SIPcharts using uniform ultra-thin and thin layers for Z-response measurements in two-photon excitation fluorescence microscopy

G. Vicidomini, J. M. Zwier, P. Bianchini, F. Cella, E. Ronzitti, S. Krol, T. Szellas, G. F. Brakenhoff, A. Diaspro

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Layer-by-Layer or self-assembly techniques can be used to prepare fluorescent polymer samples on glass coverslips serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general for sectioning microscopy.1-3 Layers can be realized as ultra-thin (≪ 100 nm) or thin (approx. 100 nm) characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized in a Sectioned Imaging property chart (SIPchart),3 we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the zresponse of the system over a full field of view area. These samples are also useful for monitoring photobleaching behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be efficiently used for real time alignment of the microscope.

Original languageEnglish
Title of host publicationProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume6442
DOIs
Publication statusPublished - 2007
EventMultiphoton Microscopy in the Biomedical Sciences VII - San Jose, CA, United States
Duration: Jan 21 2007Jan 23 2007

Other

OtherMultiphoton Microscopy in the Biomedical Sciences VII
CountryUnited States
CitySan Jose, CA
Period1/21/071/23/07

Fingerprint

Fluorescence microscopy
Microscopic examination
Photons
Microscopes
Polymers
Lighting
Scanning
Imaging techniques
Photobleaching
Molecules
Self assembly
Image processing
Fluorescence
Calibration
Derivatives
Glass
Lasers
Monitoring

Keywords

  • 4Pi microscopy
  • Optical alignment
  • Optical sectioning
  • Shift-variant system
  • Thin fluorescent layer
  • Two-photon excitation microscopy
  • Ultra-thin uniform fluorescent layer
  • z-response

ASJC Scopus subject areas

  • Engineering(all)

Cite this

Vicidomini, G., Zwier, J. M., Bianchini, P., Cella, F., Ronzitti, E., Krol, S., ... Diaspro, A. (2007). SIPcharts using uniform ultra-thin and thin layers for Z-response measurements in two-photon excitation fluorescence microscopy. In Progress in Biomedical Optics and Imaging - Proceedings of SPIE (Vol. 6442). [644224] https://doi.org/10.1117/12.714250

SIPcharts using uniform ultra-thin and thin layers for Z-response measurements in two-photon excitation fluorescence microscopy. / Vicidomini, G.; Zwier, J. M.; Bianchini, P.; Cella, F.; Ronzitti, E.; Krol, S.; Szellas, T.; Brakenhoff, G. F.; Diaspro, A.

Progress in Biomedical Optics and Imaging - Proceedings of SPIE. Vol. 6442 2007. 644224.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Vicidomini, G, Zwier, JM, Bianchini, P, Cella, F, Ronzitti, E, Krol, S, Szellas, T, Brakenhoff, GF & Diaspro, A 2007, SIPcharts using uniform ultra-thin and thin layers for Z-response measurements in two-photon excitation fluorescence microscopy. in Progress in Biomedical Optics and Imaging - Proceedings of SPIE. vol. 6442, 644224, Multiphoton Microscopy in the Biomedical Sciences VII, San Jose, CA, United States, 1/21/07. https://doi.org/10.1117/12.714250
Vicidomini G, Zwier JM, Bianchini P, Cella F, Ronzitti E, Krol S et al. SIPcharts using uniform ultra-thin and thin layers for Z-response measurements in two-photon excitation fluorescence microscopy. In Progress in Biomedical Optics and Imaging - Proceedings of SPIE. Vol. 6442. 2007. 644224 https://doi.org/10.1117/12.714250
Vicidomini, G. ; Zwier, J. M. ; Bianchini, P. ; Cella, F. ; Ronzitti, E. ; Krol, S. ; Szellas, T. ; Brakenhoff, G. F. ; Diaspro, A. / SIPcharts using uniform ultra-thin and thin layers for Z-response measurements in two-photon excitation fluorescence microscopy. Progress in Biomedical Optics and Imaging - Proceedings of SPIE. Vol. 6442 2007.
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abstract = "Layer-by-Layer or self-assembly techniques can be used to prepare fluorescent polymer samples on glass coverslips serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general for sectioning microscopy.1-3 Layers can be realized as ultra-thin (≪ 100 nm) or thin (approx. 100 nm) characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to directly measure the z-response of different scanning optical microscopes. In two-photon excitation microscopy z-sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized in a Sectioned Imaging property chart (SIPchart),3 we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the zresponse of the system over a full field of view area. These samples are also useful for monitoring photobleaching behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be efficiently used for real time alignment of the microscope.",
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AU - Zwier, J. M.

AU - Bianchini, P.

AU - Cella, F.

AU - Ronzitti, E.

AU - Krol, S.

AU - Szellas, T.

AU - Brakenhoff, G. F.

AU - Diaspro, A.

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