Abstract

We present a depth-resolved Image Mapping Spectrometer (IMS) which is capable of acquiring 4D (x, y, z, λ) datacubes. Optical sectioning is implemented by structured illumination. The device’s spectral imaging performance is demonstrated in a multispectral microsphere and mouse kidney tissue fluorescence imaging experiment. We also compare quantitatively the depth-resolved IMS with a hyperspectral confocal microscope (HCM) in a standard fluorescent bead imaging experiment. The comparison results show that despite the use of a light source with four orders of magnitude lower intensity in the IMS than that in the HCM, the image signal-to-noise ratio acquired by the IMS is 2.6 times higher than that achieved by the equivalent confocal approach.

© 2011 OSA

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2011 (1)

R. T. Kester, N. Bedard, L. Gao, and T. S. Tkaczyk, “Real-time snapshot hyperspectral imaging endoscope,” J. Biomed. Opt. 16(5), 056005 (2011).
[CrossRef] [PubMed]

2010 (3)

2009 (2)

L. Gao, R. T. Kester, and T. S. Tkaczyk, “Compact image slicing spectrometer (ISS) for hyperspectral fluorescence microscopy,” Opt. Express 17(15), 12293–12308 (2009).
[CrossRef] [PubMed]

V. Raicu, M. R. Stoneman, R. Fung, M. Melnichuk, D. B. Jansma, L. F. Pisterzi, S. Rath, M. Fox, J. W. Wells, and D. K. Saldin, “Determination of supramolecular structure and spatial distribution of protein complexes in living cells,” Nat. Photonics 3(2), 107–113 (2009).
[CrossRef]

2008 (1)

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron 39(7), 808–818 (2008).
[CrossRef] [PubMed]

2007 (1)

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(Pt 2), 240–254 (2007).
[CrossRef] [PubMed]

2005 (1)

J. W. Lichtman and J. A. Conchello, “Fluorescence microscopy,” Nat. Methods 2(12), 910–919 (2005).
[CrossRef] [PubMed]

2004 (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

2003 (2)

R. Dixit and R. Cyr, “Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy,” Plant J. 36(2), 280–290 (2003).
[CrossRef] [PubMed]

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[CrossRef] [PubMed]

2002 (1)

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[CrossRef] [PubMed]

2001 (2)

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80(4), 2029–2036 (2001).
[CrossRef] [PubMed]

1997 (2)

M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997).
[CrossRef] [PubMed]

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(Pt 1), 9–20 (1997).
[CrossRef] [PubMed]

1994 (1)

Bedard, N.

R. T. Kester, N. Bedard, L. Gao, and T. S. Tkaczyk, “Real-time snapshot hyperspectral imaging endoscope,” J. Biomed. Opt. 16(5), 056005 (2011).
[CrossRef] [PubMed]

Brady, D. J.

Choi, K.

Conchello, J. A.

J. W. Lichtman and J. A. Conchello, “Fluorescence microscopy,” Nat. Methods 2(12), 910–919 (2005).
[CrossRef] [PubMed]

Cull, C. F.

Cyr, R.

R. Dixit and R. Cyr, “Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy,” Plant J. 36(2), 280–290 (2003).
[CrossRef] [PubMed]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Descour, M. R.

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

Dixit, R.

R. Dixit and R. Cyr, “Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy,” Plant J. 36(2), 280–290 (2003).
[CrossRef] [PubMed]

Fletcher-Holmes, D. W.

Ford, B. K.

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

Fox, M.

V. Raicu, M. R. Stoneman, R. Fung, M. Melnichuk, D. B. Jansma, L. F. Pisterzi, S. Rath, M. Fox, J. W. Wells, and D. K. Saldin, “Determination of supramolecular structure and spatial distribution of protein complexes in living cells,” Nat. Photonics 3(2), 107–113 (2009).
[CrossRef]

Fung, R.

V. Raicu, M. R. Stoneman, R. Fung, M. Melnichuk, D. B. Jansma, L. F. Pisterzi, S. Rath, M. Fox, J. W. Wells, and D. K. Saldin, “Determination of supramolecular structure and spatial distribution of protein complexes in living cells,” Nat. Photonics 3(2), 107–113 (2009).
[CrossRef]

Gao, L.

Ghoneim, M.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(Pt 2), 240–254 (2007).
[CrossRef] [PubMed]

Gorman, A.

Hagen, N.

Haraguchi, T.

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[CrossRef] [PubMed]

Harvey, A. R.

Hasegawa, M.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(Pt 2), 240–254 (2007).
[CrossRef] [PubMed]

Hiraoka, Y.

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[CrossRef] [PubMed]

Hopt, A.

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80(4), 2029–2036 (2001).
[CrossRef] [PubMed]

Hoyt, C. C.

Huisken, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Jansma, D. B.

V. Raicu, M. R. Stoneman, R. Fung, M. Melnichuk, D. B. Jansma, L. F. Pisterzi, S. Rath, M. Fox, J. W. Wells, and D. K. Saldin, “Determination of supramolecular structure and spatial distribution of protein complexes in living cells,” Nat. Photonics 3(2), 107–113 (2009).
[CrossRef]

Juskaitis, R.

Karadaglic, D.

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron 39(7), 808–818 (2008).
[CrossRef] [PubMed]

Kester, R. T.

Kumazaki, S.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(Pt 2), 240–254 (2007).
[CrossRef] [PubMed]

Kusumi, A.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(Pt 1), 9–20 (1997).
[CrossRef] [PubMed]

Lichtman, J. W.

J. W. Lichtman and J. A. Conchello, “Fluorescence microscopy,” Nat. Methods 2(12), 910–919 (2005).
[CrossRef] [PubMed]

Lynch, R. M.

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

Melnichuk, M.

V. Raicu, M. R. Stoneman, R. Fung, M. Melnichuk, D. B. Jansma, L. F. Pisterzi, S. Rath, M. Fox, J. W. Wells, and D. K. Saldin, “Determination of supramolecular structure and spatial distribution of protein complexes in living cells,” Nat. Photonics 3(2), 107–113 (2009).
[CrossRef]

Morris, H. R.

Murphy, S. M.

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

Neher, E.

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80(4), 2029–2036 (2001).
[CrossRef] [PubMed]

Neil, M. A. A.

Nishiyama, M.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(Pt 2), 240–254 (2007).
[CrossRef] [PubMed]

Oh-Oka, H.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(Pt 2), 240–254 (2007).
[CrossRef] [PubMed]

Okamoto, K.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(Pt 2), 240–254 (2007).
[CrossRef] [PubMed]

Oliver, T.

Ozaki, K.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(Pt 1), 9–20 (1997).
[CrossRef] [PubMed]

Pepperkok, R.

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[CrossRef] [PubMed]

Pisterzi, L. F.

V. Raicu, M. R. Stoneman, R. Fung, M. Melnichuk, D. B. Jansma, L. F. Pisterzi, S. Rath, M. Fox, J. W. Wells, and D. K. Saldin, “Determination of supramolecular structure and spatial distribution of protein complexes in living cells,” Nat. Photonics 3(2), 107–113 (2009).
[CrossRef]

Raicu, V.

V. Raicu, M. R. Stoneman, R. Fung, M. Melnichuk, D. B. Jansma, L. F. Pisterzi, S. Rath, M. Fox, J. W. Wells, and D. K. Saldin, “Determination of supramolecular structure and spatial distribution of protein complexes in living cells,” Nat. Photonics 3(2), 107–113 (2009).
[CrossRef]

Rath, S.

V. Raicu, M. R. Stoneman, R. Fung, M. Melnichuk, D. B. Jansma, L. F. Pisterzi, S. Rath, M. Fox, J. W. Wells, and D. K. Saldin, “Determination of supramolecular structure and spatial distribution of protein complexes in living cells,” Nat. Photonics 3(2), 107–113 (2009).
[CrossRef]

Rietdorf, J.

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[CrossRef] [PubMed]

Sako, Y.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(Pt 1), 9–20 (1997).
[CrossRef] [PubMed]

Saldin, D. K.

V. Raicu, M. R. Stoneman, R. Fung, M. Melnichuk, D. B. Jansma, L. F. Pisterzi, S. Rath, M. Fox, J. W. Wells, and D. K. Saldin, “Determination of supramolecular structure and spatial distribution of protein complexes in living cells,” Nat. Photonics 3(2), 107–113 (2009).
[CrossRef]

Sekihata, A.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(Pt 1), 9–20 (1997).
[CrossRef] [PubMed]

Shimada, Y.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(Pt 1), 9–20 (1997).
[CrossRef] [PubMed]

Shimi, T.

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
[CrossRef] [PubMed]

Shimizu, Y.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(Pt 2), 240–254 (2007).
[CrossRef] [PubMed]

Stelzer, E. H.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Stoneman, M. R.

V. Raicu, M. R. Stoneman, R. Fung, M. Melnichuk, D. B. Jansma, L. F. Pisterzi, S. Rath, M. Fox, J. W. Wells, and D. K. Saldin, “Determination of supramolecular structure and spatial distribution of protein complexes in living cells,” Nat. Photonics 3(2), 107–113 (2009).
[CrossRef]

Swoger, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Terazima, M.

S. Kumazaki, M. Hasegawa, M. Ghoneim, Y. Shimizu, K. Okamoto, M. Nishiyama, H. Oh-Oka, and M. Terazima, “A line-scanning semi-confocal multi-photon fluorescence microscope with a simultaneous broadband spectral acquisition and its application to the study of the thylakoid membrane of a cyanobacterium Anabaena PCC7120,” J. Microsc. 228(Pt 2), 240–254 (2007).
[CrossRef] [PubMed]

Tkaczyk, T. S.

Treado, P. J.

Volin, C. E.

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

Wells, J. W.

V. Raicu, M. R. Stoneman, R. Fung, M. Melnichuk, D. B. Jansma, L. F. Pisterzi, S. Rath, M. Fox, J. W. Wells, and D. K. Saldin, “Determination of supramolecular structure and spatial distribution of protein complexes in living cells,” Nat. Photonics 3(2), 107–113 (2009).
[CrossRef]

Wilson, T.

D. Karadaglić and T. Wilson, “Image formation in structured illumination wide-field fluorescence microscopy,” Micron 39(7), 808–818 (2008).
[CrossRef] [PubMed]

M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997).
[CrossRef] [PubMed]

Wittbrodt, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Yamamoto, M.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(Pt 1), 9–20 (1997).
[CrossRef] [PubMed]

Yanagisawa, Y.

Y. Sako, A. Sekihata, Y. Yanagisawa, M. Yamamoto, Y. Shimada, K. Ozaki, and A. Kusumi, “Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1,” J. Microsc. 185(Pt 1), 9–20 (1997).
[CrossRef] [PubMed]

Zimmermann, T.

T. Zimmermann, J. Rietdorf, and R. Pepperkok, “Spectral imaging and its applications in live cell microscopy,” FEBS Lett. 546(1), 87–92 (2003).
[CrossRef] [PubMed]

Appl. Opt. (1)

Appl. Spectrosc. (1)

Biophys. J. (2)

B. K. Ford, C. E. Volin, S. M. Murphy, R. M. Lynch, and M. R. Descour, “Computed tomography-based spectral imaging for fluorescence microscopy,” Biophys. J. 80(2), 986–993 (2001).
[CrossRef] [PubMed]

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80(4), 2029–2036 (2001).
[CrossRef] [PubMed]

Cell Struct. Funct. (1)

Y. Hiraoka, T. Shimi, and T. Haraguchi, “Multispectral imaging fluorescence microscopy for living cells,” Cell Struct. Funct. 27(5), 367–374 (2002).
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Supplementary Material (2)

» Media 1: MOV (2294 KB)     
» Media 2: MOV (2010 KB)     

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Figures (9)

Fig. 1
Fig. 1

Depth-resolved IMS with SI.

Fig. 2
Fig. 2

Axial PSF measurement in the depth-resolved IMS.

Fig. 3
Fig. 3

Spectral imaging of a sectioned depth layer of a fluorescent microsphere by depth-resolved IMS. (a) Panchromatic display of acquired datacube. (b) The spectra of the core and ring of the microsphere. (c) – (d) Pseudo-colored core and ring components after spectral unmixing. (e) A merged image of (c) and (d). (f) Intensity profile of two fluorescent dyes across the microsphere section. (g) Measured spectrum vs. fitted spectrum at the interface of the two fluorophores.

Fig. 4
Fig. 4

Spectral channel images of mouse kidney tissue section acquired by (a) depth-resolved IMS, and (b) IMS under wide-field imaging.

Fig. 5
Fig. 5

(Media 1 and Media 2) 4D (x, y, z, λ) imaging of a mouse kidney tissue section by the depth-resolved IMS. The glomeruli and convoluted tubules were stained with Alexa Fluor 488 and the filamentous actin was stained with Alexa Fluor 568. The acquired 4D (x, y, z, λ) datacube is of size 350 × 350 × 35 × 46. A total of four out of 46 wavelength (x, y, z) datacubes are shown.

Fig. 6
Fig. 6

Signal variance vs. Intensity in (a) IMS and (b) HCM.

Fig. 7
Fig. 7

Experimental design concept for comparison study.

Fig. 8
Fig. 8

(a) Depth-resolved IMS imaging vs. (b) HCM imaging in a standard fluorescent bead imaging experiment. The color bar is in the unit of converted photons from image intensity counts.

Fig. 9
Fig. 9

Excitation laser power vs. Measured signal in HCM imaging.

Tables (1)

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Table 1 Imaging Parameters for Depth-resolved IMS and HCM in Comparison Experiment

Equations (17)

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I = ( I 1 I 2 ) 2 + ( I 1 I 3 ) 2 + ( I 2 I 3 ) 2 .
r e s z = F W H M ( P S F ) = 1.08   μ m .
g = photons / count .
N c , I M S ~ σ c = σ p / g , N c , H C M ~ 1.2 1 / 2 σ c = 1.1 σ p / g ,
σ p 2 = S p .
N c , I M S 2 ~ S p / g 2 = g S c / g 2 = S c / g , N c , H C M 2 ~ 1.2 S p / g 2 = 1.2 g S c / g 2 = 1.2 S c / g .
g I M S = 17.6  photons/count, g H C M = 0.0152  photons/count .
L = Δ λ H C M Δ λ I M S × Δ z H C M Δ z I M S / M H C M 2 M I M S 2 = 1.11.
r = S p , I M S L S p , H C M = S C , I M S g I M S L S C , H C M g H C M = 4.87 × 10 3 × 17.6 × 1.11 4.84 × 10 4 × 0.0152 = 130.
E H C M ~ 0.32  mW 1 4 π × P S F 2 = 0.32  mW 1 4 π × ( 1.22 λ / N A ) 2 = 0.32  mW 1 4 π × ( 0.44   μ m ) 2 = 2.14  mW/ μ m 2 .
E I M S ~ 40  mW 1 4 π × ( 400   μ m) 2 = 3.2   × 10 4 mW/ μ m 2 .
ξ = I ( P = ) I ( P = 0.32  mW ) = a a ( 1 e 0.32 b ) = 1.77.
r * > r / ξ = 73
S N R H C M = S p , H C M N p , H C M = S p , H C M 1.2 1 / 2 σ p = S p , H C M 1.2 S p , H C M = S p , H C M / 1.2 = 24.7.
I ¯ = 1 1000 j = 1 1000 I ( x 1 , y 1 , z 1 , t j , λ = 516 n m ) , σ I = 1 1000 1 j = 1 1000 ( I ( x 1 , y 1 , z 1 , t j , λ = 516 n m ) I ¯ ) 2 .
S N R I M S = i j I ( x i , y j ) i j σ I 2 ( x i , y j ) = i j I ( x i , y j ) 4.78 i j I ( x i , y j ) = 1 4.78 L S p , I M S = 64.7 ,
S N R I M S S N R H C M ~ 2.6

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