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Absorption microscopy

Kuliah Karakteristik Mikroskopi

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Basic absorption microscopy

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Phase constrast microscopy

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When light wave travels through a medium other than vaccum,

interaction with medium causes the wave amplitude and phase to

change in a manner dependent on the properties of medium. Changesin amplitude (brightness) arise from the scattering and absorption of 

light, which is often wavelength dependent and may give rise to colors.

Photographic equipment and human eyes are only sensitive to

amplitude variation. Without special arragements, those changes are

therefore invisible. Yet, phase change often, carry important information.(wikipedia)

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Working principle

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The images constrast is increase in two steps:

1. The background light is phase shifted -90 degree by passing it

through a phase shift ring. This eliminates the phase different

between backgorund light and the scattered light, leading to an

increase intensity between foregound and background.

2. To further increase constrast, the background is dimmed by gray

filter ring.

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Differential Interference Constrast Microscopy

Patented by Georges Nomarski at 1952.

The microscope enhances constrast by creating

artificial shadows as if the object is illuminated

from the side. This DIC microscopy uses Polarized light,

making it unsuitable when the object alter

polarization

Replaced by Hoffmann modulation contrast

microscopy. Invented by Robert Hoffmann at1975

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The introduction of digital camera in the mid 1990

Quantitative Phase Constras Microscopy

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Single molecule absorption

Direct detection of optical absorption with single-

molecule sensitivity at room temperature is difficult 

because absorption is not a background-free

measurement and is often complicated by samplescattering 

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• Celebrano et al. 8 described in detail in Nature

Photonics, allows for the direct imaging ofindividual molecules by monitoring the

attenuation induced in probe light.

The molecular absorption cross-sections, often < than afew square angströms,

a tightly focused light beam, as used in optical

microscopes, reveal that less than one photon in a

million is absorbed.

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Direct observation of a single molecule by measuring its

Attenuation (serapan) of a light beam

Three different method that allow detection of 

light absorption by single molecule

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The local heating induced by light absorption results in tiny changes

in the local refractive index, which can be probed by the enhanced

backscattering of a second, probe, beam(red). Fast intensitymodulation of the absorbed blue beam results in measureable

modulations in backscattering intensity with the same frequency.

“Double beam”

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Modulation of the blue beam influences the absorption of

the green beam by depleting (menghabiskan) the ground

state.

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The attenuated Power (P) of the incident beam (P) by the single

molecule:

hv  is the photon energy; is the absorption crosssection for the single chomophore

(single molecule) (~ 10-16 cm2 at room temperature);

S is the beam waist (pinggang) area .

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K relax 

  is k relax2   : the rate constant of the rate-limiting step in the

sequential relaxation process from the vibronic states prepared by theoptical excitation to the lowest vibrational level in the ground electronic

state.

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