Abstract

Recent advances in fluorescence confocal microscopy have focused on exciting multiple dyes, leading to the use of diode laser sources. We show that by varying a diode’s operating parameters, diode lasers can address some of the inherent problems associated with multiple dye excitation. Cooling the laser diode caused its emission wavelengths to decrease linearly, its output power to increase five times, and the noise due to reflected laser light for equal fluorescence signals to be reduced five times. We utilized these improvements to produce fluorescence confocal images that minimized the reflected laser intensity, while still efficiently exciting the fluorochrome.

© 2004 Optical Society of America

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References

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  1. J. M. Girkin; A. I. Ferguson; D. L. Wokosin; and A. M. Gurney, �??Confocal microscopy using and InGaN violet laser diode at 406nm�??, Opt. Express 7, 336-41 (2000), <a href="http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-10-336">http://www.opticsexpress.org/abstract.cfm?URI=OPEX-7-10-336</a>
    [CrossRef] [PubMed]
  2. See for example Bio-Rad�??s Radiance2100�?�, <a href="http://cellscience.bio-rad.com/products/confocal/Radiance2100/default.htm">http://cellscience.bio-rad.com/products/confocal/Radiance2100/default.htm</a>
  3. See for example Coherent Inc.�??s data sheet <a href="http://www.coherent.com/Downloads/Radius635_DSandHeNeCC_100103.pdf.">http://www.coherent.com/Downloads/Radius635_DSandHeNeCC_100103.pdf</a>.
  4. P. M. Conn, Methods in Enzymology Volume 307 Confocal Microscopy (Academic Press, San Diego, 1999).
    [CrossRef]
  5. C. J. R. Sheppard and D. M. Shotton, Confocal Laser Scanning Microscopy (Springer-Verlag, New York, 1997).
  6. C. J. Hawthorn, K. P. Weber, and R. E. Scholten, �??Littrow configuration tunable external cavity diode laser with fixed direction output beam�??, Rev. Sci. Instrum 72, 4477-4497 (2001).
    [CrossRef]
  7. S.M. Sze, Physics of Semiconductor Devices (John Wiley & Sons, New York, 1981), p. 264.
  8. S.M. Sze, Physics of Semiconductor Devices (John Wiley & Sons, New York, 1981), p. 91-92.
  9. Bio-Rad Fluorochrome database, <a href="http://cellscience.bio-rad.com/fluorescence/fluorophoreDatab.htm">http://cellscience.bio-rad.com/fluorescence/fluorophoreDatab.htm</a>

Methods in Enzymology (1)

P. M. Conn, Methods in Enzymology Volume 307 Confocal Microscopy (Academic Press, San Diego, 1999).
[CrossRef]

Opt. Express (1)

Rev. Sci. Instrum (1)

C. J. Hawthorn, K. P. Weber, and R. E. Scholten, �??Littrow configuration tunable external cavity diode laser with fixed direction output beam�??, Rev. Sci. Instrum 72, 4477-4497 (2001).
[CrossRef]

Other (6)

S.M. Sze, Physics of Semiconductor Devices (John Wiley & Sons, New York, 1981), p. 264.

S.M. Sze, Physics of Semiconductor Devices (John Wiley & Sons, New York, 1981), p. 91-92.

Bio-Rad Fluorochrome database, <a href="http://cellscience.bio-rad.com/fluorescence/fluorophoreDatab.htm">http://cellscience.bio-rad.com/fluorescence/fluorophoreDatab.htm</a>

C. J. R. Sheppard and D. M. Shotton, Confocal Laser Scanning Microscopy (Springer-Verlag, New York, 1997).

See for example Bio-Rad�??s Radiance2100�?�, <a href="http://cellscience.bio-rad.com/products/confocal/Radiance2100/default.htm">http://cellscience.bio-rad.com/products/confocal/Radiance2100/default.htm</a>

See for example Coherent Inc.�??s data sheet <a href="http://www.coherent.com/Downloads/Radius635_DSandHeNeCC_100103.pdf.">http://www.coherent.com/Downloads/Radius635_DSandHeNeCC_100103.pdf</a>.

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Figures (9)

Fig. 1.
Fig. 1.

(a) HeNe emission and Cy5 absorption spectra. The Cy5 absorbs the laser excitation with ~50% efficiency. (b) Cy5 fluorescence and 660nm barrier filter spectra show that much of the short fluorescence wavelengths are blocked by the filter, resulting in very faint fluorescence images. (c) The HeNe laser emission and 640nm barrier filter spectra show that the filter transmits a significant portion of the reflected HeNe spectrum. Only the bottom 0.1% intensity of the laser spectrum is shown, to approximately match the intensity of the Cy5 fluorescence, which was normalized to 1.0.

Fig. 2.
Fig. 2.

Diode current vs. diode voltage. The solid black curves are constant power, with the 60mW curve the upper bound on power applied to the diode.

Fig. 3.
Fig. 3.

Laser power as a function of temperature, showing a power maximum around -80°C and parabolic behavior down to -120°C. The power meter saturated at output powers above ~20mW (adjusted for fiber optic transmission losses).

Fig. 4.
Fig. 4.

(a) Spectral response of the laser diode at 20mA and three different temperatures, showing the shift to shorter wavelengths with lower diode operating temperature. Intensity is in arbitrary units. (b) Peak wavelength versus diode operating temperature is linear, demonstrating the wavelength tunability of cooled diode lasers.

Fig. 5.
Fig. 5.

(a) Diode laser emission and barrier filter spectra, showing the barrier filter almost completely blocks the emission from a diode cooled to -196°C, while transmitting more when the diode operates at room temperature. (b) Laser diode emission and Cy5 absorption spectra, showing that at -196°C the diode laser excites the fluorochrome nearly as efficiently as at 23°C.

Fig. 6.
Fig. 6.

(a) Fluorescence signals produced by exciting Cy5 with the laser diode operated at constant current, but two different temperatures: 23°C and liquid nitrogen temperature. (b) Relative noise level – for equal fluorescence signal maxima – due to reflected laser intensities after passing through the 640nm barrier filter with the diode operated at 23°C and liquid nitrogen temperature. The noise (reflected laser light) is decreased by a factor of 5 at -196°C, resulting in a factor of 5 improvement in signal-to-noise ratio.

Fig. 7.
Fig. 7.

(a) Laser power output as a function of diode current for various temperatures. The threshold current is the x-axis intercept of each line. The threshold current decreased by 75% as the temperature was decreased from room temperature to -170°C. (b) Threshold current as a function of temperature, showing the usual exponential temperature dependence.

Fig. 8.
Fig. 8.

Diode efficiency as a function of temperature at various operating currents. At around – 80°C, the diode efficiency was the greatest, independent of operating currents. The precise maximum efficiencies for the 24mA and 27mA data sets are not known, since the power meter saturated above 20mW (adjusted for fiber optic transmission losses).

Fig. 9.
Fig. 9.

Fluorescence confocal microscope images of filter paper fibers soaked in Cy5. The images were taken while operating the diode laser at different subambient temperatures ranging from 6°C (top left), to -49°C (top right), to -97°C (bottom right), to -196°C (bottom left), which shows the greatest contrast.

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