Single Molecule Spectroscopy: Single Live Cell · Dr. Subhadip Ghosh, NISER Dr. Dibyendu Das, USA...
Transcript of Single Molecule Spectroscopy: Single Live Cell · Dr. Subhadip Ghosh, NISER Dr. Dibyendu Das, USA...
Single Molecule Spectroscopy: Single Live Cell
Kankan Bhattacharyya
• 2015: Intl. Yr of Light • 1000 yrs “Optics” by Al-
Haytham
• 200 yrs Fresnel Theory
• 150 yrs of Maxwell
• 100 yrs Light-cosmology
• 1 Nobel, 3 missed
• Raman Effect, Raman-Nath
• JC Bose: Microwave
• SN Bose: BEC
• Sudarshan: Coherent State
• CKN Patel: CO2 Laser
Light & IT
Chemistry Nobel: 2014
Chemistry of Small Things
Single Molecule Spectroscopy
- Every molecule is different
E Betzig S Hell W Moerner
K Bhattacharyya, Resonance (2015)
How to see a Single Molecule?
Focus ~ /2 = 200 nm > molecule (1 nm)
Dilute solution, ONE molecule in focus
Focal Volume = 3 = 10-15 litre = 1 fL
10-9 M= 6 x 1023 x 10-9 mol. per litre
= 6 x 1014 per litre = 0.6 mol. per fL
No of mol. in focal spot = 0.6 WHY??
Molecule in & out of focus: Diffusion
Confocal Microscopy: How to see a single molecule?
W Moerner
Stanford, (1989)
Fluorescence
Confocal
pinhole
Live Cell Imaging: Seeing inside a cell
• Cell: ~20,000 nm ~ 100 times bigger than focus
• Label different parts of a cell with fluorescent dye
• Cancer Cell: How different from a normal cell?
cell Space & time resolution
>100 ns
1.7 ns 1.3 ns
Chattoraj, KB J Phys. Chem C 118 (2014) 22339
• Au25 is characterized by very long >100 ns life time
• Au13 ~ 1 ns life time
• Short life time (1.3 & 1.7 ns) inside cell proves Au25
• Au25 in bulk water becomes Au13 inside cell
Fluorescent Gold Nanocluster Inside a Human Breast Cell
Au25 Au13
Bulk
water
Live
cell
Emission energy = Ef / N1/3
Cancer
N = number of Au atoms in cluster
Normal
Breast Cell
MCF10A Breast
Cancer Cell
MCF 7
• Why higher uptake in cancer cell?
• Cancer cell contains 7-fold higher Thiol (glutathione)
• Thiol (Glutathione) Stabilizes gold nano-clusters (Au-NC) more
• Au-NC with anti-cancer drug for selective killing of cancer cells?
Chattoraj, Bhattacharyya, J. Phys. Chem. C 118 (2014) 22339
Fig. 10 Evaluation and comparition of anticancer activity of doxorubicin loaded Au-NC and
free doxorubicin by MTT assay: (A,B) breast cells, (C,D) lung cells.
(A) (B)
(C) (D)
• Doxorubicin
• Alone kills both
cancer & normal
• On Loading to
Au-NC, normal
Cell survives
Selective killing
of cancer cells
by doxorubicin
loaded Au-NC
Breast cancer Breast normal
Lung cancer
Lung normal
Shyamtanu Chattoraj, KB,
• Warburg Effect (1956) Predicted that Cancer cells contain
more lipid droplets than normal cell
• Cancer causes glycolysis i.e. non-oxidative breakdown of
glucose to pyruvate, instead of respiration (oxidation to CO2)
• Pyruvate: precursor of fatty acid & cytoplasmic lipid droplets
• Can we show that cancer cell has more lipid droplets?
Normal
Cancer Diagnosis
Lung Cancer Cell
Cancer
Normal
Lung Cancer Cell and Lipid Droplets
Rajdeep, Surajit (IICB) KB
Med Chem Comm 5 (2014) 536
2000 downloads in 5 months!!
Normal Lung Cell
• Lipid droplets: ~20 times more in a cancer cell
• Lipid droplets: low polarity of ( ~ 6), slow solvation (1700 ps)
• Cytosol: ~ 24, solvation time ~ 1000 ps
• Cancer cell: Hydrophobics accumulate in lipid droplets &
Favors non-polar reactions
Solvation Dynamics & Intermittent Oscillation of Cell Membrane: Live CHO Cell
CHO Cell:
Cell surface thiols most reactive
Shirsendu, KB,
JPC B 118 (2014) 2949
Labeling of thiols (SH) by coumarin maleimide (CPM dye)
H
S -SH
Cysteine
o
Oxidation
Reduction
S H S H
S S
H S
H
Cytoplasm
Membrane
Cytoplasm
S S S
S
S
Cytoplasm
Membrane
Nucleus Nucleus
• Internalization: rupture of S-CPM bond. Unbound, non-
fluorescent CPM goes inside
• Membrane Potential fluctuates in seconds (due to red-ox)
• Can we see the fluctuations through fluorescence oscillations
• Possible because the ps diode lasers are very stable
(2014 Physics Nobel)
Shirshendu, KB JPC B 118 (2014) 2949
CPM H
CPM
• Origin of Oscillations (10-20%)
a) Red-Ox cycle & environment
b) Unbinding & rebinding of CPM
Shirshendu, KB,
JPC B 118 (2014) 2949
H
S -SH
Non-
fluorescent Fluorescent
Red-Ox Oscillations in Mitochondria of a Live Cell:
Breast Cancer & normal cell
Shyamtanu, KB
J. Phys. Chem. B
119 (2015, in press)
• How different is cancer & normal cell?
• Programmed Cell death: Apoptosis
Human Normal Breast Cell: MCF 10A
Shyamtanu, KB
J. Phys. Chem. B
119 (2015) 8842
• In MCF 10A breast cells : thiols in mitochondria most reactive
• Confirmed by co-localization of CPM with mitotracker green dye
Human Breast Cancer Cell: MCF-7
• CPM labels clustered mitochondria in perinuclear region of cancer cell
Scheme 1. Thiol-disulfide exchange in Mitochondria
Shyamtanu, KB
J. Phys. Chem. B
119 (2015) 8842
Normal Breast Cell
Cancer cell
• Oscillations in Normal Breast Cell
• No oscillation in Breast Cancer cell !
• Red-ox cycle less active in cancer cell
• During red-ox oscillation, cytochrome C
comes out of mitochondria and kills cell
• Normal cell dies, shows oscillation
• Cancer cell never dies, No Oscillation!!
Normal cell
CPM in MCF 10A
Shyamtanu, KB
J. Phys. Chem. B
119 (2015) 8842
Gene Silencing in a Cancer Cell by siRNA
Shyamtanu Chattoraj, S. Saha, SS Jana, KB
J. Phys. Chem. Lett. 5 (2014) 1012−1016
• siRNA= short interfering double-stranded RNA (21-23 nucleotides)
• siRNA: degrades (“silence’) disease related gene in mRNA
• siRNA: binds with ribo-nucleo-proteins to form RNA induced
silencing complex (RISC) & then guides RISC to target gene
• Once siRNA binds target gene, other agents in RISC cleave mRNA
Gene Silencing by siRNA siRNA
Formation of RNA induced Silencing complex (RISC)
Kinetics??
Degradation of mRNA i.e. silencing
Kinetics?? Degraded
mRNA
mRNA
Signaling molecules
mRNA
RISC
Specific binding of siRNA to mRNA
mRNA-siRNA
Confocal image: siRNA labeled with a
fluorescence probe (alexa 488) in MCF-7 cell
• Specific siRNA bound to mRNA:
high fluorescence intensity, many regions
• Free siRNA (in culture medium):
low fluorescence intensity
• Non-specific siRNA does not bind
to mRNA: Low fluorescence intensity
Binding of siRNA to mRNA causes
increase in fluorescence intensity
of Alexa 488
• Normal Breast Cell (MCF 10A)
• 60 sec : searching of gene
• 60-125 sec: high intensity, bound
• >125 sec: low intensity after silencing
• Red : Specific siRNA, bound, high intensity & fluctuations
• Black: non-specific siRNA (control): low intensity
High Intensity Period: bound
• At intermediate times (60-160 sec):
high fluorescence intensity & fluctuations
• Several cycles of unbinding & rebinding
Low Intensity Period
• siRNA not bound to target in m-RNA
• Initial period (0 – 60 s): searching
• Long time (>160 s): After degradation
Cancer Cell MCF-7
Fluctuations in Fluorescence Intensity
Shyamtanu, KB
JPC Lett. 5 (2014) 1012
• On-time= residence or dwell time during which
specific siRNA remains bound to mRNA
• On-time (high intensity periods):
• Multiple Values
• 16273 on-time periods detected
• All are odd-integral multiples of 5.5 ms
• Odd-integral multiples:
Evidence of Stochastic Resonance !!
On-time Histogram & Stochastic Resonance:Cancer Cell MCF-7
Shyamtanu, KB
JPC Lett. 5 (2014) 1012
• Random Noise, (t): causes transition from one well to another
• Periodic Noise, Q(): periodically lower & raise two wells, creates bias
• For Q() = sin t, x= positive & highest for t =/2, A-to-B transition
• x = negative & highest for t =3/2, B-to-A transition
• Time in well B (Bound) : 3/2 - /2 = i.e. t=/= T0/2
• If B-to-A does not occur at t =3/2, next one at + 2=3T0 /2
• Time in well B (Bound) : odd-integral multiples of T0 /2
• Stochastic Resonance:
• Noise assisted crossing: Double well
• Langevin Equation:
)()()(
Qtdx
xdUx
Free
Bound
Bound
Free
t = 0-T0/2 t = T0/2-T0
A
B A
B U(x)
x
What did we learn? siRNA
Formation of RNA induced Silencing complex (RISC)
Searching time ~ 60 s Unbinding
& rebinding
Degradation of mRNA i.e. silencing
Silencing time ~ 160 sec
On-time = N x 5.5 ms N=1,3,5,… Stochastic Resonance!
mRNA
Signaling molecules
mRNA
RISC
mRNA-siRNA
Degraded mRNA
Shyamtanu, KB
JPC Lett. 5 (2014) 1012
• Normal Breast Cell
• Faster (~70 s) silencing
• No stochastic resonance
• No unbinding/rebinding
Normal
cell
Cancer
Cell
Normal cell
Cancer Cell • Breast cancer cell
• Silencing difficult (120 sec)
• Stochastic resonance
• Cancer has a signal (sin t)!
Fluctuating Bio-molecules: smFRET & D-A distance
D + A*
A + h
D* + A
A*
D + h D*
FRET
• Forster Theory of FRET
• Rate of FRET 1/ [RDA]6
• Acceptor (Alexa) attached to Microtubules
• Donor (EGFP) bound to end-binding protein Mal3
• Mal3 binds to microtubule: only in Polymerized state
• When microtubule polymerized: FRET occurs, A em high
• When microtubule depolymerized: NO FRET, D em high
Thread Like
microtubule
Cytoskeleton network
= skeleton of
cytoplasm
• Microtubules are dynamic polymers of & tubulins
• Length of microtubule fluctuates: a few nm to 1000 nm
• D (EGFP) – A (Alexa) Distance fluctuates 28-80 Å
Prasenjit, Surajit Ghosh, KB PCCP 17 (2015) 6687
Fluorescence fluctuation of an antigen–antibody complex:
Do they have a rigid structure?
• Antigen: enhanced GFP (EGFP)
DONOR
• Antibody: anti-EGFP VHH-His6
Alexa labeled ACCEPTOR
• smFRET: D-A distance
• D-A distance fluctuates with time
• Structure not RIGID
Surajit Ghosh (IICB) & KB
PCCP 17 (2015) 25250
• Single Molecule: Each Different & organelle specific
• Etching & higher uptake of Au-NC in a live cancer cell
• Lipid droplets: implications in cancer
• Structural oscillations at cell surface
• Oscillations at Mitochondria: Normal vs Cancer Cell
• Stochastic Resonance in a Live Cancer cell
• Viewing Jumping Microtubules by smFRET
• Antigen-Antbody Complex is not Rigid
“Everything which living things do can be understood
in terms of jiggling & wiggling of atoms.”
Feynman, 1963
Acknowledgements
FUNDS DST (IRHPA)
“Centre for Ultrafast
Spectroscopy & Microscopy”
JC Bose Fellowship
STUDENTS
• Shirshendu Ghosh
• Rajdeep Chowdhury
• Shyamtanu Chattoraj
Collaborator
• Dr. S. S. Jana, IACS
• Dr. Surajit Ghosh, IICB
Thank You Very Much
Gold Nanoclusters: Next Generation Fluorophore
for Cell Imaging
Chattoraj, KB,
JPC C
118 (2014) 22339
Au25 Au13
Em energy
= Ef / N1/3
100 ns ~2 ns
Prasenjit, Surajit Ghosh, KB
PCCP 17 (2015) 6687
• Signal: Fluorescence from bound state (specific siRNA bound to mRNA)
• x = combined distance of ~21 nucleotides (siRNA-to-mRNA)
• Random Noise, (t): Random polymer chain dynamics of siRNA &
mRNA, & fluctuations in concentration of signaling molecules in RISC
• Periodic Noise, Q(): Periodic oscillations in structure & concentration
Structural Oscillation: Co-operative motion of siRNA & mRNA
Concentration oscillations of signaling molecules in RISC
• Stochastic Resonance: Bistable
• Enhancement of signal by noise
)()()(
Qtdx
xdUx
Free
Bound
Bound
Free
t = 0-T0/2 t = T0/2-T0
A
B A
B U (x)
x
Effect of Alcohol on Protein:
Size & Dynamics of Lysozyme
• Fluorescence Correlation Spectroscopy (FCS)
• Diffusion Coefficient, Dt=kT/6r (for a sphere)
• r gives size, changes when a protein unfolds
• (local friction) may vary in heterogeneous medium e.g.
in a vesicle
Dr. Subhadip
Ghosh, NISER
Dr. Dibyendu
Das, USA
Dr. Dibyendu
Sasmal, JNU
Fluorescence Intensity Fluctuation in a microscope
24.0 24.8 25.60
20
40
60
80
Time (s)
Cou
nts
• Conformational Dynamics of Protein
Quenching by NH2 (0.1 nm) OR by FRET (5 nm)
P
NH2 High intensity
Low Intensity P
Variation of size
Variation of
Relaxn time
Lysozyme
Effect of Alcohol on Size & Dynamics of Lysozyme
Shyamtanu, KB
J. Chem. Phys. 140 (2014) 115105
k+
k-
G
Shyamtanu, KB
J. Chem. Phys. 140 (2014) 115105
Oscillatory variation of folding dynamics and size of
a protein on Alcohol concn
Western Blot analysis
Synthesis of protein, NMHC-II-A (translation) by
mRNA in MCF-7 Cells
Specific siRNA stops protein synthesis by 98%
by degrading MYH9 gene in mRNA
Non-specific siRNA does not stop synthesis
Real time PCR 90% mRNA degradation within 48 hours
Indicates transcriptional inhibition
Specific
siRNA
Non-Specific
Solvation Dynamics
• Solvation : stabilization of an ion or dipole by solvent
• How long does it take in water?
Answer: <10-12 sec = 1 ps
• How long does it take in biological system?
Answer: ~100-1000 ps
• Biological water is much slower!!
• KB, Chem Comm Feature (2008), Acc Chem Res
(2003); Bagchi, JPC Feature &Chem Rev (2000)
Real biological water is inside a live cell!!
4 m
4
5 6
1 2
3
Z = 10 m above surface
A
• Lipid Vesicles
• Dye C153 only in membrane
• dia ~20 m
• focus ~0.2 m
• 4-6: near water
• 1-3: deep in wall
Supratik, KB,
Langmuir 28 (2012) 10230
• Diffusion coefficient (Dt ) of C153 in water 550 m2 s-1
• Dt of C153 in vesicle 3-21 m2 s-1
• Diffusion in vesicle (membrane) slower
• Friction not uniform : inhomogeneous
• Diffusion controls rate of reaction : varies
0 3 6 9 12 15 18 21 240
3
6
9
12
15
Dt (m2/s)
No o
f O
ccu
rren
ce • Dt in different regions
of a single vesicle
• Dt=kT/6r
• Friction in regions of
200 nm
• Solvation dynamics in live CHO cell
• Rise at long wavelength: in Microscope
0 2 4 6
0.0
0.2
0.4
0.6
0.8
1.0 DCM in cytoplasm
510 nm
570 nm
720 nm
IRF
No
rma
lize
d C
ou
nts
Time (ns)
0.0 0.8 1.6 2.4 3.2 4.00.0
0.2
0.4
0.6
0.8
1.0
Time (ns)
No
rma
lize
d C
ou
nts
420 nm
600 nm
DAPI: nucleus
Sasmal, KB Langmuir 29 (2013) 2289
Solvation Dynamics of C153 in a live CHO cell
Shirsendu, KB Langmuir 29 (2013) 7975
• Solvation time, bulk water ~ 1 ps
• Nucleus ~ 750 ps
• Cytoplasm 1100 ps
• Lipid droplets 3600 ps, ~ 7
• Lipid droplets non-polar, slow
~ 7
40 60 80 100 120 140 160 180100
150
200
250
300
350
Inte
nsi
ty o
f sp
ikes
Time (sec)
• Initial ~60 sec: Low Fluorescence intensity, searching period
• 60-160 sec: High intensity & fluctuations, unbinding-rebinding
• 160-180 sec: Gradual decrease in intensity, silencing
Variation of peak fluorescence intensity against time
Shyamtanu, KB
JPC Lett. 5 (2014) 1012
200 nm
(/2)
Bleached
by em
Absorption
abs
Stimulated
Emission
em
</2
Super Resolution Microscopy: < /2
STED = Stimulated Emission Depletion (S. HELL)
Strategy: two lasers abs & em
1) abs excites to upper state
2) em bleaches outer periphery of ~ 200 nm spot & collect
emission from smaller spot at centre
Effect of GdnHCl in the Hydrodynamic radius of HSA
Sasmal, KB JPC B
115 (2011)13075
Supratik, …Bhattacharyya
J Phys Chem B 116 (2012) 12189
Effect of ionic liq (RTIL) on Cytochrome C
• Two molten globules (MG)
• Denatured>MG>native
• Ionic liq increases size of native
• Ionic liq reduces size of denatured
• Two MG: affected differently
Salt effect on ESPT in Niosome
Addition of NaCl causes
• Decrease in amount of free water
• ESPT: Slower
• Intensity of HA higher
• Decay of HA slower
Tridib KB JPC B 116 (2012) 8105
0 M
NaCl
4M NaCl
HA A-
HA
In what time scale proton transfer
occurs inside a live cell?
Supratik, KB
JCP 138 (2013) 215102
• Ionic dye, HPTS
• No Lipid droplet!
HPTS
CHO cell
R6G
Excited State Proton Transfer (ESPT)
O3S SO3
OHO3S
_
_
_
• Photo-acid: Acidity much higher (107 times) in excited state,
pK*=0.4 and pk=7.4
• Rate of proton transfer (ESPT): ~3 ps, 90 ps in bulk water Monitored by decay of HA emission (460 nm) or rise of A- em (520 nm)
Pyranine, HPTS
HA
A-* HA*
A-
-H+
+H+
Abs HA em
440 nm
A- em
520 nm
HA* = [H+ …. A-* ]= H+ + A-*
A-
HA
• HA emission strong: Normal Lung & CHO cell
• HA emission weak in cancer cell (A549)
• ESPT faster in cancer cell !!
CHO Cell
Suprotik, KB,
JCP (2013)
A-
HA
A-
HPTS in Live cell: Emission spectrum under microscope
Rajdeep, KB, JPC B (PPT Spl issue)
Lung Cell
WI38, Normal
A549, Cancer
Water
Rise time
• Water ~90 ps
• Normal lung cell 300 ps
• Cancer cell 200 ps
Rajdeep, KB JPC B (PPT Spl issue, 2014)
Rise & Decay of 430 nm (HA) & 510 nm (A-) emission):
In live cell under a microscope
HA
A-
Rajdeep, KB JPC B (PPT Spl issue)
AHA
HA
kk
AHA
HA
Z
Y
X
dt
d
diss
PT
rec
0
0
0k
System PT(ps) rec (ps) diss (ps)
_____________________________________
Water 5 7 50
Normal Lung Cell 40 25 120
Lung Cancer Cell 40 30 80
CHO cell 50 25 120
Niosome (no NaCl) 40 30 75
• Initial proton transfer 8 times slower in lysosome than water
• Recombination ~4 times slower in cell than water
• Dissociation ~ 2 fold slower in cell
• Dissociation in normal cell 1.5 times slower than cancer cell
• Dissociation of ion pair faster in the cancer cell: cause of faster ESPT!!
Rajdeep, KB JPC B (PPT Spl issue)
Single Molecule: Why Important?
Student X
100/100 Student Y
00/100
Average 50/100
Average does not give correct picture!
Chemistry Nobel 2013 & A Missed Indian Hero
Aneesur Rahman
Born: 1927 (Hyderabad)
Died: 1987 (Minnesota)
M. Karplus M. Levitt A. Warshel
“for the development of Computer Simulation
for complex chemical systems”
What is Fluorescence Correlation
Spectroscopy (FCS)?
0 5 10 15 20 25 30 3518.8
19.0
19.2
19.4
19.6
19.8
Time (s)
De
tect
ed
Inte
nsi
ty (
kcp
s)
t1
t2
t3
t4
t5
Fluorescence observed: t2, t3, t4
No Fluorescence detected: t1, t5
< F(0)F()> G() =
<F(0)>2
F(0)
Fluorescence Intensity Fluctuation in a microscope
• Binding/unbinding of Probe
to a Protein
24.0 24.8 25.60
20
40
60
80
Time (s)
Cou
nts
• Diffusion : in & out of focus
Picosecond Dynamics under a microscope
A few molecules: How to record ?
• Emission Spectrum
• Fluorescence Decay
• Each time collect one photon: Repeat many times
• Spectrum & decay correspond to focus ~200 nm
R6G in
Membrane
DCM in
Cytoplasm
DAPI in
Nucleus
Image of Chinese Hamster Ovary (CHO) Cell
Sasmal, KB Langmuir 29 (2013) 2289
Shirshendu, KB Langmuir 29 (2013) 7995
DAPI in Nucleus and
DCM in Cytoplasm
540 600 660 720
0
2k
4k
6k
8k
Wavelength (nm)
Em
. In
ten
sit
y
400 450 500 550 600
0.0
0.2
0.4
0.6
0.8
1.0
Wavelength (nm)
Em
. In
ten
sit
y
Emission Spectra of DCM & DAPI in CHO Cell under a Microscope
Ethanol
2-Propanol
1:1
CH3OH-
water
Cytoplasm DAPI
Water
• Polarity of Cytoplasm is ~ 2-propanol, ~ 15
• Polarity of Nucleus is ~ 1:1 CH3OH-H2O mixture, ~ 65
DCM Nucleus
Sasmal, KB Langmuir 29 (2013) 2289
Conformational Dynamics in Protein
• HSA covalently labeled at cys-34 by the dye, CPM
• During conformational dynamics quenching of CPM
emission by NH2 group (in tryptophan & other residues)
H
S -SH
Cysteine
Sasmal, KB JPC B 115 (2011) 13075
Suprotik, KB JPC B 116 (2012) 12189
0 1k 2k 3k 4k 5k
0.0
0.2
0.4
0.6
0.8
1.0
Wavenumber (cm-1)
C(t
)
Nucleus
Cytoplasm
Av. Solvation time
• Cytoplasm: 1300 ps
• Nucleus: 750 ps
• Bulk water: 1 ps
Solvation Dynamics in a live CHO cell under a Microscope
Sasmal, KB Langmuir 29 (2013) 2289
Conclusion
Gene Silencing involves
• Initial search for target gene (~ 60 s)
• does not occur in one shot
• Several cycles of unbinding & rebinding
• On-time: odd-integral multiple of 5.5 ms
• Silencing occurs in ~160 s
• Stochastic Resonance
IACS
• Cancer Cell: How Different ??
• Cancer cell: Increased glycolysis,
pyruvate, lipid
• Lipid droplet (LD): ~10-fold higher
in number in a Cancer cell, A549
450 500 550 600 6500.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 Lipid Droplet
C153 (CHO)
C153 (A549)
Inte
nsit
y
Wavelength (nm)
4 m
4
5 6
1 2
3
Z = 10 m above surface
A
• Dye C153 only
in membrane
• 4-6: near water
• 1-3: deep in wall
4 m
4
5 6
1 2
3
Z = 10 m above surface
A
• Dye C153 only in membrane
• 4-6: near water
• 1-3: deep in wall
Average solvation times
• 1200 ps in buried region 1 ( = 2000 cm-1)
• 750 ps in 6 near water pool ( = 1300 cm-1)
Solvation Dynamics under a Microscope:
Single Giant Lipid Vesicle
10 m
A few Molecule: How to record ?
• Emission Spectrum
• Fluorescence Decay
• Each time collect one photon
& then repeat many times
• C153 in DLPC (Fluid phase)
Supratik, KB, Langmuir 28 (2012) 10230
Size of a Protein in Native & Non-native States
• Cytochrome C
• Molten globules (MG): one with SDS; another with urea & SDS
• Effect of Ionic liquid on size of Native, denatured & MG
Conformational Dynamics in Protein
• HSA covalently labeled at cys-34 by the dye, CPM
• During conformational dynamics quenching of CPM
emission by NH2 group (in tryptophan & other residues)
H
S -SH
Cysteine
Sasmal, KB JPC B 115 (2011) 13075
Suprotik, KB JPC B 116 (2012) 12189
I║ ┴
Basic Design of Confocal Microscope
TSUNAMI
Pratik
IIT Kanpur
Prof GS
Agarwal
Kalyan
IIT Guwahati
Saptarshi
IISER-Bhopal Durba
BITS/
Presidency
Sudip, Visva-Bharati
University
(A)
N N
Br
(B) (C)
O
N
O
N
O
CH3
N
CH3
CH3
CPM
CPM-HSA
Ionic liquid
Rh (Ǻ)
native 38
1.5 M Ionic liquid 61
6 M GdnHCl 61
1.5 M RTIL + 6M GdHCl 41
• Ionic liquid unfolds native HSA: size increase
• Ionic liquid reduces size of HSA denatured
by GdnHCl
• Hydrophobic Collapse in presence of two
Denaturing agents
Sasmal, KB J Phys. Chem. B 115 (2011)13075
Effect of Ionic liquid
on Size of HSA
Lysosomes
• waste disposal system of the cell, digests biomolecules
• spherical vesicles containing > 50 hydrolytic enzymes
• acidic environment of about pH 5
• discovered by Christian de Duve, Nobel in 1974.
LYOSOSOME Cytoplasm Nucleus
Cargo
Building
Blocks
Lytic
Enzymes
Lung Cancer cell (A 549)
• HPTS and lysotracker dye localizes in same place
• HPTS located in lysosome
• Dt (in bulk water) = kT/6r =350 m2 s-1
• Normal Lung cell, Dt = 12 m2 s-1 ~ 30 cP
• Lung Cancer cell, Dt = 8.5 m2 s-1 ~ 40 cP
• CHO (cytoplasm) Dt = 15 m2 s-1 =~25 cP
• Viscosity of Lysosome: First measurement
HPTS
Bulk
water
CHO Cell
Cytoplasm
Fluorescence Correlation Spectroscopy
HPTS
Rajdeep, KB
JPC B (PPT Spl issue)
• Rotational Relaxn. in water (200 ps)
• Rot. Relaxn. in Cancer cell (1350 ps)
• Rotational Motion ~7 times slower
• FCS: transl. diffusion 40 times slower
• FCS & anisotropy decay probe
different motions & length scale
HPTS in live cell
under microscope
Rajdeep, KB
JPC B (PPT Spl issue)
Anisotropy Decay in a live Cell under a microscope
• No Power Law !!
Rajdeep, KB JPC B (PPT Spl issue, 2014)
Solvation Dynamics in a Live Cell: Under a Microscope
• Pure water: ~ 1 ps
• Lung Cells: Med Chem Comm (2014)
Cytoplasm: Normal (800 ps); Lung cancer cell (1000 ps)
Lipid droplets: Normal (1700 ps); Lung Cancer cell (3700 ps)
Nucleus: Normal (450 ps); Lung Cancer cell (300 ps)
• Similar slow solvation in Mitochondria, Membrane of other cells
Langmuir (2013 & 2013), JPC B (2014 & 2014)
Lung Cancer Cell
Normal Lung Cell
Factors controlling Proton Transfer
• Solvation Dynamics: slow inside a cell
• Number of “free” water molecules: Few inside a Cell
• Slow ESPT inside lysosome (compared to bulk water):
caused by slow solvation & lack of “free” water
Chemistry Nobel 2013 & A Missed Indian Hero
Aneesur Rahman Born: 1927 (Hyderabad) Died: 1987 (Minnesota)
M. Karplus M. Levitt A. Warshel
“for the development of multiscale models (Computer
Simulation) for complex chemical systems”
• Tripos, Cambridge: Math (1948), Phys („49)
• DSc: 1953, Leuven, Belgium
• Osmania Univ: 1953-57, Lecturer
• TIFR: 1957-60, Fellow
• Argonne: 1960-1985
• Father of Molecular Dynamics Simulation (PR A, 1964)
“Motion of atoms in liquid Ar” 864 Ar atoms on a CDC 3600
• Collaborators: Parinello, Klein, ….
• Micro-canonical ensemble approach to lattice gauge theory
• 1985: shifted to Minnesota for better treatment of cancer
“Accurate simulation of protein dynamics in solution,”
LEVITT* & SHARON PNAS 85 (1988) 7557-7561
“The first simulation of crystals of bovine pancreatic trypsin inhibitor (BPTI)
lasted for only a few ns(7)”
7. Hermans, J. & Rahman, A. (1976) in Models for Protein Dynamics, ed.
Berendsen, H. J. C. (CECAM, France), pp. 153-158.
Scientists who missed Nobel for Single Molecule
• Rotman (1961): hydrolysis of sugar by single enzyme
under microscope by fluorescence of dye liberated
• Magde, Elson & Webb (1972): FCS
• Keller (1983) : LIF to detect single molecule
• Bjorklund (1980): Frequency-Modulation Spectroscopy
used by Moerner (1989) for single molecule absorption
• Oritt (1990): Single molecule by fluorescence
PULSED LASER Powerful, TSUNAMI!!
Peak Power
= Pulse energy/duration
= 10-9 J/10-15 sec
= 106 (Mega) Watt !
CW LASER
Time Time
I
MODE LOCKING
RAMAN-NATH 1935
Amplified,
x 106 1012
(Tera watt)
Indian Pioneer in Laser
• invented carbon dioxide laser in 1963
• used for cutting & welding metal
• as a laser scalpel in surgery
• laser skin resurfacing
• military range finding C. Kumar N. Patel
(1938-)
TSUNAMI
Prof GS
Agarwal
2014 Physics Nobel
“Invention of efficient blue LEDs (for) white light sources”
“Incandescent light lit 20th century; 21st century will be lit by LED”
Akasaki, Amano (Nagoya), Nakamura (Nichia-Santa Barbara)
• Red & green LEDs (1960), but NO white light without blue LED
• Amano’s 1500 failures to grow GaN crystals
• Thin layer (30 nm) of AlN nucleated on sapphire at 500 C & then
heated up to 1000 C (growth temp of GaN, APL, 1986)
Prasenjit, Surajit Ghosh, KB PCCP 17 (2015) 6687
220 ms 430 ms