Histotech Lecture Flashcards
1
Q
Histology Definition
A
- Studying the microscopic form of cells, tissues, organs
- Relates form to function
2
Q
Pathology Definition
A
- Studying abnormal cells, tissues, and organs that could cause disease
- Must understand “normal” to identify the abnormal
3
Q
Histotechnology Definiton
A
- Making slides
- Technology behind histology/pathology
- Various processing, embedding, sectioning, and staining techniques
4
Q
Microscopy Definition
A
- Using various imaging modalities to study objects (cells, tissues, organs, particles) that cannot be resolved with the human eye
- Three branches: optical, electron, scanning probe
5
Q
What is great about Histology?
A
- Basic informative technology (size, shape, number, location, interaction, composition)
- Pictures say a lot
- Applicable in clinical and research
- Continuous advancement of technology
6
Q
What goes in the Planning Stage?
A
- Must define the endpoint (# of cells, cell size, presence/absence of molecule, elemental analysis)
- Each imaging modality requires specific processing, embedding, sectioning, and staining techniques
- Samples aren’t interchangeable, so multiple modalities without compromising endpoint quality
7
Q
Types of Optical Microscopy
A
- Bright Field Microscope = Light Microscopy (LM)
- Phase Contrast Microscope
- Fluorescent microscope
8
Q
Bright Field Microscope (LM)
A
- Halogen/LED bulb is light source
- Compound upright obj. turret = most common
- Must have stained specimen
9
Q
Phase Contrast Microscope
A
- Halogen/LED bulb is light source
- Found on Inverted microscope
- Image cell cultures
- No staining necessary
10
Q
Fluorescent microscope
A
- UV bulb is light source
- Special objectives are available
- Filter cubes for various wavelengths are required
- Microscope can have upright or inverted objective turret
- Plethora of fluorescent probes are available
- Staining can be antibody or non-antibody format
11
Q
Confocal Laser Scanning Probe Microscopy
A
- Laser is light source
- Fluorescent imaging only
- 3D image is created by scanning multiple focal planes = optical sectioning
- VERY high resolution and contrast
- Software is critical to this modality, because the software compiles all the images
12
Q
Types of Electron Modalities
A
- Scanning Electron Microscopy
2. Transmission Electron Microscopy
13
Q
Scanning Electron Microscope (SEM)
A
- NOT the same as confocal laser scanning
- Light source is electron beam
- Creates a 3D topgraphical image of sample
- No sample stain
- Sample preparation very different than other modalities
14
Q
Transmission Electron Microscopy (TEM)
A
- Light source is electron beam
- Can resolve ultrastructure of organelles = black/white image
- Requires special fixation: glutaraldehyde
- Requires special processing and embedding: resin
- Requires special staining: lead (electron dense)
15
Q
Fixation Stage in Sample Preparation
A
- based on embedding medium
- Chemical process that preserves/stabilizes proteins, structures, and organelles (freezes the proteins when you add fixative)
- MOST CRITICAL STEP
- Embedding medium must match fixation
- Every step after fixation relies on the fixation step for success
- FRESH fixative is the best
- Must fix your sample with the correct fixative at the ideal concentration for the optimal amount of time
- Depending on the sample, fixative can be administered by cardiac perfusion (animals) or immersion (cell cultures)
- The fixative is replacing the water in the cells and tissues
16
Q
Fixative Procedure and Embedding Media for 2-4% paraformaldehyde (PFA)
A
Immunostaining
Freezing Media
17
Q
Fixative Procedure and Embedding Media for 10% NBF
A
Routine stains
Paraffin
18
Q
Fixative Procedure and Embedding Media for 2% PFA 3% glutaraldehyde
A
TEM
Resin/Epoxy
19
Q
Processing Stage in Sample Preparation
A
- Based on embedding medium
- Protocol that dehydrates tissue (removes water) and replaces it with some type of embedding medium
- Specifics of protocol depends on embedding media
- Replacing fixative with embedding media further stabilizes cells/tissues of sample for sectioning
- Embedding media must match imaging modality
- Fixative is removed by dehydration through graded series of ethanol
- Dehydration is followed by infiltrating tissue with embedding media; solvent is used as a vehicle
20
Q
Embedding Stage in Sample Preparation
A
- Based on imaging modality
- Placing the sample in a form and covering with embedding medium (end product = block; tissue orientation is critical!)
- Must confirm which surface will be sectioned to achieve proper sample alignment/ orientation
21
Q
Sectioning Stage in Sample Preparation
A
- Based on imaging modality
- Cutting the sample block to desired thickness
- Special equipment is needed to section the blocks
- Different EMs need different blades
- Section thickness ranges from 60nm to 20microm; depends on EM & application
- Most time consuming and challenging step
- Size of target helps define section thickness
22
Q
Microtome Definition and Types
A
- instrument used to cut tissue
- Cryotome: frozen sections
- Microtome: paraffin sections
- Ultramicrotome: resin sections
- Combo microtomes are available for paraffin & resin sections*
23
Q
Staining Stage in Sample Preparation
A
- Based on imaging modality
- Choosing a stain for desired endpoint; specific or general
- There are general stains that are used routinely (H&E)
- Stains can be based on sample chemistry
- pH=acid/base
- content=lipid; collagen; glycoproteins
- Some stains are component specific
- Luxol fast blue = myelin diseases
- Wright stain = hematology
- Periodic Acid Schiff = carbohydrates
- Masson’s trichrome = collagen, wound healing/fibrosis
