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 “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
1 nanometer = 10 -9 m Molecules are of nanometers (0 000001 mm) dimensions (0.000001 mm) dimensions
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!
Need a powerful magnifying glass !!! To see very small objects like Mr. Homer’s brain, y j , we need very very powerful magnifying glasses! Arindam Chowdhury IITB 2010
The eye of fly : Microscopy at Microscopy at various orders of magnification These spikes or balls are made of balls are made of many millions of molecules !!! Arindam Chowdhury IITB 2010
Molecules are 10,000 TIMES smaller than Red Blood Cells (5 micrometers) than Red Blood Cells (5 micrometers) Arindam Chowdhury IITB 2010
What are molecules? A mesh of electrons/protons attached to each other More number of bonded atoms, larger the size of molecules (no upper limit) NOT ALL MOLECULES CAN BE “SEEN” Arindam Chowdhury IITB 2010
To see such small specimens… A normal microscope will not do… Arindam Chowdhury IITB 2010
Even with very high powered microscopes Only large molecules can be observed Only large molecules can be observed Fullerene Fullerene Graphite Graphite Pentacene Large macromolecules: polymers proteins DNA can also be Large macromolecules: polymers, proteins, DNA can also be seen, but many a time, we have to play some tricks to see them! Arindam Chowdhury IITB 2010
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 Use forces exerted by electrons f t d b l t “ 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 can see large biological macromolecules like DNA Arindam Chowdhury IITB 2010
I. Feeling S urfaces by (AFM) Arindam Chowdhury IITB 2010
Arindam Chowdhury IITB 2010
Seeing graphite surface with AFM Distance between Two layers ~ 0.35 nm Height difference Between consecutive layers ~ 0.35 nm
Graphite surface at atomic resolution Distance between atoms observed matches perfectly with the theoretical values with the theoretical values
Visualizing S ingle Pentacene Molecules Science, August 30, 2009 Arindam Chowdhury IITB 2010
Using light to see single macromolecules l l • Some molecules emit intense light of different S l l i i li h f diff 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 Emitted depends on the type of molecule. d d d h f l l Arindam Chowdhury IITB 2010
Seeing Molecules with light: Using Fluorescence of colored dyes as probes Fluorescence of colored dyes as probes Arindam Chowdhury IITB 2010
Many naturally occurring minerals are fluorescent and emit various colors of light g Arindam Chowdhury IITB 2010
Some living creatures also do the same! also do the same! Arindam Chowdhury IITB 2010
Basic Concept of Fluorescence Microscopy Arindam Chowdhury IITB 2010
Arindam Chowdhury IITB 2010
Need to work in a dark room since outside light can overwhelm weak fluorescence light can overwhelm weak fluorescence Arindam Chowdhury IITB 2010
Individual cells and routinely imaged using multi ‐ color fluorescence microscopy Arindam Chowdhury IITB 2010
Cellular and Nuclear dynamics Arindam Chowdhury IITB 2010
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)!!!
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
DNA is a double helix: Two strings intertwined Double Helix S bl l S tructure DNA can resemble a loose spring Or something like a rubber band g Lengh of the DNA can be controlled by adding monomer Units so that it looks like a long one-dimensional wire Arindam Chowdhury IITB 2010
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
It is possible to see long individual DNA molecules on surface using AFM DNA molecules on surface using AFM DNA molecules are not chemically attached, but are adsorbed on surface Arindam Chowdhury IITB 2010
Coiling and packing of DNA molecules Arindam Chowdhury IITB 2010
S ingle long DNA molecules labeled with “ green” fluorescent dye molecules with green fluorescent dye molecules Difficulty: If DNA is not anchored to the surface, they will wander off! Arindam Chowdhury IITB 2010
DNA molecules can be attached to a surface and imaged with adsorbed fluorescent dyes g y Images of Single DNA Molecules in solution Molecules in solution Arindam Chowdhury IITB 2010
Isolated DNA Dynamics in S olution Lipid bila er bilayer provides natural environment No Buffer Flow Flow Stop Direction With Buffer Flow Flow D Flow Start DNA “Curtain” DNA Labeled with YoYo1 Dye (Green): Images in False Color Arindam Chowdhury IITB 2010
S tretched-DNA anchored at both ends Arindam Chowdhury IITB 2010
Rubberband like behavior of stretched DNA Arindam Chowdhury IITB 2010
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! Thank you for being patient! Arindam Chowdhury IITB 2010
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