Abstract

We report development of a continuous scanning procedure and the use of a time delay integration (TDI) line scan camera for a light-sheet based microscope called a thin-sheet laser imaging microscope (TSLIM). TSLIM is an optimized version of a light-sheet fluorescent microscope that previously used a start/stop scanning procedure to move the specimen through the thinnest portion of a light-sheet and stitched the image columns together to produce a well-focused composite image. In this paper, hardware and software enhancements to TSLIM are described that allow for dual sided, dual illumination lasers, and continuous scanning of the specimen using either a full-frame CCD camera and a TDI line scan camera. These enhancements provided a ~70% reduction in the time required for composite image generation and a ~63% reduction in photobleaching of the specimen compared to the start/stop procedure.

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  1. P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques 46(4), 287–294 (2009).
    [PubMed]
  2. A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170(Pt 3), 229–236 (1993).
    [PubMed]
  3. A. H. Voie, “Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy,” Hear. Res. 171(1-2), 119–128 (2002).
    [CrossRef] [PubMed]
  4. H. Siedentopf and R. Zsigmondy, “Über Sichtbarmachung und Groessenbestimmung ultramikroskopischer Teilchen, mit besonderer Anwendung auf Goldrubinglaesern,” Annalen der Physik 10, 1–39 (1903).
  5. E. Fuchs, J. S. Jaffe, R. A. Long, and F. Azam, “Thin laser light sheet microscope for microbial oceanography,” Opt. Express 10(2), 145–154 (2002).
    [PubMed]
  6. 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]
  7. H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
    [CrossRef] [PubMed]
  8. J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
    [CrossRef] [PubMed]

2009

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques 46(4), 287–294 (2009).
[PubMed]

2007

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[CrossRef] [PubMed]

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
[CrossRef] [PubMed]

2004

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]

2002

E. Fuchs, J. S. Jaffe, R. A. Long, and F. Azam, “Thin laser light sheet microscope for microbial oceanography,” Opt. Express 10(2), 145–154 (2002).
[PubMed]

A. H. Voie, “Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy,” Hear. Res. 171(1-2), 119–128 (2002).
[CrossRef] [PubMed]

1993

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170(Pt 3), 229–236 (1993).
[PubMed]

1903

H. Siedentopf and R. Zsigmondy, “Über Sichtbarmachung und Groessenbestimmung ultramikroskopischer Teilchen, mit besonderer Anwendung auf Goldrubinglaesern,” Annalen der Physik 10, 1–39 (1903).

Azam, F.

Becker, K.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[CrossRef] [PubMed]

Burns, D. H.

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170(Pt 3), 229–236 (1993).
[PubMed]

Buytaert, J. A. N.

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
[CrossRef] [PubMed]

Deininger, K.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[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]

Deussing, J. M.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[CrossRef] [PubMed]

Dirckx, J. J. J.

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
[CrossRef] [PubMed]

Dodt, H.-U.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[CrossRef] [PubMed]

Eder, M.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[CrossRef] [PubMed]

Fuchs, E.

Glass, T. J.

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques 46(4), 287–294 (2009).
[PubMed]

GrandPre, P. Z.

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques 46(4), 287–294 (2009).
[PubMed]

Hillenbrand, M.

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques 46(4), 287–294 (2009).
[PubMed]

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]

Jaffe, J. S.

Jährling, N.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[CrossRef] [PubMed]

Johnson, S. B.

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques 46(4), 287–294 (2009).
[PubMed]

Leger, J. R.

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques 46(4), 287–294 (2009).
[PubMed]

Leischner, U.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[CrossRef] [PubMed]

Long, R. A.

Mauch, C. P.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[CrossRef] [PubMed]

Santi, P. A.

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques 46(4), 287–294 (2009).
[PubMed]

Schierloh, A.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[CrossRef] [PubMed]

Siedentopf, H.

H. Siedentopf and R. Zsigmondy, “Über Sichtbarmachung und Groessenbestimmung ultramikroskopischer Teilchen, mit besonderer Anwendung auf Goldrubinglaesern,” Annalen der Physik 10, 1–39 (1903).

Spelman, F. A.

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170(Pt 3), 229–236 (1993).
[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]

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]

Voie, A. H.

A. H. Voie, “Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy,” Hear. Res. 171(1-2), 119–128 (2002).
[CrossRef] [PubMed]

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170(Pt 3), 229–236 (1993).
[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]

Zieglgänsberger, W.

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[CrossRef] [PubMed]

Zsigmondy, R.

H. Siedentopf and R. Zsigmondy, “Über Sichtbarmachung und Groessenbestimmung ultramikroskopischer Teilchen, mit besonderer Anwendung auf Goldrubinglaesern,” Annalen der Physik 10, 1–39 (1903).

Annalen der Physik

H. Siedentopf and R. Zsigmondy, “Über Sichtbarmachung und Groessenbestimmung ultramikroskopischer Teilchen, mit besonderer Anwendung auf Goldrubinglaesern,” Annalen der Physik 10, 1–39 (1903).

Biotechniques

P. A. Santi, S. B. Johnson, M. Hillenbrand, P. Z. GrandPre, T. J. Glass, and J. R. Leger, “Thin-sheet laser imaging microscopy for optical sectioning of thick tissues,” Biotechniques 46(4), 287–294 (2009).
[PubMed]

Hear. Res.

A. H. Voie, “Imaging the intact guinea pig tympanic bulla by orthogonal-plane fluorescence optical sectioning microscopy,” Hear. Res. 171(1-2), 119–128 (2002).
[CrossRef] [PubMed]

J. Biomed. Opt.

J. A. N. Buytaert and J. J. J. Dirckx, “Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution,” J. Biomed. Opt. 12(1), 014039 (2007).
[CrossRef] [PubMed]

J. Microsc.

A. H. Voie, D. H. Burns, and F. A. Spelman, “Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens,” J. Microsc. 170(Pt 3), 229–236 (1993).
[PubMed]

Nat. Methods

H.-U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing, M. Eder, W. Zieglgänsberger, and K. Becker, “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat. Methods 4(4), 331–336 (2007).
[CrossRef] [PubMed]

Opt. Express

Science

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]

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Figures (5)

Fig. 1
Fig. 1

Top-view and side-view of TSLIM’s light-sheet pathway. A solid state laser beam is converted in an optical assembly (beam expander, cylindrical lens and microscope objective) and passes through the specimen chamber containing the tissue. The thinnest portion of the light-sheet is at focal point of the microscope objective. The illuminated plane in the tissue is collected by a microscope objective and recorded using a digital camera. Upper left panel: the specimen is moved through the beam waist to produce a well-focused imaged across the width of the specimen.

Fig. 2
Fig. 2

Optical section of a mouse cochlea before and after stitching. (a). Double white lines indicate the confocal region of the light-sheet where the specimen is in focus. Note that to the left and right of this region the tissue appears to be out-of-focus due to a thicker beam. (b). After stitching of image columns focus appears improved across the width of the specimen. Bar = 250 µm for panels a,b. (c). A higher magnification of 2b (above scale bar) showing a stitching artifact (arrowhead) and two less noticeable artifacts to the left of the arrowhead. Insert in upper left is a 3X magnification of the region outlined in main Fig. and shows the stitching artifact in detail. Bar = 50 µm.

Fig. 3
Fig. 3

Hardware triggering and scanning with both, the Retiga 2000R and the Dalsa HS-40-04K40 camera. The encoder signal of Newport’s LTA-HS servomotor in x-direction of the ESP 301 motion controller is bypassed over a custom made card and an RS422-to-TTL converter and goes to a National Instrument NI PCIe – 6321 Counter/Timer/Trigger Card. This card is programmed to divide the rising edge of the received signal by a constant factor and the result is an output signal. The signal triggers the Retiga 2000R and the Dalsa HS-40-04K40 by a connection to the camera’s trigger input channel.

Fig. 4
Fig. 4

Comparison between full frame (a) and line scan camera (b) with a higher magnification. Due to increased resolution by the line scan camera the image can be enlarged more without showing pixelation. Bar = 20µm. (c) Comparison between size of a Retiga CCD image in the center panel with the image using the Dalsa camera (full image) at the same magnification. The Dalsa camera allows for collection of a larger view of the specimen. The dashed lines indicate that the width of the image is not limited because it can be scanned in that dimension. Bar = 150µm.

Fig. 5
Fig. 5

Photobleaching of a specimen following 50 repeated imaging of the same plane of an optical section. The start/stop procedure using the CCD camera takes 1550 sec to image 50 sections and retains 65% of the fluorescence. A continuous scan using the CCD takes only 750 sec and retains 80% of the fluorescence. A continuous scan with the line scan camera takes only 475 sec and retains 87% of the fluorescence making it the best choice for TSLIM imaging.

Equations (5)

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m = w R O I p M
i = p w R O I M s i
V max C A M = p 80 H z w R O I M 1000
V max E X P = p M t E
t p i c = 1200 t S t r i p w R O I = 1200 30 m s 5 = 7.2 s

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