L1 - Light Microscopy

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Last updated 8:11 PM on 5/6/26
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34 Terms

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<p>Light Microscope</p>

Light Microscope

knowt flashcard image
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Type of Light Microscopy

  1. Brightfield = visible light passes thru the specimen

  • often need stains + living cells

  1. Phase contrast = visible light passes thru the specimen

  • no stains + a halo + living cells

  • makes darks darker and lights lighter

  1. DIC = visible light passes thru the specimen

  • can be alive + no stain + shadows

  • makes darks darker and lights lighter

  1. Widefield Fluorescence

  • uses fluorescent molecules (DAPI, FITC, rhodamine)

  • short wavelength (high E) → longer wavelength (low E)

  1. Composite Images = figures made from multiple images

  • red + green = yellow

  • purple + green = white

  • visible light is ADDITIVE (RGB)

= more colours → lighter

  • pigments are subtractive (CMYK)

= more colours → darker

  1. Confocal Fluorescence = shows a 2D optical plane

  • using a laser to focus on MIDDLE of specimen

  • shows more detail than wide field

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<p>Brightfield microscopy</p>

Brightfield microscopy

= visible light passes thru specimen

  • often need stains

ex. looking at chloroplasts or mito

<p>= visible light passes thru specimen</p><ul><li><p>often need stains</p></li></ul><p>ex. looking at chloroplasts or mito</p><p></p>
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<p>Phase Contrast microscopy</p>

Phase Contrast microscopy

= visible light passes thru specimen

  • lenses make darks darker + lights lighter

  • NO stains + get halo

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<p>Differential Interference Contrast (DIC) microscopy</p>

Differential Interference Contrast (DIC) microscopy

= visible light passes thru specimen

  • can be alive + NO stain + get shadows

  • lenses make darks darker + lights lighter

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<p>Widefield Fluorescence microscopy</p>

Widefield Fluorescence microscopy

  • uses fluorescent molecules (DAPI, FITC, rhodamine)

  • shine short wavelength (high E) on specimen → see longer wavelength (low E)

ex. DAPI absorbs UV → give off blue

ex. FITC absorbs blue → gives off green

(-) : fuzzy images → thus we use confocal fluorescence microscopy to fix issue

<ul><li><p>uses fluorescent molecules (DAPI, FITC, rhodamine) </p></li><li><p>shine short wavelength (high E) on specimen → see longer wavelength (low E) </p></li></ul><p></p><p>ex. DAPI absorbs UV → give off blue</p><p>ex. FITC absorbs blue → gives off green </p><p></p><p>(-) : fuzzy images → thus we use confocal fluorescence microscopy to fix issue</p><p></p>
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Fluorescent Synthetic Molecules

ex. DAPI absorbs UV → give off blue

ex. FITC absorbs blue → gives off green

<p>ex. DAPI absorbs UV → give off blue</p><p>ex. FITC absorbs blue → gives off green </p>
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Fluorescent Proteins

ex. GFP

  • CFP

  • YFP

  • RFP

<p>ex. GFP</p><ul><li><p>CFP</p></li><li><p>YFP</p></li><li><p>RFP</p></li></ul><p></p>
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example of wide field

knowt flashcard image
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Newt Cell Image

knowt flashcard image
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<p>what is the blue?</p>

what is the blue?

DNA

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<p>what is the green?</p>

what is the green?

Microtubules

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<p>Stage of Newt Cell?</p>

Stage of Newt Cell?

G2 or prophase (interphase)

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<p>Composite Images</p>

Composite Images

= figures made from multiple images

  • red + green = yellow

  • purple + green = white

ex. Newt image = 2 images superimposed

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Visible light

  • additive = more colours → lighter

<ul><li><p>additive = more colours → lighter </p></li></ul><p></p>
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Pigments

  • Subtractive = more colours → darker

<ul><li><p>Subtractive = more colours → darker</p></li></ul><p></p>
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Red + green =

YELLOW - thus red and green (two proteins) are in the same place

<p>YELLOW - thus red and green (two proteins) are in the same place</p>
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Purple + green =

WHITE

<p>WHITE</p>
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<p>Confocal Fluorescence microscopy</p>

Confocal Fluorescence microscopy

= shows a 2D optical plane within a 3D specimen

  • uses a laser to focus on MIDDLE of specimen

  • shows more detail than wide field + more $$$

<p>= shows a 2D optical plane within a 3D specimen</p><ul><li><p>uses a laser to focus on MIDDLE of specimen</p></li><li><p>shows more detail than wide field + more $$$</p></li></ul><p></p>
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<p>Confocal vs. Widefield microscopy images</p>

Confocal vs. Widefield microscopy images

knowt flashcard image
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<p>ex. EVOS FL microscope </p>

ex. EVOS FL microscope

has: brightfield, phase contrast, and widefield fluorescence

<p>has: brightfield, phase contrast, and widefield fluorescence</p>
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Steps to image cells

  1. Put specimen on slide

  • whole cells : put 3 microliters of cell culture on slide + coverslip

  • thin sections of cell/tissue : immobilize in wax/plastic → section w a microtome

  1. Make things visible

a). via natural colour (ex. chloroplast - green, mitochondria - brown from pigments in ETC)

b). via coloured stains = synthetic molecules with 2 properties: affinity for target + absorbs light

ex. Hematoxylin (nucleic acids) & Eosin (proteins)

c). fluorescent stains = synthetic molecules with 2 properties: affinity for target + absorbs light

ex. DAPI (DNA)

d). fluorescent probes = 2 molecules attached together

  • used when stains WONT stick

  • properties : affinity for target, covalent bond, fluoresces light

e). specialized fluorescent stains

ex. zombie violent (dead cells purple)

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Putting whole cells on slide:

  • put 3 micrometers of cell culture on slide + add coverslip

ex. yeast

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Putting thin section of cells or tissues on slide:

  • immobilize cells/tissue in wax or plastic → section w a microtome

ex. human intestine biopsy

<ul><li><p>immobilize cells/tissue in wax or plastic → section w a microtome</p></li></ul><p>ex. human intestine biopsy</p><p></p>
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<p>what microscope was used?</p>

what microscope was used?

DIC

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Make things Visible: Natural Colour

  • some organelles have a natural colour

ex. chloroplasts : green

ex. mitochondria : brown (bc of pigments in ETC)

<ul><li><p>some organelles have a natural colour</p></li></ul><p>ex. chloroplasts : green</p><p>ex. mitochondria : brown (bc of pigments in ETC)</p><p></p>
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<p>Make things Visible: Coloured stains</p>

Make things Visible: Coloured stains

= synthetic molecules with 2 properties : affinity for target & absorbs light

ex. Hematoxylin - binds to nucleic acids (DNA/RNA)

ex. Eosin - binds to proteins

→ H&E staining:

  • by using their natural affinities for staining, it makes nuclei blue & cytoplasm pink (proteins)

<p>= synthetic molecules with 2 properties : affinity for target &amp; absorbs light</p><p></p><p>ex. Hematoxylin - binds to nucleic acids (DNA/RNA)</p><p>ex. Eosin - binds to proteins</p><p></p><p>→ H&amp;E staining:</p><ul><li><p>by using their natural affinities for staining, it makes nuclei blue &amp; cytoplasm pink (proteins)</p></li></ul><p></p>
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H&E Staining : Steps

  1. cell/tissue on slide

  2. add stain #1 (H)

  3. wash

  4. add stain #2 (E)

  5. wash

  6. coverslip

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<p>Make things Visible: Fluorescent stains</p>

Make things Visible: Fluorescent stains

= synthetic molecules with 2 properties : affinity for target & absorbs light

ex. DAPI (sticks to DNA) → makes nuclei (DNA) fluroesce blue

  • DAPI is excited by UV light

<p>= synthetic molecules with 2 properties : affinity for target &amp; absorbs light</p><p>ex. DAPI (sticks to DNA) → makes nuclei (DNA) fluroesce blue </p><ul><li><p>DAPI is excited by UV light</p></li></ul><p></p>
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<p>Make things Visible: Fluorescent Probes</p>

Make things Visible: Fluorescent Probes

= 2 molecules attached together (antibody + fl. dye)

  • used when stains WONT stick

  • antibody sticks to target, then fl. dye sticks to antibody via covalent bond

  • each microtubule is coated with fl. dye (a newt cell would be labelled w anti-microtubule antibodies w fl. dye attached)

Antibody = small proteins made by WBC (b-cells)

  • high affinity for microtubules → thus used for immunofluorescence

Fluroescent dyes = synthetic molecules that require an antibody to bind to target

ex. Alexa Fluor 488 (green)

<p>= 2 molecules attached together (antibody + fl. dye)</p><ul><li><p>used when stains WONT stick</p></li><li><p>antibody sticks to target, then fl. dye sticks to antibody via covalent bond</p></li><li><p>each microtubule is coated with fl. dye <em>(a newt cell would be labelled w anti-microtubule antibodies w fl. dye attached)</em></p></li></ul><p></p><p>Antibody = small proteins made by WBC (b-cells)</p><ul><li><p>high affinity for microtubules → thus used for immunofluorescence</p></li></ul><p></p><p>Fluroescent dyes = synthetic molecules that require an antibody to bind to target</p><p><span style="color: rgb(255, 255, 255);">ex. Alexa Fluor 488 (green)</span></p><p></p>
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Tubulin proteins are?

heterodimers

<p>heterodimers </p>
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<p>Do you have to use a green fluorescent dye to label a Newt cell</p>

Do you have to use a green fluorescent dye to label a Newt cell

No, bc images are captured in B&W → then coloured

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<p>Make things Visible: Specialized Fluorescent stains</p>

Make things Visible: Specialized Fluorescent stains

ex. Zombie Violet = only stains dead cells purple

  • used to tell if a cell is dead or alive

<p>ex. Zombie Violet = only stains dead cells purple</p><ul><li><p>used to tell if a cell is dead or alive</p></li></ul><p></p>
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<p>L1 Summary</p>

L1 Summary

Testable Content:

  • which method(s) was used?

  • which method(s) would you use?