Microscopy and tissue preparation
Đã giới thiệu| English | Ý nghĩa |
|---|---|
| fixation/fɪkˈseɪʃn/ | fixation |
| dehydration/ˌdiːhaɪˈdreɪʃn/ | The removal or loss of water from a substance or organism. |
| embedding | embedding |
| sectioning | sectioning |
| staining | staining |
| resolution/ˌrezəˈluːʃn/ | The ability to distinguish fine detail or closely separated features. |
The picture is manufactured
- A histology slide is not a photograph of tissue. It is made, step by step, and each step can create or destroy evidence.
- To read a slide, you must know what the preparation did to it.
Five steps to a slide
- Fixation 固定 (formalin cross-links proteins), dehydration 脱水 (alcohols remove water) and embedding 包埋 (paraffin supports the tissue).
- Sectioning 切片 (a microtome cuts 3–5 µm slices) and staining 染色, usually with haematoxylin and eosin (H&E).
Put the steps of tissue preparation in order.
Độ khó 3/5 · 200 xu + phần thưởng nhỏ khi hoàn thành lần đầu tiên hôm nay
Fix to preserve, remove water so paraffin can enter, embed for support, cut thin sections, then stain.
What can be seen
- A section is 3–5 µm thick but a cell is 10–30 µm across, so each section cuts through only part of a cell.
- The light microscope's resolution 分辨率 is about 200 nm, half the wavelength of visible light. Magnifying beyond about 1500× only enlarges the blur.
A cell is 20 µm across. How many 4 µm sections does it take to cut all the way through it?
Độ khó 2/5 · 140 xu + phần thưởng nhỏ khi hoàn thành lần đầu tiên hôm nay
20 / 4 = 5, so any one section shows only a fifth of the cell; a nucleus may be missing from a cell's section.
Why H&E works
- Haematoxylin acts as a basic dye and stains acidic structures blue-purple: nuclei, full of DNA and RNA. Eosin is acidic and stains basic structures pink: cytoplasm and collagen.
- The logic is charge pairing. Special stains follow the same idea: PAS stains carbohydrates magenta, so basement membranes and glycogen light up.
Why do nuclei stain blue-purple with H&E?
Độ khó 1/5 · 100 xu + phần thưởng nhỏ khi hoàn thành lần đầu tiên hôm nay
Acidic nucleic acids bind the basic dye; basic cytoplasmic proteins bind the acidic eosin.
Worked example: the resolution limit
- Two mitochondria 150 nm apart cannot be told apart, because they are closer than 200 nm. A 2 µm bacterium and a 10 µm nucleus are easy to see.
- The electron microscope's wavelength is about 0.005 nm, so it resolves about 1000 times finer detail, but the specimen must be dead and in a vacuum.
Two structures are 100 nm apart. Can a light microscope show them as separate?
Độ khó 1/5 · 100 xu + phần thưởng nhỏ khi hoàn thành lần đầu tiên hôm nay
Resolution is set by the wavelength of light, about 200 nm; more magnification only enlarges the blur. An electron microscope is needed.
Worked example: the frozen section
- (Fictional vignette) A surgeon waits, mid-operation, to know if the tumour's edge is clear. The tissue is frozen and cut within minutes, skipping dehydration and embedding.
- Ice crystals tear the tissue and detail is blurred, but an answer within the hour beats a perfect answer tomorrow.
A frozen section gives the same quality of detail as a paraffin section, only faster.
Độ khó 1/5 · 100 xu + phần thưởng nhỏ khi hoàn thành lần đầu tiên hôm nay
Ice crystals tear the tissue and detail is blurred; the frozen section trades quality for an answer within minutes.
Three common slips
- Every gap is real. Shrinkage during dehydration creates false spaces.
- Stain colour is the tissue's colour. Colour comes from the dye chemistry.
- Forgetting the cut is one plane. A tube cut at an angle looks like an oval.
Match each appearance to its cause.
Độ khó 2/5 · 140 xu + phần thưởng nhỏ khi hoàn thành lần đầu tiên hôm nay
An artefact you can name is an artefact you can discount.
Recap
- Fix, dehydrate, embed, section (3–5 µm), stain.
- Light microscopy resolves about 200 nm.
- H stains acidic nuclei blue; E stains basic cytoplasm pink. Frozen sections trade quality for speed.