength emit light with a longer wavelength
Fluorescence
True or false, phase contrast microscopy can be used with living cultured cells
True
This pattern makes use of basic dyes and stain structure BLUE?
Basophilic
They are initiated directly from the cells, tissues and organs of animals/ human typically used for experiment for a few days.
Primary cell culture
Measurement of Tissue section for Electron microscopy
< 1 microns
Infiltration of tissue sample by placing it into a melted parrafin
Infiltration
Antigen found in nucleus
Estrogen receptors Progesterone receptors
T/F, cytology general involves looking at a single cell type
True
T/F, primary cell culture propagated repeatedly gives rise to cell lines
True
Steps in Tissue preparation?
a. Fixation b. Dehydration c. Clearing d. Infiltration E. Embedding
What structure are acidophilic?
Mitochondria Collagen Secretory granules
How does cell create an organism?
Cells + assoc. matrix (highly specialized & functionalized) —> Tissue (Fundamental)–> Organs –> System –> organism
T/F, in situ hybridization can be used to identify the cells containing specific messenger RNAS (MRNA) in which the corresponding gene is being transcribed
True
Collects & focuses a cone of light that illuminates the tissue slide on the stage
Condenser
Clinical applications cell & tissue culture
a. Used to study molecular changes that occur in cancer. b. Could analyze viruses, mycoplasma, & some protozoans c. Used to prepare tissues for routine genetic chromosomal analysis
This process infuses dyes to different cell type structures that has different staining pattern
Staining
Step of embedding where alcohol is removed thru miscible agents (Toluene & Xylene).
Clearing
What is the most common PROCEDURE used in histological research?
Preparation of tissue section to be studies in LIGHT MICROSCOPE
Placing a small piece of tissue in a solution of chemical fixatives that preserves by cross-lining & inactivating degradative enzyme
Fixation
T/F, TEM can magnify up to 400,000 times
True
How long does overall sample prep be ready?
12-2.5 days
What are fixatives?
Solution of stabilizing/ cross-linking compounds
How does tissue processing occur?
Tissue sections are processed into thin sections which is best for microscopy
What does specific cytokeratin diagnose
Tumors of epithelial origin
What is used to embed tissue?
Melted paraffin
What are the 2 basic staining pattern?
a. Basophilic SP b. Acidophilic SP
A single dye applied separately to distinguish additional features of a tissue
Counter-stain (i.e Eosin)
Appearance of highly oriented molecules (birefringence) under polarizing light microscopy
Bright red or Yellow Apple green- amyloid
A type of Electron microscopy that provides high resolution view of surfaces of cells, tissues, and organs
Scanning electron microscopy (SEM)
This exposes tissue for sectioning or slicing on a microtome
Trimming
Substances that exhibits Birefringence
Cellulose Collagen Microtubules Actin filament
Study of the tissues of the body and how they are arranged to constitute organs
Histology
A light wave vibrating in more than one plane
Unpolarized light
A method of localizing newly synthesized macromolecules in cells or tissue sections
Autoradiography
Utilizes the hexose rings of polysaccharides and other carb-rich tissues and stains each macromolecules distinctly
Periodic acid-Schiff (PAS) reaction
A method of using cover slip to completely cover the tissue section and subject it to mounting medium
Mounting
This pattern makes use of acidic dyes and stains structure PINK
Acidophilic sp
Why use paraffin in tissue infiltration?
It makes tissue sample more rigid and easier to cut
What is an Ideal microscopic preparation?
a. Should be well-preserved tissue b. Has the same structure as the molecular composition in the body
Processes of embedding
a. Dehydration b. Clearing c. infltration
Resolution of TEM microscope
3 nm
True or False, In fluorescence microscopy, the emission of light upon tissue irradiation is in the visible portion of the spectrum.
True
Acidophilic stains?
a. Eosin b. Orange G c. Acid fuchsin
How to get total magnification?
Magnification power of objective lense x Magnification power of ocular lense
This uses a lens system that produces visible images from transparent objects
Phase contrast microscopy
The process of transforming unpolarized lt. to polarized lt.
Polarization
Explain direct method of IHC
a. Makes use of a single Ab b. Application of reagent containing specific Ab for binding to target antigen (Anti-A) c. Development of Antigen-Ab reaction indicates presence of target antibody d. Color change upon reaction indicates the presence of target antigenp
Enzymes that can be detected using histochemistry:
a. Phosphatase b. Dehydrogenase c. Peroxidase
How tissues are collected for histological study
a. Scraping or brushing the tissues (papsmear) b. Collecting body fluids (urine / resp secretion) c. Fine needle aspirations (abdominal fluid: ascites, pleural fluid: lungs, CSF: spinal canal)
Examination of a single cell type as often found in fluid specimen.
Cytology
Fixative of choice for Electron microscopy?
Glutaraldehyde solution followed by buffered Osmium tetroxide
A process of fixing tissue-embedded wax piece into a supporting wooden block (plastic embedding cassette) to be fixed for sectioning to microtome
Blocking of sections
Importance of Fixation
a. Prevents Auto-lysis b. Prevents bacterial degradation of Tissue c. Preserve cell & tissue structure
Difference of typical staining procedure vs IHC
In IHC, no staining medium is incorporated but the Ab-antigen complex will stain the slide
Temperature needed to maintain melted paraffin
52~60 C
What is Tissue?
Cellular organizational level in-between a cell & complete organs. Group of similar cells with their EC matrix from the same origin & carry out specific fxn.
Optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out of focus light (scattered light) in image formation
Confocal microscopy
Visual markers used in IHC
a. Fluorescent dye b. Colloidal metal c. Hapten d. Radioactive markerw e. Enzyme (peroxidase)
They act as catalyst to bring about a specific type of biochemical reaction
Enzyme
A microscopy method based on the interaction of tissue components with beams of electrons.
Electron microscopy
A technique in which a single stranded DNA or RNA sequence called probe is allowed to form complementary base pairs with DNA or RNA present in a tissue or chromosome sample
In situ hybridization
T/F, usual cut used in TEM is 40-90nm
True
Fixative for electron microscopy
Glutaraldehyde / formaldehyde
A microscopy method which uses polarized light to visualize highly organized subunit
Polarizing microscopy
Two main types of electron microscope
a. Transmission electron microscope (TEM) b. Scanning electron microscope (SEM)
A method for localizing cellular structures using a specific enzymatic activity present in the structures
Enzyme histochemistry
Breast duct cell tumor can be diagnosed by what specific antigen?
Steroid hormone receptor
Magnification used to observe high magnification of more detailed areas
X40
Type of Ab-antigen binding
Direct
Determining the localization of a gene in a specific chromosome cannot be applied in In situ hybridization
False In situ is ideally applied
Describe extracellular matrix?
Contains many kinds of molecules (Collagen)
Identify this staining in IHC [insert pic]
Cytoplasmic staining
Antigen found in Cytoplasm
Cytokeratin (present in epith cells)
In autoradiograph, they indicate region of cells in which specific macromolecules where synthesized prior to fixation
Silver grains
A machine used for tissue embedding & sectioning
Tissue processor
Measurement of Tissue section for Light microscopy
1-10 microns
Commonly used visual markers for IHC light microscopy
Peroxidase (enzymes)
A microscope that has a strong UV light source w/ special filters that select traces of different wavelengths emitted by a certain substance
Fluorescence Microscopy
Methods for localizing cellular structure in tissues using enzyme activity present in these structure
Histochemistry & cytochemistry
How does manual tissue processing works?
Tissues usually undergo chains of reaction
Objective of trimming tissues?
To expose full face of tissue section to microtome knife
What to with the paraffin infiltrated tissue?
Placed in a tissue block with melted paraffin then hardened.
This magnification is used for observing a large area field at low magnification
X4
What does protein polypeptide hormone diagnose?
Certain endocrine tumors
How does Glutaraldehyde & Formaldehyde prevent degradation?
Interaction with Amine groups of Proteins
This method reveal locations of newly synthesized glycoproteins containing a specific sugar
Autoradiography
What is cytology?
Study of STRUCTURE & FUNCTION of cell
What color does PAS reaction produce?
Purple or magenta
Clinical application of cytology
a. Used to dx or screen for CA b. Screen for fetal abnormalities c. Papsmears to dx infectious organisms
Who selects tissue sections?
Pathologist or medical officer
Explain the indirect method of IHC
a. Makes use of two Ab b. Ab1 binds to target Antigen c. Ab2 binds to Ab1 d. Color change upon reaction indicates the presence of target antigenp
Identify this staining in IHC [insert pic]
Membranous staining
Combines histologicals, immunologicals, & biochemical techniques for identification of specific tissue component by means of specific antigen/ antibody tagged with a visible label
IHC
T/F, in SEM, surface is coated with a very thin layer of heavy metal
True
What are the basic steps of tissue processing?
A. Fixation of specimen B. Selection of tissue processing C. Processing of tissue D. Tissue embedding E. Blocking of tissue section F. Trimming G. Section cutting H. Routing & staining w/ hemotoxylin & eosin I. Mounting
What embedding materials used? & where are they used?
a. Paraffin: light microscopy b. Plastic resins: light & electron microscopy
How does electron microscopy allows for 1000 fold increase in resolution?
The wavelength in an electron beam is much shorter than that of light
Refers to structures being observed under electron microscopy
Ultra structures
Colors produced in electron microscopy
Black, white, shades of gray *lighter region- area less dense (easy electron passage) *darker region- area highly dense
Magnification used to observe medium magnification & smaller field
X10
Identify this staining in IHC [insert pic]
Nuclear staining
What is the specimen in phase contrast microscopy?
Unstained cells and tissue section
What should be obtained in section cutting?
A thin slice of tissue less than one cell layer thick w/o changing its architecture of tissue section.
What protein is readily stained by Basic dye?
Nucleic acid
What is tissue embedding?
Tissue infiltration with embedding materials to impart rigid consistency to the tissue
Cultures of animal cells that can be propagated repeatedly & sometime indefinitely, also arises from primary cell culture
Cell line
Type of Ab-Antigen binding
Indirect
A special cutting device for tissue preparation
Microtome
Resolution of TEM
3 nm resolution
Implies the specific binding between two single strands of nucleic acid which occurs under appropriate condition if the strands are complimentary
Hybridization techniques
Commonly used mounting medium?
D.P.X (distrene, plasticizer, xylene)
What are the two interacting component of tissues?
Extracellular matrix & cell
Functions of EC matrix
a. Supports the cell b. Contains fluid that transport nutrients to the cell and carry away waste & secretory products
Why record tissue specimen?
Prevent specimen mixed up
Objective of Tissue embedding
Facilitate sectioning by embedding tissue in a solid medium.
A method for localizing specific antigen in tissues or cells based on antigen-antibody reaction
Immunohistochemistry (IHC)/ Immunocytochemistry
Light waves vibrate in a single plane
Polarized light
What does carcinoembryonic antigen diagnose?
Glandular tumors
Application of polarizing microscopy
Myelin Osteoid Crystals
A critical factor in obtaining a crisp detailed image w/ light microscope
Resolving power
How does Tissue specimen recorded?
a. By numbering them like DBH/1256 b. BY RECORDING them in register
Method of removing cells by drawing them thru a fine needle
Method of removing cells by drawing them thru a fine needle
Live cells and tissue are maintained and studied outside the body
Cell and tissue culture
T/F, exam of the entire tissue block
True
Fixative for light microscopy
Buffered isotonic solution of 37% formaldehyde
Enlarge & project the illuminated image of the object toward the eyepiece
Objective lense
Smallest distance b/n two particles in w/c they can be seen as separate objects
Resolving power
Color produced in peroxidase reactions
Brown
T/F, in SEM, you can only visualize surfaces as beam does not pass thru specimens
True
True or false, phase contrast microscopy follows the principle that light changes its speed when passing through the cellular and extracellular structures with different refractive indices
True
A microscopy technique that Uses high intensity light often from a laser
Confocal microscopy
What is fixation?
Placing a small piece of tissue in a solution of chemical fixatives that preserves by cross-lining & inactivating degradative enzyme
Most commonly used microscopic method?
Brightfield microscopy
Microscopy method that allows examination of cells without fixation or staining hence prominently used in all cell culture laboratories. It can also be used for living cells
Phase-contrast microscopy
How does labeled antibody in IHC visualized?
Using light microscopy in the presence of a suitable colorogenic substrate system
It will magnify the image for another x10 & project it to the viewer
Eyepieces or ocular
Procedures done in IHC staining
[insert pic taken from module 1 part 3, 10:09]
Final step before microscopic observation of tissue
Mounting a protective glass cover on the slide with clear adhesive
More commonly used Immunohistochemistry method? Why
Indirect Labeling the protein of interest is amplified by indirect method
Why does fixation carried out ASAP?
To prevent putrefaction & autolysis
Step of embedding w/c transfers tissue to a series of increasing alcohol concentration until 100% to fully remove all water
Dehydration
Example of Basophilic dye?
a. Toluidine Blue b. Alcian blue c. Methylene blue d. Hematoxylin
Heavy metal used for surface coating in SEM (and how?)
Osmium tetroxide Lead citrate Bind to macromolecules increasing electron density & visibility
Ability to rotate the direction of polarized light
Birefringence
Produces an image of living cells with a more apparent 3D aspect.
Differential interference contrast microscopy (DICM)
T/F, in situ hybridization is ideal for determining if a cell has a specific sequence of DNA such as a gene or part of a gene
True
Antigen found in membrane staining in IHC
HER2/ neu
