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Seeing Single Molecules Seeing Single Molecules Dr. Arindam - - PowerPoint PPT Presentation

Nov 02, 2022 285 likes •660 views

Seeing Single Molecules Seeing Single Molecules Dr. Arindam Chowdhury Department of Chemistry IIT-Bombay Mumbai IIT Bombay, Mumbai arindam@ chem.iitb.ac.in Outline Molecular length scales and magnification How to see or

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Seeing Single Molecules Seeing Single Molecules

  • Dr. Arindam Chowdhury

Department of Chemistry IIT-Bombay Mumbai IIT Bombay, Mumbai

arindam@ chem.iitb.ac.in

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Outline

  • Molecular length‐scales and magnification
  • How to “see” or “feel” individual molecules
  • Examples related to chemistry and biology

– S

eeing molecules using light and electrons

– S

eeing DNA molecules and their dynamics

Please stop me whenever you are uncomfortable or getting lost

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Molecules are of nanometers (0 000001 mm) dimensions

1 nanometer = 10-9 m

(0.000001 mm) dimensions

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Why do we want to see single molecules?

  • Seeing is believing: “know that they exist” !
  • To understand their behaviors (properties)

as individual molecules

  • To see how a chemical/biological reaction occurs
  • It is challenging and exciting and often quite fun!
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Need a powerful magnifying glass !!!

To see very small objects like Mr. Homer’s brain,

Arindam Chowdhury IITB 2010

y j , we need very very powerful magnifying glasses!

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The eye of fly: Microscopy at Microscopy at various orders

  • f magnification

These spikes or balls are made of

Arindam Chowdhury IITB 2010

balls are made of many millions of molecules !!!

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Molecules are 10,000 TIMES smaller than Red Blood Cells (5 micrometers) than Red Blood Cells (5 micrometers)

Arindam Chowdhury IITB 2010

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What are molecules? A mesh of electrons/protons attached to each other

More number of bonded atoms, larger the size

  • f molecules

(no upper limit)

Arindam Chowdhury IITB 2010

NOT ALL MOLECULES CAN BE “SEEN”

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To see such small specimens…

Arindam Chowdhury IITB 2010

A normal microscope will not do…

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Even with very high powered microscopes Only large molecules can be observed Only large molecules can be observed

Fullerene Graphite Fullerene Graphite

Large macromolecules: polymers proteins DNA can also be

Pentacene

Arindam Chowdhury IITB 2010

Large macromolecules: polymers, proteins, DNA can also be seen, but many a time, we have to play some tricks to see them!

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What do we need to observe Single Molecules ?

  • Microscopes or rather Nanoscopes that uses either

light or electrons which can “see” or “feel” molecules

  • Blind man’s way of looking is to “ feel” things:

U f t d b l t Use forces exerted by electrons “ ATOMIC FORCE Microscopy” (AFM)

  • Direct electron densities and position of atoms:

Electron Microscopy such as S TM and TEM (not to be discussed in today’s lecture) (not to be discussed in today s lecture)

  • Using light to visualize: Fluorescence microscopy

can see large biological macromolecules like DNA

Arindam Chowdhury IITB 2010

can see large biological macromolecules like DNA

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  • I. Feeling S

urfaces by (AFM)

Arindam Chowdhury IITB 2010

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Arindam Chowdhury IITB 2010

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Seeing graphite surface with AFM

Distance between Two layers ~ 0.35 nm Height difference Between consecutive layers ~ 0.35 nm

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Graphite surface at atomic resolution

Distance between atoms

  • bserved matches perfectly

with the theoretical values with the theoretical values

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Visualizing S ingle Pentacene Molecules

Arindam Chowdhury IITB 2010

Science, August 30, 2009

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Using light to see single l l macromolecules

S l l i i li h f diff

  • Some molecules emit intense light of different

Color upon shining light on them This property is called “Fluorescence” which This property is called Fluorescence which Is commonly observed in nature. Color of light E i d d d h f l l Emitted depends on the type of molecule.

Arindam Chowdhury IITB 2010

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Seeing Molecules with light: Using Fluorescence of colored dyes as probes Fluorescence of colored dyes as probes

Arindam Chowdhury IITB 2010

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Many naturally occurring minerals are fluorescent and emit various colors of light g

Arindam Chowdhury IITB 2010

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Some living creatures also do the same! also do the same!

Arindam Chowdhury IITB 2010

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Basic Concept of Fluorescence Microscopy

Arindam Chowdhury IITB 2010

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Arindam Chowdhury IITB 2010

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Need to work in a dark room since outside light can overwhelm weak fluorescence light can overwhelm weak fluorescence

Arindam Chowdhury IITB 2010

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Individual cells and routinely imaged using multi‐color fluorescence microscopy

Arindam Chowdhury IITB 2010

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Cellular and Nuclear dynamics

Arindam Chowdhury IITB 2010

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Each cellular nucleus contains all genetic information wrapped up as chromosomes pp p

A full length 5 meter long human DNA is packed within a nucleus the size of few micrometers (10‐6 m)!!!

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Can we see the DNA Molecule ?

DNA does not emit light! So we need to make it emit light somehow, in order to see these individual macromolecules. But anyways, AFM can be used to see individual DNAs

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DNA is a double helix: Two strings intertwined

bl l S Double Helix S tructure DNA can resemble a loose spring Or something like a rubber band g

Arindam Chowdhury IITB 2010

Lengh of the DNA can be controlled by adding monomer Units so that it looks like a long one-dimensional wire

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Long DNA molecules are not like rigid rods, but like long strings which can bend rods, but like long strings which can bend

Arindam Chowdhury IITB 2010

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It is possible to see long individual DNA molecules on surface using AFM DNA molecules on surface using AFM

Arindam Chowdhury IITB 2010

DNA molecules are not chemically attached, but are adsorbed on surface

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Coiling and packing of DNA molecules

Arindam Chowdhury IITB 2010

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S ingle long DNA molecules labeled with “ green” fluorescent dye molecules with green fluorescent dye molecules

Arindam Chowdhury IITB 2010

Difficulty: If DNA is not anchored to the surface, they will wander off!

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DNA molecules can be attached to a surface and imaged with adsorbed fluorescent dyes g y

Images of Single DNA Molecules in solution

Arindam Chowdhury IITB 2010

Molecules in solution

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Isolated DNA Dynamics in S

  • lution

Lipid bila er bilayer provides natural environment No Buffer Flow Flow Stop With Buffer Flow Direction Flow Start Flow D

Arindam Chowdhury IITB 2010

DNA “Curtain”

DNA Labeled with YoYo1 Dye (Green): Images in False Color

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S tretched-DNA anchored at both ends

Arindam Chowdhury IITB 2010

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Rubberband like behavior of stretched DNA

Arindam Chowdhury IITB 2010

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Summary Summary

  • It is possible to see individual molecules and

It is possible to see individual molecules and image them at atomic resolution

  • Seeing large biological macromolecules can

provide valuable information about their structure, reactivity and dynamic behavior Thank you for being patient!

Arindam Chowdhury IITB 2010

Thank you for being patient!