Blue laser diode excitation on the BD LSR II
Blue laser diodes (BLDs) emitting at 440 nm were originally developed for the telecommunications industry for fiber optic signal transmission. Several laser manufacturers have subsequently developed modules that are applicable for integration into flow cytometers. While the selection of fluorochromes that are well-excited by a blue diode laser line are limited, they in fact do emit near the excitation maxima of Cyan Fluorescence Protein. Normally a violet (400-410 nm) or a argon-ion blue (457 nm) line is used for CFP excitation. These excitation lines are less-than-optimal and are therefore compromises, since there was previously no easily obtainable laser line in the 430 to 440 nm excitation maxima of CFP. CFP is now broadly used in CFP-YFP FRET systems; therefore, it was therefore of interest whether the blue diode line would excite CFP more efficiently than the more commonly available violet laser diode line.
(Below). Blue laser diodes (BLD). A blue laser diode from Power Technology, Inc., mounted on the BD LSR II. This particular laser emitted at 440 nm at approximately 4 mW power level. Versions emitting at 20 mW are becoming available.
BLD mounting scheme on the BD LSR II. Since the BLD emits at a wavelength longer than the Vioflame VLD, it cannot be mounted in the UV laser position as the near-UV laser diodes have. A 460 LP laser dichroic mounted in a three-axis gimbaled holder was suspended at a 45 degree angle from a section of optical rail mounted above the optical bench, intersecting the red and blue-green laser lines. The blue diode was then mounted on the LSR II between the UV and violt laser positions, and the beam aligned to the 460 LP dichroic. The blue beam was then steered to the flow cell, and the resulting signal aligned to the violet diode pinhole (position 2). The newly inserted dichroic effectively reflected and steered the blue beam, with minimal interference with the blue-green and red beams.
(Below). BLD mounting scheme. A 460 LP dichroic was mounted downstream of the red laser dichroic, and the blue diode mounted between the UV and violet positions and its beam aligned to this new dichroic.
(Below). BLD mounting scheme. The BLD is visible on the left side of the photograph. The 460 LP dichroic is visible on the right, suspended from a section of optical rail mounted over the optical bench. Red an blue-green laser beams were able to pass through this dichroic with minimal attentuation.
(Below). BLD alignment. The BLD beam is shown reflecting off the 460 LP dichroic, passing through the focusing optics and the flow cell.
(Below). Alignment of BLD on the BD LSR II. Polyscience 2 micron yellow-green beads were used to align the BLD into the violet laser pinhole. BLD-excited signals were detection in the violet laser trigon (usually in the Cascade Yellow detector position).
BLD excitation of Cyan Fluorescence Protein. Although the selection of fluorochrome candidates for BLD excitation is small, its potential ability to excite Cyan Fluorescence Protein (CFP) is a significant application for this laser. CFP is now widely used as a donor fluorochrome for CFP-YFP FRET systems; unfortunately, the 430 - 440 nm excitation maxima of CFP has not been covered by any laser source that was either easily obtainable or readily integrated into a flow cytometer. Violet laser lines (i.e. 407 and 413 nm from krypton-ion sources, and 400 to 410 nm lines from diode sources) have been most commonly used to excite CFP - however, these lines are low on the shoulder of the CFP excitation maxima. Similarly, the 457 nm line used for simultaneous CFP-GFP-YFP excitation is to long and less than optimal. SP2/0 and NIH 3T3 cell lines constituitively expressing ECFP (Bob and Teresa Hawley, American Red Cross Holland Laboratory) were therefore analyzed on the BD LSR II equipped with the BLD described above. BLD excitation at 4 mW gave approximately a 3-4-fold increase in CFP fluorescence over a 25 mW violet diode source (see below).
(Below). BLD and VLD excitation of CFP. VLD (top row) or BLD (bottom row) excitation of SP2/0 (left column) or NIH 3T3 cells (right column) constituitively expressing ECFP. A 495/20 nm narrow bandpass filter sandwiched with a 488 notch filter was used to detect ECFP.
(Below). BLD excitation of CFP with different laser dichroics. BLD excitation with the 460 LP dichroic described above (top row) or a 480 LP dichroic (bottom row) of SP2/0 (left column) or NIH 3T3 cells (right column) constituitively expressing ECFP. A 495/20 nm narrow bandpass filter sandwiched with a 488 notch filter was used to detect ECFP in both cases
Although the BLD excited CFP at a higher signal-to-noise ratio than the VLD, there are some practical problems with its use. It is likely the 440 nm line excites a wide variety of endogenous proteins, resulting in elevated cellular autofluorescence. In addition, the 440 nm line is likely to excite YFP to some extent, potentially complicating CFP-YFP FRET analysis. These issues are currently being assessed.
BLD excitation of Quantum Dots. Since the BLD emits at wavelengths below the currently available panel of Quantum Dots (Qdots), the laser was assessed for its ability to excite these fluors. EL4 cells were labeled with biotin-conjugated anti-CD44 or CD90, followed by streptavidin conjugates of Qdot 525, 565, 585 or 605. BLD excitation of Qdot labeled cells was only slightly lower than wih the standard VLD.
(Below). BLD or VLD excitation of Qdot 525 and Qdot 565. EL4 cells were labeled with CD44 or CD90 via indirect labeling with Qdot 525 (left histograms) or Qdot 565 (right histograms) as described above and analyzed with either BLD (top row) or VLD (bottom row) excitation.
(Below). BLD or VLD excitation of Qdot 585 and Qdot 605. EL4 cells were labeled with CD44 or CD90 via indirect labeling with Qdot 585 (left histograms) or Qdot 605 (right histograms) as described above and analyzed with either BLD (top row) or VLD (bottom row) excitation.
