Dr. David L. Wokosin Profile

Dr. David L. Wokosin

Research Associate Professor at Northwestern Univ

SPIE Involvement:

Author

Proceedings Article | 14 February 2008 Paper

Nonlinear excitation fluorescence microscopy: source considerations for biological applications

David Wokosin

Proceedings Volume 6871, 687128 (2008) https://doi.org/10.1117/12.773610

KEYWORDS: Imaging systems, Pulsed laser operation, Fiber lasers, Absorption, Luminescence, Microscopy, Tissues, Laser systems engineering, Second-harmonic generation, Laser sources

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Proceedings Article | 30 March 2005 Paper

Pulse duration spectra and measurements for laser scanning microscope systems

David Wokosin

Proceedings Volume 5700, (2005) https://doi.org/10.1117/12.591167

KEYWORDS: Objectives, Pulsed laser operation, Lenses, Laser systems engineering, Photodiodes, Absorption, Mirrors, Laser scanners, Diodes, Microscopes

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Proceedings Article | 10 May 2001 Paper

Confocal microscopy using a InGaN laser diode at 406 nm

John Girkin, Allister Ferguson, David Wokosin, A. Gurney

Proceedings Volume 4260, (2001) https://doi.org/10.1117/12.426782

KEYWORDS: Semiconductor lasers, Confocal microscopy, Objectives, Argon ion lasers, Head, Ultraviolet radiation, Imaging systems, Prisms, Microscopes, Image resolution

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Proceedings Article | 24 April 2001 Paper

Novel compact sources for multiphoton microscopy

John Girkin, David Wokosin

Proceedings Volume 4262, (2001) https://doi.org/10.1117/12.424553

KEYWORDS: Multiphoton microscopy, Laser sources, Semiconductor lasers, Imaging systems, Absorption, Pulsed laser operation, Mirrors, Femtosecond phenomena, Sapphire lasers, Computing systems

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Proceedings Article | 24 April 2001 Paper

Full systems considerations for multiphoton microscopy

David Wokosin, John Girkin

Proceedings Volume 4262, (2001) https://doi.org/10.1117/12.424558

KEYWORDS: Signal detection, Lenses, Imaging systems, Objectives, Confocal microscopy, Multiphoton microscopy, Luminescence, Near infrared, Microscopy, Sensors

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Proceedings Article | 24 April 2001 Paper

Multiphoton microscopy: a retrospective focused on the visualization of embryo division dynamics

David Wokosin

Proceedings Volume 4262, (2001) https://doi.org/10.1117/12.424544

KEYWORDS: Confocal microscopy, Laser scanners, Luminescence, Microscopy, Multiphoton microscopy, Two photon imaging, Imaging systems, Visualization, Tissues, Laser development

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Proceedings Article | 4 June 1999 Paper

Ultrafast infrared lasers and H2O2 production in living cells

Philip Hockberger, David Wokosin, Hans-Florian Zeilhofer, Dieter Swandulla

Proceedings Volume 3616, (1999) https://doi.org/10.1117/12.351835

KEYWORDS: Luminescence, Sapphire lasers, Ultrafast phenomena, Microscopy, Infrared lasers, Microscopes, Neodymium lasers, Absorption, Ultrafast lasers, Ultrafast imaging

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Proceedings Article | 8 July 1998 Paper

Multiphoton excitation fluorescence imaging applied to the study of embryo development

David Wokosin, John White

Proceedings Volume 3269, (1998) https://doi.org/10.1117/12.312334

KEYWORDS: Luminescence, Microscopy, Confocal microscopy, Multiphoton fluorescence microscopy, Multiphoton microscopy, Ultraviolet radiation, Ultrafast lasers, Green fluorescent protein, Two photon imaging, Laser scattering

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Proceedings Article | 29 April 1998 Paper

Pulse-width considerations for multiple-photon excitation laser scanning fluorescence imaging

David Wokosin, John White, Michael Dymott

Proceedings Volume 3260, (1998) https://doi.org/10.1117/12.307082

KEYWORDS: Luminescence, Dispersion, Microscopes, Absorption, Fiber lasers, Pulsed laser operation, Head, Microscopy, Multiphoton microscopy, Picosecond phenomena

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Fluorescence microscopy is a ubiquitous and powerful tool for the biologist mainly due to the availability of a wealth of highly specific fluorescent probes. Multiphoton (two or more photon) excitation fluorescence microscopy is an optical sectioning technique that offers significant advantages over other optical sectioning techniques in terms of improved viability of living material and the ability to penetrate deeper into specimens. The use of a longer excitation wavelength (typically twice that of the excitation peak of the fluorophore) increases the penetration of the excitation into the sample, yet essentially eliminates single-photon excitation in the bulk of the sample. In order to attain the high peak-power densities necessary for the production of multiphoton events while keeping mean power levels below damaging levels, ultrashort-pulsed excitation sources are used. Some sources, such as mode-locked, Ti:sapphire lasers, can produce pulses less than 100 fs. Pulses this short need to be pre-chirped in order to compensate for the group velocity dispersion of the microscope optics so that the pulse width is maintained at the sample. Without such pre-compensation we show that the average power required to produce a fixed level of two-photon excited signal, using typical microscope optics, is fairly constant from 60fs to 250fs. We argue that the choice of pulse width is an important consideration for a biological imaging system since varying the source pulse width may be used to change the relative amounts of two- and three- photon excitation. With a pre-chirped (compensated) system, if the pulse length is quadrupled then twice the power will be required to attain the previous level of two-photon excited fluorescence, but only half the three-photon excitation (or absorption) will be produced. Pulse widths may be varied on compensated systems by adjusting the pre-compensation. This may be used to favor three-photon excitation of UV-excited fluorophores, or, on the other hand, it may be desirable to reduce levels of three-photon excitation during two-photon imaging of live samples using 700 nm - 800 nm radiation as deleterious excitation of endogenous fluorophores or absorption by (and therefore damage to) proteins and nucleic acids could occur. Variable pulse widths may therefore prove to be an important parameter for live cell studies. Alternatively, for a given range of applications, a simpler and cheaper fixed-pulse length source with the desired characteristics may be chosen.

Proceedings Article | 10 April 1997 Paper

Optimization of the design of a multiple-photon excitation laser scanning fluorescence imaging system

David Wokosin, John White

Proceedings Volume 2984, (1997) https://doi.org/10.1117/12.271272

KEYWORDS: Luminescence, Confocal microscopy, Microscopy, Multiphoton microscopy, Multiphoton fluorescence microscopy, Imaging systems, Mirrors, Objectives, Head, Neodymium lasers

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Multi-photon (two or more photon) excitation imaging offers three significant advantages compared to laser-scanning confocal fluorescence microscopy for 3-D and 4-D fluorescence microscopy: considerable reduction in total sample excitation, increased depth penetration, and increased detection sensitivity. All-solid-state ultra-fast lasers offer tremendous potential for affordable, reliable, 'turn-key' multi-photon excitation sources. We have been developing a multi-photon system that utilizes an all-solid- state Nd:YLF excitation source. We have been evaluating the potential of this source for biological microscopy and have been optimizing system parameters for this application area. We have found that the 1047 nm radiation from these lasers can excite by two-photon fluorescence many commonly used fluorophores that are normally excited from blue to yellow light. In addition, we have found that this wavelength readily excites several normally UV excited fluorophores by the mechanism of three-photon excitation. The Nd:YLF laser has proven reliable in operation with nearly 6000 hours logged without significant loss of power. However, the original system produced rather long pulses for multi-photon excitation (300 fs) and a beam shape that was not ideal. We have recently commissioned the development of an improved pulse compressor from the manufacturers that gives narrower pulses (120 fs), improved beam shape, and a smaller insertion loss. This optimized excitation system has 6 times more potential two-photon excited fluorescence and 22 times more potential three-photon excited fluorescence than the prototype system. In addition, by optimizing coatings in the excitation and signal paths, we have improved the descanned detection sensitivity by 20% for two-photon excited fluorescence and 315% for three-photon excited fluorescence. The excitation optical transfer efficiency (1047 nm) of our imaging system is currently 60% to the back aperture of the objective. The emission optical transfer efficiency (670 nm) is currently 47% for descanned detection and 83% for non- descanned detection; both from the objective back aperture. Surprisingly, we find there is a signal-to-background increase of a factor of 17 between descanned and non- descanned modes of detection using a Nile Red solution sample.

Proceedings Article | 10 May 1996 Paper

Multiple-photon excitation imaging with an all-solid-state laser

David Wokosin, Victoria Centonze, John White, Steven Hird, S. Sepsenwol, Graeme Malcolm, Gareth Maker, Allister Ferguson

Proceedings Volume 2678, (1996) https://doi.org/10.1117/12.239552

KEYWORDS: Confocal microscopy, Luminescence, Pulsed laser operation, Second-harmonic generation, Head, Optical filters, Infrared imaging, Infrared radiation, Imaging systems, Signal detection

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Two-photon excitation imaging is a recently described optical sectioning technique where fluorophore excitation is confined to--and therefore defines--the optical section being observed. This characteristic offers a significant advantage over laser-scanning confocal microscopy; the volume of fluorophore excited in the minimum necessary for imaging, thereby minimizing the destructive effects of fluorophore excitation in living tissues. In addition, a confocal pinhole is not required for optical scattering--thus further reducing the excitation needed for efficient photon collection. We have set up a two-photon excitation imaging system which uses an all-solid-state, short-pulse, long-wavelength laser as an excitation source. The source is a diode-pumped, mode-locked Nd:YLF laser operating in the infrared (1047nm). This laser is small, has modest power requirements, and has proven reliable and stable in operation. The short laser pulses from the laser are affected by the system optical path; this has been investigated with second harmonic generation derived from a nonlinear crystal. The system has been specifically designed for the study of live biological specimens. Two cell types especially sensitive to high-energy illumination, the developing Caenorhabditis elegans embryo and the crawling sperm of the nematode, Ascaris, were used to demonstrate the dramatic increase in viability when fluorescence is generated by two-photon excitation. The system has the capability of switching between two-photon and confocal imaging modes to facilitate direct comparison of theory of these two optical sectioning techniques on the same specimen. A heavily stained zebra fish embryo was used to demonstrate the increase in sectioning depth when fluorescence is generated by infrared two-photon excitation. Two-photon excitation with the 1047nm laser produces bright images with a variety of red emitting fluorophores, and some green emitting fluorophores, commonly used in biological research. Fortuitously, we have found that at least four blue emitting fluorophores normally excited by UV light are excited by the pulsed 1047nm laser, by what we believe to be three-photon excitation. Multi-photon excitation is demonstrated by a double labelled C. elegans embryo.

Showing 5 of 11 publications

Conference Committee Involvement (6)

Multiphoton Microscopy in the Biomedical Sciences VIII

20 January 2008 | San Jose, California, United States

Multiphoton Microscopy in the Biomedical Sciences VII

21 January 2007 | San Jose, California, United States

Multiphoton Microscopy in the Biomedical Sciences VI

22 January 2006 | San Jose, California, United States

Multiphoton Microscopy in the Biomedical Sciences V

23 January 2005 | San Jose, CA, United States

Multiphoton Microscopy in the Biomedical Sciences IV

25 January 2004 | San Jose, CA, United States

Showing 5 of 6 Conference Committees

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