Bio150 Report1 Fluorescence Microscopy

8/6/2019 Bio150 Report1 Fluorescence Microscopy

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Biology 150

Estonilo | Gascon


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property of some atoms and molecules to

absorb light of a particular wavelength and

after

a brie

f inter

val (fluore

sce

nce

life

time)

to re-emit light at longer wavelengths


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Allows quantitative spatial and temporal

visualization of fluorescent material in

microscope specimens that areeither

intrinsically fluorescent (primary or

autofluorescence) or which have been

coupled to extrinsic fluorescent molecules

(secondary fluorescence)


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To deliverexcitation energy to the fluorescing

species in the specimen and to separate the

much weakeremitted fluorescence light

from the brighterexcitation light. (filter

fluorometer)


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Specificity

Sensitivity

Environmental sensitivity High Temporal and Spatial Resolution


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The mercury lampemits ultraviolet (UV) light

o directed to a specific fluorescence filter cube located in the mirror

turret unit.

Th

eex

citer

filter

in the

cube

is de

signe

d to allow spe

cificwavelengths of UV light to pass though

o those that are absorbed by the dye in use

o reflected by the dichroic mirror

o pass through the objective lens to illuminates the specimen

(violet rays).


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The light emitted from the specimen (multiple colorrays) is

directed upwards through the objective and pass through

the dichroic mirror

o removes the incident UV light

o allows the longer wavelength visible light to pass through

The light emitted is viewed afterpassing through the

emission filter

o selects which wavelength of light passes through to theeyepieces

(only the green ray).

o Bright green glowing dye-stained cell or organelle against a dark

background.


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Excitation light sources

Wavelength selection devices

Objectives

Detectors


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- consist of tungsten or halogen lamps (incident

illumination) and mercury, xenon or metal halide arc lamps

or lasers (epi-illumination)

- selection depends on the fluorescent probes being used.


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o Include tunable lasers, monochromators,

AOTF and LCTF

o

Used to select a certain part of theelectromagnetic spectrum for

transmission, whilepreventing therest of

theelectromagnetic spectrum from

passing through the filter

o Excitation filter and barrier filter


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o Transmitted-light and reflected-light

versions

o

Depends on wavelength of lightoQuartz objectives for use in UV; Fluars for

wide spectral range


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o Allow visualization of low levels ofemitted

fluorescence without photobleaching to

the specimen.

o Allow realtime recording of living cell and

tissuephysiology


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TIRFM (Total Internal Reflection Fluorescent

Microscopy) uses a completely different concept.

Very thin layer of the specimen (around 200 nm) is

used to create the image. Therefore, TIRFM is ideal

to analysee.g. single molecule interactions or

membraneprocesses. To achieve this target, a light

beam is directed within a critical angle towards the

cover slip.

Because of the higherrefractive index of the cover

slip compared to the specimen,


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total internal reflection occurs

o almost no direct light enters the specimen

o evanescent wave travels in the specimen direction

o only strongenough to excite fluorochromes within the

first few hundred nanometres close to the cover slip.

o The fluorescent image is restricted to this small depth

cannot be driven into deeper areas of the specimen, and also

does not contain out of focus blur from deeper areas.


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Bio150 Report1 Fluorescence Microscopy

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