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Hyperspectral Microscope based on

Imaging Fourier transform Spectrometry

Dr. Li Jianping, Claude

jp.li@siat.ac.cn

June 2017

The Claude Research Group

Copyright: Claude

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Outline

Spectral Imaging

Fourier transform Imaging Spectroscopy

A general-purpose FTIS instrument

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• 2D-Imaging

• 1D-Spectroscopy

Spectral Imaging (Imaging Spectroscopy)

Acquisition timeTime Resolution

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Why Spectral Imaging?

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Applications of Spectral Imaging

• Astronomy

• Remote sensing

• Meteorology

• Oceanography

• Mining geology

• Mineralogy

• Agriculture

• Surveillance

• ……

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Applications in Medicine, Biology and Chemistry

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Modalities of Spectral Imaging

Imaging Spectroscopy

• Microscopy

• Photography

• Telescopy

• Reflection

• Absorption

• Transmission

• Scattering

• Raman

• Fluorescence

• Electroluminescence

• Chemoluminescence

• Bioluminescence

• ……

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Methods for Spectral Imaging

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Fourier Transform Spectrometer (FTS)

0

0

( ) ( ) ( ) ( ) cos 2I x I x I x B xd

2 exp 2B I x j x dx

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Fourier Transform Imaging Spectrometer (FTIS)

( , ) ( ) cos(2 cos ')p pI x B x d d

2 , exp 2c pB I x j x dx

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Throughput (Jacquinot) Advantage

• FTSs can have more than 60 times higher energy-

gathering capability than grating spectrometers for the

same resolving power and similar instrument size.

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Multiplex (Fellgett) Advantage

• FTS simultaneously observes all the spectral information from the

entire range of a given spectrum during a scan period.

FT

Mixed

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Wavenumber (Connes) Advantage

• The wavenumber scale of an FTIR is derived from a He-Ne laser fringe that

acts as internal references for sampling positions in each scan. Therefore,

the wavenumber calibration of FTS is much more accurate and has much

better long-term stability than the calibration of dispersive instruments.

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High and Variable Spectral Resolution

Instrumental lineshape:

11/ 2 (cm )L

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Wide Spectral Range

• Nyquist limit wavelength

• System spectral transmission response

▫ Detector spectral response

▫ Optics spectral characteristics

min 2 x

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A UV-Vis-NIR FTIS

Setup of the UV-Vis-NIR FTIS based on the beam-folding position-tracking technique

Jianping. Li, Robert. Chan, and Xuzhu. Wang, Tests of a practical visible-NIR imaging Fourier transform spectrometer for biological and chemical fluorescence emission measurements. Opt. Express, 2009. 17(23).

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Prototyping and testing (Gen1)

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Testing Summary

Mode Determinants/Typical value

Spectral Resolution

Spectral Range

ImageResolution

Spatial Resolution

Temporal

Hyp

ersp

ectr

al-

imag

ing

Determinant Max. OPDΔδx + Camera QE

Camera speed + PZT speed

Diffraction-limitedCamera specs

Camera speedImage resolutionSpectral resolutionSNR requirement

Typical valueMax.~10cm-1

Tunable~360-900nm

Max.300x300pixels

500nm50x, NA=0.55

20s200x200pixels, 512frames

On

ly-i

mag

ing Determinant Camera QE Camera Pixel #

Diffraction-limitedCamera specs

Camera Speed

Typical value 200-1100nm1004x1002pixels (Cascade 1k, Photometrics)

500nm50x, NA=0.55

10fps@ full frame

Jianping. Li, Robert. Chan, and Xuzhu. Wang, Tests of a practical visible-NIR imaging Fourier transform spectrometer for biological and chemical fluorescence emission measurements. Opt. Express, 2009. 17(23).

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Instrumental Reconfigurability

Transmission Reflection

EPI-Fluorescence TIR-Fluorescence

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Prototype Gen2

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Absorption

0

( )( ) log[ ( )] log[ ]

( )I

A TI

Beer’s law:

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Reflection

Mixed SCCBs with red, green and blue color and common glass beads

JP. Li and Robert Chan. Towards a UV-Vis-NIR Hyperspectral Imaging Platform for Fast Multiplex Reflection Spectroscopy, Opt. Lett., 2010. 35(20): p. 3330-3332.

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502nm, 550nm and 630nm

JP. Li and Robert Chan. Towards a UV-Vis-NIR Hyperspectral Imaging Platform for Fast Multiplex Reflection Spectroscopy, Opt. Lett., 2010. 35(20): p. 3330-3332.

512 frames, 100×100pixels, binning =8. total acquisition time is only about 10s.

SCCB decoding speed of about 100beads/s.

Reflection: SCCBs

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Fluorescence

Li, J., R.K.Y. Chan, and X. Wang, Tests of a practical visible-NIR imaging Fourier transform spectrometer for biological and chemical fluorescence emission measurements. Opt. Express, 2009. 17(23).

50x, 100x100 pixels, binning=8, 5ms exposure time, 1k frames, total acquisition time ~40s

Dye-loaded fluorescent polystyrene nano-beads (φ200nm)

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TIRF

Jianping Li and Robert Chan, “Two-Mode Total Internal Reflection Fluorescence Hyperspectral Microscopy”, Focus on Microscopy, March, 2010, Shanghai.

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Towards a general-purpose Hyperspectral Microscopy System (Gen3)

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Design Considerations

Integration with standard microscopes1

Equip with faster and larger-chipped camera2

Equip with GPU based parallel FFT processing3

User-friendly software GUI development4

Versatile SI platform for analytical sciences5

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The real HSM system

Jianping Li and Yi Xiao, “GPU accelerated parallel FFT processing for Fourier transform hyperspectral imaging”, Applied Optics (IF: 1.784), 54(13): p. D91-D98, 2015.

SoftwareHardware

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Optical Physical

Performance and specs

• Spectral resolution (max)：~0.4nm

• Spatial resolution (max)：~0.5m

• Spectral range：~360-1000nm

• Pixel number: 2560x2160

• Instrument size: • 30×50×28mm （L×W×H）

• Objective lenses：• 5x,10x,20x,50x,80x BDF-RT-PL• 10x, 20x, 40x Pha

• Host computer: • Intel i7-3820CPU• NVIDIA GeForce GTX650 GPU• 64GB RAM• 2TB HDD

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ApplicationsBright-field (transmission)

Bright-field (phase-contrast)

Bright-field (reflection)

Dark-field (scattering)

Electroluminescence

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Technological Comparison

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FeaturesExcellent spatial & spectral resolving

High & adjustable spectral resolution and wide spectral range

High & adjustable spatial multiplex and resolution

No spatial resolution degradation

Good accuracy

High measurement throughput

High optical throughput

Rapid image acquisition

Parallel FFT processing

Operational

convenience

Multi-modality imaging

Flexible hardware adaptability

“WYSIWYG” preview function

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Acknowledgement• Dr. Robert Chan,

▫ Dr. XZ Wang

▫ Dr. JX Fu,

▫ Mr. R Li

▫ Mr. ZN Xu @HKBU

• Dr. XW Zhao and Prof. ZZ Gu @SEU

• Prof. JF Wang @CUHK

• Prof. B Ren @XMU