HYPERSPECTRAL MICROSCOPE PLATFORM FOR HIGHLY MULTIPLEX BIOLOGICAL IMAGING. Marc Verhaegen

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1 HYPERSPECTRAL MICROSCOPE PLATFORM FOR HIGHLY MULTIPLEX BIOLOGICAL IMAGING Marc Verhaegen CMCS, MONTREAL, MAY 11 th, 2017

2 OVERVIEW Hyperspectral Imaging Multiplex Biological Imaging Multiplex Single Particle Tracking of Different Neuronal Receptor Subtypes NIR Imaging in the 2nd Biological Window 2

3 Hyperspectral Imaging

4 WHY HYPERSPECTRAL IMAGING? Color image (Reflectance, Absorbance, Fluo, Raman, ) Add the spectral dimension Hyperspectral Cube 4

5 HYPERPSECTRAL IMAGING ACQUISITION MODES SINGLE POINT / LINE SCAN GLOBAL IMAGING GLOBAL IMAGING: Diffraction Limited MPixels Images 1 Megapixel Camera = 1 MILLION SPECTRA 5

6 HYPERPSECTRAL IMAGING LIVE 6

7 IDENTIFICATION OF CEREALS Various beer grains identified with their spectral signature Applications: - Agriculture - Sorting of cereals - Quantification of humidity and mold in cereals 7

8 IDENTIFICATION OF CANNABIS Cannabis Money tree Cannabis Cannabis plants can be distinguished from adjacent plants Applications: - Forensic - Police department 8

9 MULTIPLEX MICROSCOPY HYPERSPECTRAL IMAGING PLATFORM Spectral range: nm Spectral resolution < 2.5 nm Filtering Efficiency > 80% nonpolarized 9

10 Multiplex Biological Imaging

11 CELL FLUORESCENCE IMAGING Human osteosarcoma cells Monkey kidney fibroblast cells Relative simplicity 3-4 colors Increasing multiplexing requires fairly complex techniques Mass spectrometry Bleaching and restaining

12 COMPARING IMAGING LABELS Classic fluorescence dyes Quantum dots fluorescence dyes Fluo excitation Fluo emission Wavelength (nm) Wavelength (nm) Carbon nanotube raman labels Carbon nanotube NIR fluorescence Wavelength (nm) Wavelength (nm) 12

13 Advantages and disadvantages Advantages Classic Fluorescence Highly sensitive Versatile Energy transfer process Very small dye molecules Disadvantages Large FWHM (100 nm ) Photobleaching Energy transfer between dyes Many laser lines required when using > 2 dyes Autofluorescence can be a problem Quantum dots Fluorescence FWHM = 50 nm No photobleaching Blinking Varying size of the QD Autofluorescence can be a problem Near-IR Fluorescence with carbon nanotubes FWHM = 20 nm 2 nd near IR window exceptional penetration depth Over 12 species for multiplexing Minimal Autofluorescence No photobleaching Lateral resolution (600 nm) High cost of detectors Raman with Raman labels Narrow bands (FWHM of 0.1 nm) Molecular signature = composition information 1 laser excitation for multiplexing (5 and more) Lateral resolution 300 nm No photobleaching Autofluorescence can be a problem Lower sensitivity 13

14 Multiplex Single Particle Tracking of Different Neuronal Receptor Subtypes Paul de Koninck group, Institut universitaire en santé mental du Québec, Université Laval

15 PARTNERSHIP PROJECT FOR CNS DISEASES PLATFORM 5-10 markers Drugs for CNS 15

16 Neurotransmitters and synapses 16

17 How can we track synaptic trajectories? 17

18 Multiplexing: 5 labels Acquisition time < 3.4 s Labrecque et al. Journal of Biomedical Optics 21(4), (April 2016) 18

19 Tracking 5 labels for 5 minutes Color-coded tracking of four membrane receptors on a live neuron. (a) Stargazin-QD605, GluA2-QD655, mglur5-qd705, and GluA1-QD805 trajectories. (b) Overlay of the four receptor tracks on the GFP-CaMKII signal; scale bar 5 μm. (c) Tracking of the four receptor subtypes in a single spine region; Scale bar 5 μm. 19

20 Multi-labeling of receptors in neurons: a case study for strong glutamate stimulation 20

21 Multi-labeling of receptors in neurons: 4h Memantine incubation at 10 mm Reduced membrane diffusion of Stargazin Increased membrane diffusion of GluA2 loss of 60% of QD immunoreactivity 21

22 NIR Imaging in the 2 nd Biological Window Daniel Heller Group, Memorial Sloan-Kettering Cancer Center

23 IR IMAGING WHY? High resolution High penetration depth Detection: nm Continuously tunable filter 23

24 MARKING WITH IR NANOLABELS 8,3 6,5 7,5 10,2 9,4 7,6 8,4 8,6 12,1 11,3 10,5 8,7 9,5 10,3 IR imaging allow a deeper penetration in tissues. Spectrally sensitive to the local environment. 24

25 In-vivo imaging with carbon nanotubes D. Roxbury et al. Scientific reports 5, 2015,

26 In-vivo imaging with carbon nanotubes: Subcellular markers Januka Budhathoki-Uprety et al., ACS Nano 5, 2015, Endosome markers Courtesy of Prof. D. Heller 26

27 PRECLINICAL INFRARED IN-VIVO HYPERSPECTRAL IMAGER NIR II live animal imaging nm to 1.7 µm - Hyperspectral 100 spectral channels - ZephIR 1.7 based (512 x 650, 340 fps) - Available fluorescent tags: - DNA wrapped carbon nanotubes - Quantum dots - Small molecule dyes 27

28 Preliminary Results for small animal imaging Spectra for individual region Depth of penetration of up to 10 mm (10x greater than other optical systems) 28

29 Nanoreporter Localizes to Liver Kupffer Cells when Injected Intravenously

30 Count Count Count Hyperspectral Imaging Non-Invasively Maps Lipids In Vivo Lipid Poor Lipid Rich (8,6) in D10 on Glass Surface Mean = Add SDC Mean = Remove SDC Mean = Center Wavelength (nm)