Yellow Lasers for Flow Cytometry: An Update
William Telford1, Claudette Linton2 and Vladimir Karpov2
1National
Cancer Institute, National Institutes of Health, Bethesda, MD USA
2MPB Communications, Montreal, Quebec, Canada
Abstract
DPSS 580 nm
Yellow laser excitation (ranging from 560 to 600 nm) has been difficult to achieve in benchtop flow
cytometry due to limits on laser technology. Nevertheless, this wavelength range is potentially very useful
for the excitation of a variety of fluorochromes. DPSS 561 nm lasers have been previously shown to give
excellent excitation of PE, DsRed, lissamine rhodamine and Alexa Fluor 568, but their wavelength is too
short for optimal excitation of yellow- and red-excited fluorochromes such as Texas Red, Alexa Fluor 594,
APC and the APC tandem conjugates. Yellow HeNe lasers have been used in the past for exciting these
fluorochromes, but their inherently low power level has limited their usage in flow cytometry.
Recent advances in solid state laser technology, however, have provided several new options for yellow
excitation with increased power levels. This report evaluates several recently developed solid state yellow
lasers sources, including Yb-doped fibre laser with a PPLN frequency doubler emitting at 580 nm, and a
diode-pumped solid state 593 nm laser. Both of these units provided good excitation of Texas Red and
Alexa Fluor 594, while still giving APC and APC tandem conjugate excitation that was comparable to red
laser sources. This combined technology is therefore closing a significant gap in the laser wavelengths
available for benchtop flow cytometry.
Solid-state 580 nm fibre lasers are now
available (MPB Communications, Inc.). This
unit emitted at >500 mW but could be
attentuated using a beamsplitter to power
levels appropriate for cytometry. This
wavelength excited Texas Red, Alexa Fluor
594 and DsRed extremely well, and gave
adequate excitation of APC and its tandems.
steering
mirrors
Fibre-coupled 580 nm
laser excitation
630/22
nm
filter
Texas Red
Yellow laser excitation (between 560 and 600 nm) is potentially very useful for
flow cytometry. Excitation in the 560 to 580 nm range should give excellent
excitation of lissamine rhodamine, Alexa Fluor 568, and the fluorescent protein
DsRed. Longer wavelength yellow light can excite Texas Red and Alexa Fluor
594, while still providing good excitation of APC and its tandem conjugates.
561 nm
Lissamine
rhodamine
excitation
emission
Excitation/emission spectra for
lissamine rhodamine and Alexa Fluor
568 (left), DsRed (below) and Alexa
Fluor 594, APC and the
APC tandem conjugates in a potential
four color labeling scheme (right).
Proposed laser lines are indicated.
594 nm
580 nm laser head
90% beamsplitter
pump laser
input fiber
Cy5
InSpeck Deep Red
microspheres
Spherotech Rainbow
microspheres
APC
APC-Cy5.5
30%
2
4
3%
630/22
nm
filter
0.3%
5
1%
7
excitation
emission
DsRed
unlabeled
APC-Cy5.5
APC
emission
6
8
APCCy7
Relative fluorescence 630/22 nm filter
Alexa Fluor 594
excitation
emission
500
550
600
650
700
750
800
350
400
450
500
550
600
650
700
450
500
550
600
650
700
750
800
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Yellow laser light has nevertheless been technologically difficult to obtain using
traditional laser sources. Krypton-ion sources produce a 568 nm line, but can only
be accommodated on large-scale cytometers. Yellow HeNe lasers emitting at 594
nm have been available for years yet are relatively low in power (<8 mW). Dye
head lasers (using a high-power argon-ion pump laser and a dye head running
R6G) produce a range of yellow and orange lines, but are difficult to maintain and
are also restricted to larger cell sorters.
Kr-ion 568 nm
Dye head R6G 600 nm
DsRed fluorescence
SP2/0 cells
Above, DsRed analysis using DPSS 488 nm (left) and 580 nm
sources (middle and right). Power levels are indicated
Above, MPI InSpeck Deep Red and Spherotech
Rainbow microspheres excited with the DPSS 580
nm source.
a
v
u
fltiR
n
c
s
re
o
450
DPSS 580 nm
50 mW
3
488 nm 561 nm 580 nm
Alexa Fluor
568
DPSS 580 nm
25 mW
DPSS 488 nm
50 mW
1
10%
APC-Cy7
APC
CD90 expression
Above, EL4 cells labeled with biotin-anti-CD90 followed by the
indicated fluorochrome-conjugated streptavidin. Texas Red and
Anexa Fluor 594 were detected through a 630/22 nm filter.
580 nm
Alexa Fluor 594
Alexa Fluor 633 Alexa Fluor 647
EL4 cells
Above, the emission cavity of the DPSS 580 nm
laser mounted on a BD LSR II. Pump laser input
fiber is visible on the left.
excitation
Alexa Fluor 594
660/20
nm
filter
HeNe 594 nm
DPSS 593.5 nm
DPSS 593.5 nm lasers are not yet available
from North American or European companies,
but are obtainable from Chinese suppliers.
This unit (spec’d at 30 mW, low noise)
had significant red and infrared emission,
DPSS 593.5 nm 30 mW
requiring multistep filtering, with a final
Shanghai Dream Laser
593.5 nm output between 10 and 15 mW.
The laser beam profile was good, and the unit could be implemented on a the BD
LSR II. However, power level was still slightly unstable over long periods.
Below, Optical scheme to remove red and IR
emission from the 593.5 nm beam. The filtered
beam profile was good, but power level over
extended operation was unstable (here compared
to a HeNe 594 nm laser, at 5 sec intervals over 1
hour.
DPSS
593.5 nm
10 mW
593.5 nm
10-15 mW
Recent advances in solid state laser technology have produced several
laser sources in this wavelength range that are useful for flow cytometry.
HeNe
633 nm
17 mW
IR blocking
filter
DPSS 593.5 nm
>50 mW
APC detector
660/20 nm
High-power DPSS 561 nm
HeNe
633 nm
17 mW
DPSS 488 nm
APC detector
660/20 nm
APC-Cy5.5 detector
710/20 nm
APC-Cy7 detector
800/60 nm
IR filtered
IR filtered
DPSS
593.5 nm
10 mW
DPSS 561 nm
HeNe
594 nm
7 mW
610/20
nm
filter
Left, 561 nm excitation of Spherotech Rainbow microspheres with
the indicated emission filter.
DPSS 593.5 nm
DPSS 593.5 nm
DPSS 561 nm 50 mW
Cobolt Sweden
HeNe 594 nm
Alexa Fluor 647
660/20 nm
DsRed fluorescence
Above, DsRed fluorescence in stably transfected
SP2/0 cells, at 488 nm (left) or 561 nm (right).
APC
660/20 nm
APC-AF680
710/20 nm
APC-Cy7
800/60 nm
Above, EL4 cells labeled with biotin-anti-CD44, followed by the
streptavidin conjugated to the indicated fluorochrome. Excitation with
the DPSS 593.5 nm, with HeNe 633 and 594 nm lasers for
comparison.
visible red
and some IR
640 LP
dichroic
APC-Cy7 detector
800/60 nm
Above, MPI InSpeck Deep Red microspheres excited with the
DPSS 593.5 nm or a HeNe 633 nm laser for comparison. Emission
filters were as indicated.
DPSS 561 nm lasers have been previously
shown to give excellent excitation of PE and
DsRed, as well as the low molecular weight
fluorochromes lissamine rhodamine, Alexa
Fluor 568, Texas Red and Alexa Fluor 594.
The power level available at this wavelength
has since increased to 50 mW.
Spherotech
Rainbow
microspheres
APC-Cy5.5 detector
710/20 nm