Block 05 - Resolution

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Resolution of images
FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk
Curve resolution methods
Curve Resolution introduction. Spectral unmixing
Focused on recovering the true underlying spectroscopic model out of the raw
recorded image
It uses the most common low in the nature
Signal = constant * concentrarion
In chemistry
Intensity = m/z * concentrarion

Absorbance = 𝜺 * concentrarion
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For each pixel, in one wavelength 

Absorbance = 𝜀1 * C1 + 𝜀2 * C2 + .... + 𝜀n * Cn
For each pixel, in ALL wavelength 

Absorbance = 𝜀 * C1 + 𝜀 * C2 + .... + 𝜀 * Cn
Pure spectrum
For ALL pixel, in ALL wavelength 
Absorbance = 𝜺 C
Component 1 Component 2 Component 3
D = CST + E y y y
x x x
Mutivariate Curve Resolution (MCR)
* Figure extracted from Gendrin et al. European Journal of Pharmaceutics and Biopharmaceutics, 68 (2008) 828
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Multivariate Curve Resolution (MCR)
Multivariate Curve Resolution - MCR
Coming back to PCA 
MCR general model  D = CST + E
PCA general model  D = TPT + E
What is the difference???
Multivariate Curve Resolution – MCR vs. PCA
SCORES
MCR-ALS
PCA
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SCORES
Score value: in PCA the values are “arbitrary”. In MCR the values represent
the relative spectral intensity (influence) of that particular component. Relative
MCR-ALS
concentration.
Negativity: in PCA there are negative values. In MCR it is complicated to

find negative relative concentrations.
appearance: in PCA, a score surface does NOT have to correspond to a

pure chemical compound. It is the difference between sources of variance. In
MCR, each score surface represents one single chemical compound.
Explained variance: in PCA, the explained variance is nested and it

PCA
corresponds to a principal component. In MCR, it does not need to be nested

and we do NOT talk about individual explained variance. We talk about a
global explained variance. In this plot, the variance represents the relative
importance of that compound IN THE MODEL.
LOADINGS
MCR-ALS
PCA
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LOADINGS
Loading value: in PCA the values are “arbitrary”. In MCR the values
MCR-ALS
represent the relative molar absorptivity. It represents what is called “pure

spectrum”.
Negativity: in PCA there are negative values. In MCR there might be

negative values, depending on the pre-processing.
appearance: in PCA, loadings do NOT have to correspond to a pure

chemical compound. It is the difference between sources of variance. In MCR,
PCA
each loading represents one single chemical compound.
PCA components are ORTHOGONAL (not correlated variability)

MCR components are based on linear dependency between concentration and
pure signal
PCA aims to explain SOURCES OF VARIABILITY  Rank
MCR aims to offer REAL physicochemical understanding of the process/kinetic

reaction by using the ALREADY KNOWN RANK  What is the chemical rank of
my system?
PCA solution is nested with sign ambiguity

MCR has many MATHEMATICAL SOLUTIONS (AMBIGUITIES)
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Workflow. MCR-ALS
MCR works by iterations: Alternating Least Squares D = CST + E

Alternating solution of CST in a least square sense:
min X PCA  ĈŜT or min X PCA  ĈŜT

Ĉ Ŝ
Before starting the iterations we need to:

Estimate the rank. Usually calculated with PCA and EWFA
Give initial estimates. Usually spectral profiles / SIMPLISMA
Workflow. MCR-ALS
MCR works by iterations: Alternating Least Squares D = CST + E
1. Given D and STinit, then calculate Ĉ 4. Convergence criteria
Ĉ  X PCA (S Tinit )  
cc  Ĉ Ŝ T  it  i 
 Ĉ Ŝ T 
it  i 1
T Depending on the quality of your data

2. Given D and Ĉ , then calculate Ŝ
Ŝ T  Ĉ  X PCA 5. Go to step (1) until convergence
3. Reproduce D̂ 6. Residuals
D̂  Ĉ Ŝ T E  D  D̂ cc
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Constraints
In practice
Iterations by approximation  Tell me two integers that their product is 99
There is no any restriction  Tell me two integers that their product is 100
AMBIGUITIES!!!
Rotational  D  CS T  CRR 1S T  C ' S'T

1 T
Intensity  D  CS T  C  S  C ' S' T

CONSTRAINTS!!!
Constraints
NONNEGATIVITY  C and ST
WITHOUT NONNEGATIVITY WITH NONNEGATIVITY

0.6 0.6
0.5 0.5
0.4 0.4
0.3 0.3
Absorbance
Absorbance
0.2 0.2
0.1 0.1
0 0
-0.1 -0.1
200 220 240 260 280 300 200 220 240 260 280 300
wavelengths (nm.) wavelengths (nm.)
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Constraints
UNIMODALITY  C and ST
WITHOUT UNIMODALITY WITH UNIMODALITY

0.6 0.6
0.5 0.5
0.4 0.4
Absorbance
Absorbance
0.3 0.3
0.2 0.2
0.1 0.1
0 0
200 220 240 260 280 300 200 220 240 260 280 300
wavelengths (nm.) wavelengths (nm.)
Constraints
CLOSURE  C (MASS BALANCE)
WITHOUT CLOSURE WITH CLOSURE

1.4 1.4
1.2 1.2
1 1
CONCENTRATION
CONCENTRATION
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0 0
0 20 40 60 80 100 0 20 40 60 80 100
TIME TIME
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Constraints
SELECTIVITY  C and S
Assessment of the results
Residuals  Depending on
Structured residuals
S/N ratio expected
Comparison with the unexplained variance by PCA
High % of unexplained variance by PCA  Not appropriate model

Check No. Components
% Var. MCR NEVER is lower than % Var. PCA
Appropriate model  % Var. MCR = % Var. PCA
If % Var. MCR much higher than % Var. PCA  OVERFITTING

Check Constraints
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Assessment of the results
Lack of fit:
I J
e 2
mn
% LOF  100  i
I J
j
d
i j
2
mn
More information 
R. Tauler, M. Maeder. Two-Way Data Analysis: Multivariate Curve

Resolution – Error in Curve Resolution. Comprehensive Chemometrics,
Chap. 2.20
Multi-set MCR
Multi-Image MCR-ALS
Possibility of analyzing many images together (like in PCA)
Minimizing a common drawback in Image Analysis  Lack of selectivity on

the surface  Specially useful in Pharmaceutics
Development of quantitative models!!! How?
What we obtain with MCR are spectral intensities!!! Still, for a ROBUST and
RELIABLE quantitative model, we DO NEED calibration!!!!
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Multi-set MCR
Multi-Image MCR-ALS
Quantitative models
a)
siT
ci
= ST T
 ci si
s.c.i  100 
D C  ci si
T  CST
T
 CS (individual signal)
(global signal)
b)
Absolute quantitation
Image 1 cik
ST
Image 2
Image 3
=
Image n Bulk reference concentrations
D C
c i1 c i2 c i3 c in
Im.1 Im. 2 Im. 3 Im. n
c i1 c i1 c i1 c i1
   
c i1 c i2 c i3 c in Relative quantitation
* Figure belonging to Prof. Anna de Juan
Multi-set MCR
MCR-ALS. Some examples
Detection of semen stains in fabrics
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Multi-set MCR
Quantifying Lorazepam in real commercial tablets
Multi-set MCR
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Multi-set MCR
Multi-set MCR
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Multi-set MCR
MCR-ALS. Benefits and drawbacks
Obtaining relative concentrations and pure spectra  Libraries to compare
Resolving mixtures
Quantitation with the multi-set images
Few samples are required for constructing a good calibration
Secon order property: Calibration in presence of interferences
It is not simple to use
Extreme care with constraints
You need to know the rank
Extreme care with the initial estimates
Not very good with NIR in Foodstuff (especially with samples with water)
Multi-set MCR
10 steps to make MCR-ALS models on images
1. Buy a new computer
2. Time and patient
3. UNDERSTAND YOUR SAMPLE and TARGET
4. Visualize your iamge and think of pre-processing
5. PCA for rank
6. Perform an MCR without constraints
7. Think about constraints (the fewer the better)
8. Check if we can publish. Otherwise, come to 4
9. SAVE ALL RESULTS and extract conclussions
10. Get a beer or wine to celebrate that we start now talking about clusters!
14

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19/11/2017

FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk

Curve resolution methods

Curve Resolution introduction. Spectral unmixing

It uses the most common low in the nature

Signal = constant * concentrarion

Intensity = m/z * concentrarion

FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk

Curve resolution methods

Curve Resolution introduction. Spectral unmixing

For each pixel, in one wavelength 

For each pixel, in ALL wavelength 

For ALL pixel, in ALL wavelength 

FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk

Curve resolution methods

Curve Resolution introduction. Spectral unmixing

Component 1 Component 2 Component 3

Mutivariate Curve Resolution (MCR)

FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk

Multivariate Curve Resolution (MCR)

Multivariate Curve Resolution - MCR

Coming back to PCA 

MCR general model  D = CST + E

PCA general model  D = TPT + E

What is the difference???

FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk

Multivariate Curve Resolution (MCR)

Multivariate Curve Resolution – MCR vs. PCA

FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk

Multivariate Curve Resolution (MCR)

Multivariate Curve Resolution – MCR vs. PCA

Negativity: in PCA there are negative values. In MCR it is complicated to

appearance: in PCA, a score surface does NOT have to correspond to a

Explained variance: in PCA, the explained variance is nested and it

corresponds to a principal component. In MCR, it does not need to be nested

FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk

Multivariate Curve Resolution (MCR)

Multivariate Curve Resolution – MCR vs. PCA

FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk

Multivariate Curve Resolution (MCR)

Multivariate Curve Resolution – MCR vs. PCA

represent the relative molar absorptivity. It represents what is called “pure

Negativity: in PCA there are negative values. In MCR there might be

appearance: in PCA, loadings do NOT have to correspond to a pure

each loading represents one single chemical compound.

FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk

Multivariate Curve Resolution (MCR)

Multivariate Curve Resolution – MCR vs. PCA

PCA components are ORTHOGONAL (not correlated variability)

PCA aims to explain SOURCES OF VARIABILITY  Rank

MCR aims to offer REAL physicochemical understanding of the process/kinetic

PCA solution is nested with sign ambiguity

FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk

Multivariate Curve Resolution (MCR)

MCR works by iterations: Alternating Least Squares D = CST + E

min X PCA  ĈŜT or min X PCA  ĈŜT

Before starting the iterations we need to:

FOOD, U. Copenhagen, Denmark. www.models.life.ku.dk www.hypertools.org jmar@life.ku.dk

Multivariate Curve Resolution (MCR)

MCR works by iterations: Alternating Least Squares D = CST + E

1. Given D and STinit, then calculate Ĉ 4. Convergence criteria

T Depending on the quality of your data