1.2 Light microscopy Flashcards
Define histology
the study of tissue structure by means of staining techniques, combined with light and/or electron microscopy
what must happen to tissue before you can view it under a microscope?
be isolated, preserved, cut into thin sections and stained with dyes to reveal the structure
what can you do if you know the normal organization of tissue?
able to assess loss of normal organization in disease states
define biopsy
isolation of tissue
how to biopsy accessible tissue (eg: skin)?
direct incision (eg: needle biopsies are used to collect breast tissue)
how to biopsy internal organs (eg: heart)?
trans-vascular methods
why must tissue be preserved?
to maintain structure and prevent putrefaction
how to preserve tissue?
treatment of the tissue with a fixative such as alcohol or formalin
what happens to tissues after they are preserved?
are embedded in a solid substrate (such as wax)
why do you embed tissue in wax/solid substrate?
it allows the preparation of thin slices using a microtome
what happens to tissues once they are preserved and embedded in wax/solid substrate?
they are transparent so can be stained to reveal micro anatomical features
what is the most common stain used for tissues?
haematoxylin and eosin
how does haematoxylin work?
it binds to negatively charged macromolecules such as DNA
how does eosin work?
it is attracted to positively changed molecules (mainly protein)
what does a good H&E stain reveal?
range of pink and blue hues
where and what is H&E stain used for?
used extensively in histopathology labs for diagnosis
what does the stain Periodic Acid Schiff (PAS) show?
glycoproteins
why is periodic acid Schiff useful?
to reveal structures such as basement membranes and to reveal pathology in disorders like diabetes where elevated plasma glucose binds to tissues and impairs function
what are trichome stains useful for?
for discriminating muscle and different types of connective tissue
what does the stain immunohistochemistry (IHC) show?
evaluation of oestrogen receptor expression in breast cancer and it identifies specific proteins
what is the process of identifying specific proteins?
tissue sections are exposed to an antibody which is specific for the protein. this antibody is tagged with a chromogenic (colored) or fluorescent dye which allows visualization of specific compartmental expression when combined with a counterstain and viewed by microscopy
give examples of counterstains.
eosin, nuclear dye for immunohistochemistry
what does a cell that actively synthesizes large quantities of protein look like under a microscope?
usually has a large, pale staining nucleus with a prominent nucleoli
why is the nucleus stained pale in cells that actively synthesize a lot of protein?
bcs of the active transcription of chromatin
what is the function of nucleoli?
sites of active ribosomal RNA synthesis
what does an inactive cell look like under a microscope?
has a deep-staining nucleus which lacks visible nucleoli
why is the nucleus dark stained in inactive cells?
bcs of little chromatin being transcribed
why do inactive cells lack visible nucleoli under the microscope?
bcs of minimal ribosome production
what does a dead cell look like under a microscope?
has an intensely staining, shrunken nucleus, but sometimes the nucleus may be fragmented or absent bcs of lysis of nuclear material
why would a pink staining cytoplasm be granular under the microscope? give an example.
often contain accumulation of organelles (eg: mitochondria or secretory granules) that take up acidic dyes. EG: abundant RER in nerve cell bodies appears as Nissl substance
why would a diffused cytoplasm appear purple tinted?
bcs of the presence of cytoplasmic RNA in the form of ribosomes and ∴ active protein synthesis
what would non-staining areas in a cell show under a microscope?
that the cell contains mucous secretory vacuoles or fat droplets
what would variations of cell structure within one cell type show under a microscope? give an example.
cell division or degree of differentiation. EG: epidermis of skin is multilayered by mainly comprised of keratinocytes
generally, how do you study a slide?
start at low power and increase magnification to inspect detail. reduce the power again when moving around the slide.
in Gleason grading for prostate cancer, what does grade 1 show?
small, uniform cells that are tightly packed
in Gleason grading for prostate cancer, what does grade 2 show?
varied cell sizes and shapes, loosely packed
in Gleason grading for prostate cancer, what does grade 3 show?
increased cell size and shape irregularity, less distinction between cells
in Gleason grading for prostate cancer, what does grade 4 show?
large, irregular, fused cells
in Gleason grading for prostate cancer, what does grade 5 show?
irregular, fused cells that have invaded surrounding connective tissue cells
looking at the histology of a normal lung, what does the tissue look like?
uniform cells, nuclei also have similar shape,
looking at the histology of a lung with cancer, what does the tissue look like?
there is nuclear moulding: characteristic of small cell lung cancer
in a patient with suspected lung cancer, what questions would you ask?
what type of lung cancer is it?
in a patient with unusual looking mole, what questions would you ask?
is it an atypical benign mole? is it a seborrheic wart? is it a malignant melanoma?
what would you look at in the biopsy of a mole?
different layers of the skin
what are the layers of the skin?
epidermis on top, the dermis below the epidermis, the dermis and epidermis are separated by stratum basale and the granular layer is the uppermost layer (last living layer of epidermis)
what is a method to measure moles?
breslow thickness
what does the breslow thickness measure?
the depth of melanoma invasion from the granular layer of the epidermis (how deep the melanoma has penetrated from the granular layer into reticular dermis)
for a breslow thickness of <1 mm, what is the % of 5 year survival?
95-100%
for a breslow thickness of 1-2 mm, what is the % of 5 year survival?
80-96%
for a breslow thickness of 2.1-4 mm, what is the % of 5 year survival?
60-75%
for a breslow thickness of >4 mm, what is the % of 5 year survival?
37-50%
what are the effective treatments for malignant melanomas?
early diagnosis and excision
why are chemotherapy and radiotherapy not treatments for malignant melanoma?
bcs they are ineffective in more advanced disease
what increases the median survival in advanced disease? give an example
targeted therapy. eg: the BRAF inhibitor vemurafenib
how to check how/if the melanoma has spread?
see if the melanoma has metastasized and if it has gone into the nodes
what to do with suspected malignant melanoma patient?
do a primary surgical excision of a suspected lesion performed with 2mm margin. if diagnosis = positive, rapid further excision is required (with 2cm excision around original scar)
what are the steps to study tissues?
- collect the tissue
- fixation
- embedding and processing
- sectioning
- staining
what is fixation of tissue?
fix the tissue in a chemical preservative to prevent it from degrading and also to try maintain snapshot of the cell architecture, so that it can represent what state the tissue was in that given moment.
how to embed and process the tissue?
dehydrate the tissue and solidification of it most commonly by embedding in paraffin wax as it gives a solid medium from which the tissue can be cut
what is sectioning of the tissue?
cutting of the tissue
why do you stain tissues?
bcs tissues are translucent/colourless thus difficult to differentiate between them under the microscope
what does haematoxylin stain?
the nuclei
what does eosin stain?
proteins, collagen, everything other than the nuclei
