1 / 25

Multiplexed Fluorescence Unmixing

Multiplexed Fluorescence Unmixing. Marina Alterman , Yoav Schechner. Technion , Israel. Aryeh Weiss. Bar- Ilan , Israel. Natural Linear Mixing. i. c. i. c. Raskar et al. 2006. i. c. ImageJ image sample collection. Natural Linear Mixing. ?. + noise. i. c. i. + noise.

asa
Download Presentation

Multiplexed Fluorescence Unmixing

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Multiplexed Fluorescence Unmixing Marina Alterman, YoavSchechner Technion, Israel Aryeh Weiss Bar-Ilan, Israel

  2. Natural Linear Mixing i c i c Raskar et al. 2006. i c ImageJ image sample collection.

  3. Natural Linear Mixing ? + noise i c i + noise c Raskar et al. 2006. i + noise How do you measure i? c ImageJ image sample collection.

  4. a1 1 0 i 1 1 a = 0 1 1 i 2 2 a1 0 1 i 3 3 Single Source Excitation demultiplex Multiplexed Excitation i1 2 1 a1 1 3 i2 a2 2 1 3 i3 a3 3 Beam combiner 2

  5. Why Multiplexing? Trivial Measurements Multiplexed Measurements i + noise SNR SNR Intensity vector Same acquisition time

  6. i – single source intensities η - noise Multiplexing - Look closer Estimate c noti Xc i acquisition estimation Minimum  W=?

  7. Multiplexing: a=Wi, Mixing: i=Xc Common Approach This Work Acquired multiplexed intensities Single source intensities Concentrations ˆ ˆ ˆ ˆ c c i i a a Wi≠Wc Wi Wc Ndyes=3 Nsources=7 Nmeasure=3 size(i)=7 efficient acquisition Nmeasure=7 Alterman, Schechner & Weiss, Multiplexed Fluorescence Unmixing

  8. Fluorescence Cell structure and processes Fluorescent Specimen Horse Dermal Fibroblast Cells Corn Grain Intestine Tissue Flea http://www.microscopyu.com/galleries/fluorescence, http://www.microscopy.fsu.edu/primer/techniques/fluorescence/fluorogallery.html

  9. Linear Mixing i More molecules per pixel Brighter pixel c i c Molecules per pixel i = x∙c Alterman, Schechner & Weiss, Multiplexed Fluorescence Unmixing

  10. Linear Mixing i {cd} i = x x ∙ ∙ ∙ x For each pixel: c c ∙ ∙ ∙ c 1 2 Ndyes 1 2 Ndyes vector of concentrations (spatial distribution) Alterman, Schechner & Weiss, Multiplexed Fluorescence Unmixing

  11. Linear Mixing i i 1 2 s=1 s=2 {cd} {cd} For each pixel: i = x x ∙ ∙ ∙ x i = x x ∙ ∙ ∙ x i = x x ∙ ∙ ∙ x c c ∙ ∙ ∙ c 1 2 s 2,1 2,2 2,Ndyes 1,1 1,2 1,Ndyes s,1 s,2 s,Ndyes 1 2 Ndyes ∙ ∙ ∙ vector of concentrations (spatial distribution) vector of intensities Mixing matrix

  12. Linear Mixing i i 1 2 s=1 s=2 {cd} {cd} For each pixel: vector of concentrations (spatial distribution) vector of intensities Mixing matrix

  13. Fluorescent Microscope Intensity image e(λ) Emission Filter s = 1 s = 2 s = 3 e(λ) s = 4 Dichroic Mirror L2(λ) s = 5 Excitation Excitation Fluorescent Filter Sources λ λ λ 300 400 500 600 700 300 400 500 600 700 300 400 500 600 700 Specimen s: illumination sources Blue α(λ)

  14. Fluorescent Microscope Intensity image (mixed) Intensity image e(λ) Unmixing required Emission Filter s = 1 s = 2 s = 3 e(λ) s = 4 Dichroic Mirror L2(λ) s = 5 Excitation Excitation Fluorescent Filter Sources λ λ λ 300 400 500 600 700 300 400 500 600 700 300 400 500 600 700 Specimen s: illumination sources Green Blue Cross-talk α(λ) Cross-talk

  15. Unmix Fluorescent specimen Problem Definition Intensity image (mixed) + noise noise How to multiplex for least noisy unmixing? Alterman, Schechner & Weiss, Multiplexed Fluorescence Unmixing

  16. Sum up the concepts Man made Nature Acquired multiplexed image intensities Single source Image intensities W X a i Concentrations multiplexing mixing c unmixing demultiplexing W-1 X-1 multiplexed unmixing Alterman, Schechner & Weiss, Multiplexed Fluorescence Unmixing

  17. Look closer - again i – single source intensities η - noise Xc i Estimate c noti Alterman, Schechner & Weiss, Multiplexed Fluorescence Unmixing

  18. Multiplexed Unmixing acquisition For each pixel i acquired measurements noise estimation a X W c WX is not square + = Weighted Least Squares Other estimators OR multiplexing matrix OR mixing matrix Minimum Variance in c  W=?

  19. Generalizations Minimum Var W=? η - noise Image intensities i =? var(η) =constant Details in the paper concentrations c =? var(η) =constant c =? var(η) ≠constant Alterman, Schechner & Weiss, Multiplexed Fluorescence Unmixing

  20. Generalized Multiplex Gain What is the SNR gain for unmixing? Only Unmixing VS. Unmixing + Multiplexing Alterman, Schechner & Weiss, Multiplexed Fluorescence Unmixing

  21. 2.2 2 1.8 1.6 1.4 1.2 1 3 4 5 6 7 Nsources=Nmeasure Significance of the Model ˆ ˆ c GAINc ˆ ˆ c i i a a VS. Wi≠Wc Wi Wc Alterman, Schechner & Weiss, Multiplexed Fluorescence Unmixing

  22. 2.2 2 1.8 1.6 1.4 1.2 1 3 4 5 6 7 Nsources=Nmeasure Significance of the Model ˆ ˆ c GAINc i a Wc Alterman, Schechner & Weiss, Multiplexed Fluorescence Unmixing

  23. Significance of the Model ˆ ˆ 2.2 c GAINc ˆ ˆ c i i a 2 a 1.8 1.6 Wi 1.4 1.2 1 Wc 3 4 5 6 7 GAIN < 1 Nsources=Nmeasure For specific 3 dyes, camera and filter characteristics

  24. Natural Linear Mixing ? + noise i c i + noise c Raskar et al. 2006. i + noise c ImageJ image sample collection.

  25. Multiplexed Unmixing Xc i Generalization of multiplexing theory The goal is unmixing SNR improvement Efficient Acquisition Exploit all available sources a X + = c η W Alterman, Schechner & Weiss, Multiplexed Fluorescence Unmixing

More Related