Abstract

We describe a new method to generate thin (thickness > 200 nm) and ultrathin (thickness < 200 nm) fluorescent layers to be used for microscope optical characterization. These layers are obtained by ultramicrotomy sectioning of fluorescent acrylic slides. This technique generates sub-resolution sheets with high fluorescence emission and uniform thickness, permitting to determine the z-response of different optical sectioning systems. Compared to the state of the art, the here proposed technique allows shorter and easier manufacturing procedure. Moreover, these fluorescent layers can be employed without protective coverslips, allowing the use of the Sectioned Imaging Property (SIP)-chart characterization method with coverslip-uncorrected objectives, water immersion objectives and micro-endoscopes.

© 2014 Optical Society of America

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References

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  1. M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge University Press, 1999).
  2. M. Abramowitz, Microscope Basics and Beyond (Olympus America Inc., 2003), Vol.1.
  3. R. W. Cole, T. Jinadasa, and C. M. Brown, “Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control,” Nat. Protoc. 12, 1929–1941 (2011).
  4. A. R. Hibbs, G. MacDonald, and K. Garsha, “Practical confocal microscopy,” in Handbook of Biological Confocal Microscopy, 3rd ed., J.B. Pawley, ed. (Springer, 2006), Chap. 36.
  5. R. P. Barretto and M. J. Schnitzer, “In vivo optical microendoscopy for imaging cells lying deep within live tissue,” Cold Spring Harb Protoc 2012(10), 1029–1034 (2012).
    [CrossRef] [PubMed]
  6. S. Wilhelm, B. Gröbler, M. Gluch, and H. Heinz, “Confocal Laser Scanning Microscopy” (Zeiss Jena Inc.). http://zeiss-campus.magnet.fsu.edu/referencelibrary/laserconfocal.html .
  7. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  9. M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191(2), 135–140 (1998b).
    [CrossRef] [PubMed]
  10. J. M. Zwier, G. J. Van Rooij, J. W. Hofstraat, and G. J. Brakenhoff, “Image calibration in fluorescence microscopy,” J. Microsc. 216(1), 15–24 (2004).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
  14. R. P. J. Barretto, B. Messerschmidt, and M. J. Schnitzer, “In vivo fluorescence imaging with high-resolution microlenses,” Nat. Methods 6(7), 511–512 (2009).
    [CrossRef] [PubMed]
  15. N. Vischer, M. Savenije, and G. J. Brakenhoff, “SIPchart plugin for ImageJ,” University of Amsterdam (2014) http://simon.bio.uva.nl/sipcharts/SIPchart.html .
  16. C. A. Schneider, W. S. Rasband, and K. W. Eliceiri, “NIH Image to ImageJ: 25 years of image analysis,” Nat. Methods 9(7), 671–675 (2012).
    [CrossRef] [PubMed]
  17. B. Amos, G. McConnell, and T. Wilson, “Confocal microscopy,” in Handbook of Comprehensive Biophysics (Elsevier, 2012).
  18. C. Matthews and F. P. Cordelieres, “œMetroloJ: an ImageJ plugin to help monitor microscopes' health,” in Proceedings of ImageJ User & Developer Conference (2010).€
  19. S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169(3), 391–405 (1993).
    [CrossRef]
  20. C. Wang and N. Ji, “Characterization and improvement of three-dimensional imaging performance of GRIN-lens-based two-photon fluorescence endomicroscopes with adaptive optics,” Opt. Express 21(22), 27142–27154 (2013).
    [CrossRef] [PubMed]

2013

2012

C. A. Schneider, W. S. Rasband, and K. W. Eliceiri, “NIH Image to ImageJ: 25 years of image analysis,” Nat. Methods 9(7), 671–675 (2012).
[CrossRef] [PubMed]

R. P. Barretto and M. J. Schnitzer, “In vivo optical microendoscopy for imaging cells lying deep within live tissue,” Cold Spring Harb Protoc 2012(10), 1029–1034 (2012).
[CrossRef] [PubMed]

2011

R. W. Cole, T. Jinadasa, and C. M. Brown, “Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control,” Nat. Protoc. 12, 1929–1941 (2011).

2009

R. P. J. Barretto, B. Messerschmidt, and M. J. Schnitzer, “In vivo fluorescence imaging with high-resolution microlenses,” Nat. Methods 6(7), 511–512 (2009).
[CrossRef] [PubMed]

2008

M. A. Model and J. L. Blank, “Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope,” J. Microsc. 229(1), 12–16 (2008).
[CrossRef] [PubMed]

2007

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc. 225(1), 88–95 (2007).
[CrossRef] [PubMed]

2005

G. J. Brakenhoff, G. W. H. Wurpel, K. Jalink, L. Oomen, L. Brocks, and J. M. Zwier, “Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts,” J. Microsc. 219(3), 122–132 (2005).
[CrossRef] [PubMed]

2004

J. M. Zwier, G. J. Van Rooij, J. W. Hofstraat, and G. J. Brakenhoff, “Image calibration in fluorescence microscopy,” J. Microsc. 216(1), 15–24 (2004).
[CrossRef] [PubMed]

2003

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

1998

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191(2), 135–140 (1998b).
[CrossRef] [PubMed]

1997

G. Decher, “Fuzzy nanoassemblies: Toward layered polymeric multicomposites,” Science 277(5330), 1232–1237 (1997).
[CrossRef]

1993

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169(3), 391–405 (1993).
[CrossRef]

Barretto, R. P.

R. P. Barretto and M. J. Schnitzer, “In vivo optical microendoscopy for imaging cells lying deep within live tissue,” Cold Spring Harb Protoc 2012(10), 1029–1034 (2012).
[CrossRef] [PubMed]

Barretto, R. P. J.

R. P. J. Barretto, B. Messerschmidt, and M. J. Schnitzer, “In vivo fluorescence imaging with high-resolution microlenses,” Nat. Methods 6(7), 511–512 (2009).
[CrossRef] [PubMed]

Bianchini, P.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc. 225(1), 88–95 (2007).
[CrossRef] [PubMed]

Blank, J. L.

M. A. Model and J. L. Blank, “Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope,” J. Microsc. 229(1), 12–16 (2008).
[CrossRef] [PubMed]

Brakenhoff, G. J.

G. J. Brakenhoff, G. W. H. Wurpel, K. Jalink, L. Oomen, L. Brocks, and J. M. Zwier, “Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts,” J. Microsc. 219(3), 122–132 (2005).
[CrossRef] [PubMed]

J. M. Zwier, G. J. Van Rooij, J. W. Hofstraat, and G. J. Brakenhoff, “Image calibration in fluorescence microscopy,” J. Microsc. 216(1), 15–24 (2004).
[CrossRef] [PubMed]

Brocks, L.

G. J. Brakenhoff, G. W. H. Wurpel, K. Jalink, L. Oomen, L. Brocks, and J. M. Zwier, “Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts,” J. Microsc. 219(3), 122–132 (2005).
[CrossRef] [PubMed]

Brown, C. M.

R. W. Cole, T. Jinadasa, and C. M. Brown, “Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control,” Nat. Protoc. 12, 1929–1941 (2011).

Cole, R. W.

R. W. Cole, T. Jinadasa, and C. M. Brown, “Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control,” Nat. Protoc. 12, 1929–1941 (2011).

Cremer, C.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169(3), 391–405 (1993).
[CrossRef]

Decher, G.

G. Decher, “Fuzzy nanoassemblies: Toward layered polymeric multicomposites,” Science 277(5330), 1232–1237 (1997).
[CrossRef]

Diaspro, A.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc. 225(1), 88–95 (2007).
[CrossRef] [PubMed]

Eliceiri, K. W.

C. A. Schneider, W. S. Rasband, and K. W. Eliceiri, “NIH Image to ImageJ: 25 years of image analysis,” Nat. Methods 9(7), 671–675 (2012).
[CrossRef] [PubMed]

Hell, S.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169(3), 391–405 (1993).
[CrossRef]

Hell, S. W.

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191(2), 135–140 (1998b).
[CrossRef] [PubMed]

Hofmann, U. G.

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191(2), 135–140 (1998b).
[CrossRef] [PubMed]

Hofstraat, J. W.

J. M. Zwier, G. J. Van Rooij, J. W. Hofstraat, and G. J. Brakenhoff, “Image calibration in fluorescence microscopy,” J. Microsc. 216(1), 15–24 (2004).
[CrossRef] [PubMed]

Jalink, K.

G. J. Brakenhoff, G. W. H. Wurpel, K. Jalink, L. Oomen, L. Brocks, and J. M. Zwier, “Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts,” J. Microsc. 219(3), 122–132 (2005).
[CrossRef] [PubMed]

Ji, N.

Jinadasa, T.

R. W. Cole, T. Jinadasa, and C. M. Brown, “Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control,” Nat. Protoc. 12, 1929–1941 (2011).

Krol, S.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc. 225(1), 88–95 (2007).
[CrossRef] [PubMed]

Messerschmidt, B.

R. P. J. Barretto, B. Messerschmidt, and M. J. Schnitzer, “In vivo fluorescence imaging with high-resolution microlenses,” Nat. Methods 6(7), 511–512 (2009).
[CrossRef] [PubMed]

Model, M. A.

M. A. Model and J. L. Blank, “Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope,” J. Microsc. 229(1), 12–16 (2008).
[CrossRef] [PubMed]

Oomen, L.

G. J. Brakenhoff, G. W. H. Wurpel, K. Jalink, L. Oomen, L. Brocks, and J. M. Zwier, “Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts,” J. Microsc. 219(3), 122–132 (2005).
[CrossRef] [PubMed]

Rasband, W. S.

C. A. Schneider, W. S. Rasband, and K. W. Eliceiri, “NIH Image to ImageJ: 25 years of image analysis,” Nat. Methods 9(7), 671–675 (2012).
[CrossRef] [PubMed]

Reiner, G.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169(3), 391–405 (1993).
[CrossRef]

Schneider, C. A.

C. A. Schneider, W. S. Rasband, and K. W. Eliceiri, “NIH Image to ImageJ: 25 years of image analysis,” Nat. Methods 9(7), 671–675 (2012).
[CrossRef] [PubMed]

Schneider, M.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc. 225(1), 88–95 (2007).
[CrossRef] [PubMed]

Schnitzer, M. J.

R. P. Barretto and M. J. Schnitzer, “In vivo optical microendoscopy for imaging cells lying deep within live tissue,” Cold Spring Harb Protoc 2012(10), 1029–1034 (2012).
[CrossRef] [PubMed]

R. P. J. Barretto, B. Messerschmidt, and M. J. Schnitzer, “In vivo fluorescence imaging with high-resolution microlenses,” Nat. Methods 6(7), 511–512 (2009).
[CrossRef] [PubMed]

Schrader, M.

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191(2), 135–140 (1998b).
[CrossRef] [PubMed]

Stelzer, E. H. K.

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169(3), 391–405 (1993).
[CrossRef]

Szellas, T.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc. 225(1), 88–95 (2007).
[CrossRef] [PubMed]

Van Rooij, G. J.

J. M. Zwier, G. J. Van Rooij, J. W. Hofstraat, and G. J. Brakenhoff, “Image calibration in fluorescence microscopy,” J. Microsc. 216(1), 15–24 (2004).
[CrossRef] [PubMed]

Vicidomini, G.

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc. 225(1), 88–95 (2007).
[CrossRef] [PubMed]

Wang, C.

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Wurpel, G. W. H.

G. J. Brakenhoff, G. W. H. Wurpel, K. Jalink, L. Oomen, L. Brocks, and J. M. Zwier, “Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts,” J. Microsc. 219(3), 122–132 (2005).
[CrossRef] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Zwier, J. M.

G. J. Brakenhoff, G. W. H. Wurpel, K. Jalink, L. Oomen, L. Brocks, and J. M. Zwier, “Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts,” J. Microsc. 219(3), 122–132 (2005).
[CrossRef] [PubMed]

J. M. Zwier, G. J. Van Rooij, J. W. Hofstraat, and G. J. Brakenhoff, “Image calibration in fluorescence microscopy,” J. Microsc. 216(1), 15–24 (2004).
[CrossRef] [PubMed]

Cold Spring Harb Protoc

R. P. Barretto and M. J. Schnitzer, “In vivo optical microendoscopy for imaging cells lying deep within live tissue,” Cold Spring Harb Protoc 2012(10), 1029–1034 (2012).
[CrossRef] [PubMed]

J. Microsc.

M. A. Model and J. L. Blank, “Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope,” J. Microsc. 229(1), 12–16 (2008).
[CrossRef] [PubMed]

S. Hell, G. Reiner, C. Cremer, and E. H. K. Stelzer, “Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index,” J. Microsc. 169(3), 391–405 (1993).
[CrossRef]

G. J. Brakenhoff, G. W. H. Wurpel, K. Jalink, L. Oomen, L. Brocks, and J. M. Zwier, “Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts,” J. Microsc. 219(3), 122–132 (2005).
[CrossRef] [PubMed]

M. Schrader, U. G. Hofmann, and S. W. Hell, “Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy,” J. Microsc. 191(2), 135–140 (1998b).
[CrossRef] [PubMed]

J. M. Zwier, G. J. Van Rooij, J. W. Hofstraat, and G. J. Brakenhoff, “Image calibration in fluorescence microscopy,” J. Microsc. 216(1), 15–24 (2004).
[CrossRef] [PubMed]

G. Vicidomini, M. Schneider, P. Bianchini, S. Krol, T. Szellas, and A. Diaspro, “Characterization of uniform ultrathin layer for z-response measurements in three-dimensional section fluorescence microscopy,” J. Microsc. 225(1), 88–95 (2007).
[CrossRef] [PubMed]

Nat. Biotechnol.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[CrossRef] [PubMed]

Nat. Methods

C. A. Schneider, W. S. Rasband, and K. W. Eliceiri, “NIH Image to ImageJ: 25 years of image analysis,” Nat. Methods 9(7), 671–675 (2012).
[CrossRef] [PubMed]

R. P. J. Barretto, B. Messerschmidt, and M. J. Schnitzer, “In vivo fluorescence imaging with high-resolution microlenses,” Nat. Methods 6(7), 511–512 (2009).
[CrossRef] [PubMed]

Nat. Protoc.

R. W. Cole, T. Jinadasa, and C. M. Brown, “Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control,” Nat. Protoc. 12, 1929–1941 (2011).

Opt. Express

Science

G. Decher, “Fuzzy nanoassemblies: Toward layered polymeric multicomposites,” Science 277(5330), 1232–1237 (1997).
[CrossRef]

Other

A. R. Hibbs, G. MacDonald, and K. Garsha, “Practical confocal microscopy,” in Handbook of Biological Confocal Microscopy, 3rd ed., J.B. Pawley, ed. (Springer, 2006), Chap. 36.

M. Born and E. Wolf, Principles of Optics, 7th ed. (Cambridge University Press, 1999).

M. Abramowitz, Microscope Basics and Beyond (Olympus America Inc., 2003), Vol.1.

B. Amos, G. McConnell, and T. Wilson, “Confocal microscopy,” in Handbook of Comprehensive Biophysics (Elsevier, 2012).

C. Matthews and F. P. Cordelieres, “œMetroloJ: an ImageJ plugin to help monitor microscopes' health,” in Proceedings of ImageJ User & Developer Conference (2010).€

N. Vischer, M. Savenije, and G. J. Brakenhoff, “SIPchart plugin for ImageJ,” University of Amsterdam (2014) http://simon.bio.uva.nl/sipcharts/SIPchart.html .

S. Wilhelm, B. Gröbler, M. Gluch, and H. Heinz, “Confocal Laser Scanning Microscopy” (Zeiss Jena Inc.). http://zeiss-campus.magnet.fsu.edu/referencelibrary/laserconfocal.html .

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Figures (10)

Fig. 1
Fig. 1

Profilometer measurement of a yellow/green fluorescent layer which was cut at a nominal thickness of 100 nm.

Fig. 2
Fig. 2

Uniformity test of layer’s fluorescence; a) and b) are maximal intensity distributions emitted over the FOV by one fluorescent layer (thickness: 70 nm), in two different regions; c) represents the histogram distribution obtained by dividing the intensity maps displayed in a) and b).

Fig. 3
Fig. 3

Fluorescence intensity decay due to photo-bleaching in a yellow-green layer (thickness: 70 nm). Bleaching is induced by two-photon excitation (λ = 920 nm) on 6 different points which were randomly located on the thin layer.

Fig. 4
Fig. 4

SIP chart for the Olympus MPLFLN 100X objective over a field of view of 95 × 95µm2, obtained by using a 100 nm-thick fluorescent layer and two-photon excitation (λ = 920nm).

Fig. 5
Fig. 5

Collected fluorescence intensity z-profile and fitting parameters for the Olympus MPLFLN 100X objective. Fitted FWHMz = 1.79 μm. Theoretical FWHM z resolution was 1.12 µm.

Fig. 6
Fig. 6

SIP chart and z profile of Olympus LUMPLFLN 60XW over a field of view of 200 × 200 µm2, obtained using an ultrathin layer (thickness:100 nm) and two-photon excitation by pulsed laser at 920 nm. FWHMz = 2.8 μm (theoretical FWHMz resolution was 1.8 µm).

Fig. 7
Fig. 7

SIP chart and z profile of Grintech micro-endoscope GT-MO-080-018-810 over a 38 × 38 µm2 FOV, obtained by using a 100 nm-thick layer and two-photon excitation by a pulsed laser at 920 nm.

Fig. 8
Fig. 8

SIP chart and z profile of Grintech micro-endoscope NEM-050-06-00-520-S over a 175 × 175 µm2 FOV, obtained by using a 200 nm-thick layer and two-photon excitation (λ = 920 nm).

Fig. 9
Fig. 9

SIP chart and z profile of Olympus MPFLN 100X over a 125 × 125 µm2 FOV, obtained by using a 100 nm-thick yellow/green layer excited in an inverted Nikon Confocal A1 microscope. Excitation beam wavelength: 488 nm.

Fig. 10
Fig. 10

SIP chart and z profile of Olympus MPFLN 100X over a 125 × 125 µm2 FOV, obtained using a 100 nm-thick red layer excited an inverted Nikon Confocal A1 microscope. Excitation beam wavelength: 550 nm.

Equations (2)

Equations on this page are rendered with MathJax. Learn more.

FWHM xy ={ 2 ln2 0.32λ 2  NA 0.38λ  NA        NA 0.7, 2 ln2 0.325λ 2 N A 0.91 0.383λ N A 0.91    NA 0.7,   FWHM z =2 ln2 0.532λ 2   1 n n 2 N A 2  0.626λ 1 n n 2 N A 2 .
FWHM z 0.95 λ 1 n n 2 N A 2 .

Metrics