Rayleigh criterion for spatial resolution - NSRRC User Portal

Rayleigh criterion for spatial
resolution
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The Rayleigh criterion is the generally accepted criterion for the
minimum resolvable detail - the imaging process is said to be
diffraction-limited when the first diffraction minimum of the image of
one source point coincides with the maximum of another.
Circular aperture
Single slit
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=1.22λ/(2N. A. )=0.61λ/nsinθ
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Calculating Diffraction:
r=1.22λ/(2N. A. )=0.61λ/nsinθ
d
For 1469 cm
(1469 cm -1
d
1.22 λ
N.A.obj.
-1
1.22(6.8 μm)
0.58 0.71
,
N.A.cond.
6.8 μm)
6.4 μm
When calculating diffraction the Numeric Aperture of the objective and condenser as
well as the wavelength of interest. The Numerical Aperture is calculated as:
N.A = n sin (u) (where n = refractive index of the medium and u = the semi-field
angle of the optical device.
In general, the higher the Numerical Aperture, the better. The drawback of high
Numeric Aperture is a small working distance and a very small depth of view.
49

Focal Plane Array (FPA) Detectors
for Full-Field Imaging
There are a variety of Detector arrays, including
MCT, InSb, PtSi, Si, Si:As etc.
The array sizes vary from 16 x 16, up to 1024 x
1024 pixels
320 x 240 Si:As
128 x 128 MCT
1024 x 1024 InSb
64 x 64 MCT
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Full-Field FT-IR imaging system
Hyperion 3000
FFT
Optical layout of FPA-base FT-IR imaging system
51

Focal Plane Array (FPA) Detectors
for Full-Field Imaging
“The missile is equipped with an imaging
infrared seeker which is based on mercury
cadmium telluride (HgCdTe) Focal Plane
Array technology in the long wave infra-red
band at wavelength 8 to 12 microns of the
electromagnetic spectrum.”
The MCT 64 x 64 element array was developed for the Javelin anti-tank
missile program
These arrays are only a ‘reasonable’ price because of the number being
manufactured for this program
52

II. Background and Motivation
Infrared spectra are obtained from individual living cells.
• Lipids (cell walls)
• Nucleic acids (DNA, RNA)
• Proteins
• Each of these major
classes of cellular
components have
distinct IR markers
53

Chemical Features of Biological Components
Amide A
Amide B
54

DNA/RNA
n s
(C-O-C)
55

II. Background and Motivation (Cont.)
1. Higher An as CH 2 / An as CH 3 (lipid/
protein ratio) was found in the
cancer tissues than that of Normal
tissues.
2. The ratio could be affected by
different method of fixation for
tissue sample.
3. IR spectrum marker is also
strongly disturbed by different
sample preparation.
Normal
Cancer
R. K. Sah, Journal of Biomedical Optics, 10(5), 054017 (2005)
J. G. Wu, Biospectroscopy 62, 185 (2001)
56

II. Background and Motivation(Cont.)
Fig.1
Fig.3
Fig.2
(iii)
(ii)
(i)
Fig. 1. Raman of pure paraffin wax
Fig. 2. Raman spectra comparing (i) paraffin
wax, (ii) frozen tissue section of cervical
cancer and (iii) dewaxed FFPP (Formalin
Fixed Paraffin Processed) tissue section of
cervical cancer.
Fig. 3 Raman spectra after each subsequent
dewaxing cycle using xylene.
57

2.4
2 hr xylene washing
Malignant part of tissue section
T 1
T 2
Normal colon tissue
N 1
Absorbance
Colon cancer tissue
T 1
0.0
N 2
N 3
Absorbance
1.8
1.2
0.6
N 1
2.4
Normal part of tissue section
Wavenumbers/ cm -1 N 2
1.8
N 3
1.2
0.6
0.0
3500 3000 2500 2000 1500 1000
T 3
T 2
T 3
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Construction of Spectral Image
0 . 3
0 .1 5
P a ra ffi n
B e e s w a x
Absorbance
0 . 2
0 . 1
Absorbance
0 .1 0
0 .0 5
0 .0 0
IR spectrum of cell sample
3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0
W a v e n u m b e r/c m -1
0 . 0
3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0
W a v e n u m b e r s / c m
- 1
Reference Image
Before Treatment
Cell sample
Cells image
After Treatment
The area beneath the spectral curve was integrated in the range of 3000-
2800 cm -1 , being the absorbance of wax residual of cell sample and
collected to construct spectral image to be identify disease stage of cell.
59

III. Experimental Strategies:
Part 1. Bias-assisted Wax Physisorption using
FTIR imaging for Cancer Diagnosis
Part 2. Cancer Therapy by using Alternating
Electric Fields
60

III. Part 1. Bias-assisted Wax Physisorption
Kinetics using FTIR imaging for Cancer Diagnosis
nHOK /OECM-1 /SCC-15 /SCC-25 /OC-2 / OC-3
@ G1 phase
Step 2 Step 3
A
Alkanes(C n H 2n+2 )
(N=22,23,24,25,26,28,
30,32,34)
Cells fixed on Low-e silde
Step 1
B
Paraffin
(C 25 H 52 )
Beeswax
(C 46 H 92 O 2 )
C. Deglycosylation
D. Hydrolysis
Enzyme
Waxing
for 2 min
Waxing and Dewaxing
Stand and dry
for 10 min
Dewaxing
for 5 s
FT-IR imaging
128 scans, 8 cm -1
Xylene
Washing
E. + - Bias
+
E E = V/d
d=0.5 cm
V= 10..300
Focal-plane-array-based Synchrotron-based
FT-IR imaging system
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+
+++++
+
+++++
Part II. Innovative Cancer Therapy by using
Alternating Electric Fields
Interfering biological
processes within cancer cells
Apoptosis
FTIR analysis of AEF
treated cancer cell
_ _ _ _ _ _ _ _ _ _ _
Absorbance
1.0
0.8
0.6
0.4
0.2
AEF treatment of SCC-15
25o kHz,E= 2 V/ cm treatment of SCC-15
Amide A
Lipid
(V S CH and V as CH)
Amide B
Vs C=O
Amide I
Amide II
DNA/RNA
FTIR imaging
apoptotic cell
0.0
3500 3000 2500 2000 1500 1000
Wavenumber/ cm -1
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VI. Results and Discussion
Treatment A:
Wax Residual for Oral Cells by using alkane with different chain length as
diagnostic agent (22C~34C) to pick out a suited alkane for differentiating
malignancy from sample
Aborbance (ABS)
400
320
240
160
80
nHOK SCC-25 SCC-15
OC-2 OC-3 OEC-M1
Pentacosane
Triacontane
Beeswax
0
C22H46 C23H48 C24H50 C25H52 C26H54 C27H56 C28H58 C30H62 C46H92O2 C32H66 C34H70 --
The chain length of alkanes
*Absorbance of averaged wax residue is divided by no. of carbon atom and cells
63

Treatment B:
Paraffin and Beeswax as diagnostic agents
Normal Oral Cells
Reference image
Oral Cancer Cells
The results of kinetic FT-IR imaging of Normal Oral cells showed a stronger
capability of physisorption with paraffin than that of beeswax.
The Oral Cancer cells showed a greater capability for adsorbing beeswax than
that of paraffin.
1. Lee et al., Method for detecting cancer and reagents for use therein, USA patent,
US-8-354,222 B2 (2013); Japan patent, 119309 (2013)
2. Chiu et al., Anal. Bioanal. Chem. 405 , 1995 (2013)
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Relative Paraffin Residues used as diagnostic agent
for differentiating Colorectal Normal from Malignancy
CCD-18Co
Xylene washing
60 min
5 s
Deparaffining
10 s
50 mm
SW-480
Paraffining
2 min
SW-403
65

Relative Beeswax Residues used as diagnostic agent
for differentiating Colorectal Malignancy from Normal
CCD-18Co
Xylene washing
60 min
5 s
Debeeswaxing
10 s
50 mm
SW-480
Beeswaxing
2 min
SW-403
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