Simultaneous Multiple Single Molecule Detection through Fluorescence Imaging

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Simultaneous Multiple Single Molecule Detection
through Fluorescence Imaging
Alan I Lee University of California, Irvine The
Henry Samueli School of Engineering Department of
Biomedical Engineering February 16, 2005
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Motivation
Most of the single molecule detection can only
detect one or a few molecules (n lt 10)
simultaneously
Statistical study provides the missing link
between single molecule study and ensemble bulk
study
Time consuming for gathering a population large
enough for statistical studies
A method for observing multiple (n gt 30)
simultaneous single molecule is needed
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Why Single Molecule studies?
Individual statistics are hidden within the
ensemble value
Detection of reaction intermediates
Determination of relationship between structure
and function
Determination of reaction dynamics
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Various Types of Single Molecule Detection
Atomic force microscope
Confocal fluorescence microscope
Fluorescence resonance energy transfer (FRET)
Fluorescence correlation spectroscopy (FCS)
Capillary electrophoresis based
Emulsion based
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Experimental Concept for Emulsion Based Assay

Aqueous phase with substrate but no enzyme
Oil Phase
Aqueous phase with enzyme and substrate
1 mm
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Advantage of the Emulsion-based Assay
Large number of individual reaction droplets
simultaneously
Observation of multiple individual molecules over
time
Easy setup, no costly equipments necessary
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Distribution of enzyme molecules at 1mm droplet,
enzyme concentration _at_ 0.38nM
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Bovine a-chymotrypsin
Homodimer, has a molecular weight of 25 kD
Belongs to the family of serine proteases
Cleaves the polypeptide chain after a tyrosine
(Y), phenylalanine (F), or tryptophan (W) residue
Use as a model enzyme for studying the
effectiveness of the single molecule assays
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The Substrate for a-chymotrypsin
Bis-(succinoyl-L-alanyl-L-alanyl-L-propyl-L-phenyl
alanyl amide) Rhodamine 110
Excitation wavelength 498 nm Emission
wavelength 521 nm
http//www.probes.com/media/pis/mp06501.pdf
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Experimental Setup
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MetaMorph 6.0
LabView 6.0
Image from each time point are aligned for
position
A simple algorithm used to match the position of
the spots from each time point
Each image is thresheld through intensity
Only spots presented in all time points will be
kept
The thresheld spots are counted and the spots
with sizes larger than 1mm are filtered
The intensities for the spots are organized
according to time
The position of the spots and the intensity of
the spots are exported to MicroSoft Excel for
recording
The rearranged data are exported back to Excel
and ready for further analysis
The Excel file is ready for further sorting
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Traces of 96 individual spots
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Mean 89.43 Standard Deviation 6.01 Kurtosis
2.29 Coeff. Var. 6.73
Mean 95.11 Standard Deviation 8.21 Kurtosis
0.88 Coeff. Var. 8.63
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1550 individual spots counted by MetaMorph 6.0
160 individual spots counted by MetaMorph 6.0
Only the droplets contain enzyme (20S
proteasome) will also have blue fluorescence due
to substrate cleavage
All droplets contains green fluorescent dye
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Colocalization image (160 of 1550 droplets
contain 20S proteasome reaction as indicated by
blue fluorescence)
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Conclusion
The individuality for each single molecule is
averaged out in a bulk population
The kinetics of a bulk population can be
represented with the average of a small amount of
individual kinetics
Emulsion based single molecule enzymatic assay is
efficient and suitable for large scale screening
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Acknowledgement
James P. Brody, Ph.D. Wajeeh Saadi Mike
Yung-Chieh Tan
Funding
NSF CCR-0304612