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Lasers for Confocal Microscopy All Colors of the Rainbow © Anja Kaiser – Fotolia.Com © Lasers for Confocal Microscopy All Colors of the Rainbow © Anja Kaiser – Fotolia.com © Easy-to-use new lasers are more and more replacing bulky legacy laser systems. This new generation of Keywords lasers provides flexibility, easy operation and is all in all more convenient to use. These novel approaches Laser, Microscopy, Fluorescence, Multicolor offer also new possibilities – supporting the rapid development of new microscopy techniques. Here we will review different laser types and their fields of application. scanning confocal microscope used an ser diodes to directly produce the output air-cooled argon laser for excitation. In wavelength; however for years, it was im- comparison with other light sources la- possible to directly generate wavelengths sers provide a high brightness, a small di- in the blue and green spectral range. In vergence and they are highly monochro- 1998 the emergence of the semiconduc- matic, properties which make them ideal tor material Gallium Nitride (originally light sources for confocal microscopy. developed for the optical data storage in- Gas Lasers such as Ar-ion, Kr-ion and dustry) facilitated the direct generation others are still widely used for micros- of blue wavelengths. These new diode la- Dr. Marion Lang, copy as for a long time only these were sers, above all 488 nm, were quickly es- Technical Marketing Manager, Toptica able to provide the colors and beam tablished. Figure 1 shows a confocal im- Photonics, Gräfelfing, Germany quality required. Occasionally dye lasers age of rat brain axons acquired with the are employed which consist of a solution iBeam smart 488 nm diode laser. This In conventional wide-field microscopes of fluorescent dye pumped by a suitable year another big step was taken: 515 nm arc lamps (e.g. Xe, Hg) are used for illu- laser source. These are able to cover a is now available as direct diode technol- mination. They cover the complete visible broad spectral range but are rather cum- ogy, finally filling the “green gap”. range and thus can excite a broad range bersome to handle. of dyes. Although they have powers of Since a few years diode and DPSS la- Intensity Modulation 100 Watt and more, their radiance (flux sers are available, that fulfill the require- density per unit solid viewing angle) is ments for microscopy such as spectral Intensity modulation or fast wavelength not high enough to give satisfying results stability and beam quality. These com- switching with gas or DPSS lasers re- when applied to point scanning or spin- pact and easy to use lasers are currently quires additional acousto- or electroop- ning disk confocal microscopy. Interest- replacing the antiquated laser systems. tical modulators. In contrast, diode la- ingly, when Marvin Minsky invented the DPSS lasers are diode-pumped solid state sers can be directly modulated at high confocal microscope in 1957, he did not lasers with red or near infrared laser di- frequencies (e.g. the iBeam smart with use a laser for illumination (it was still to odes integrated in a resonator. SHG con- up to 250 MHz). Fast switching is gen- be invented) but a zirconium arc lamp. verts the (infra) red light into the visible erally required to blank the laser dur- The first commercially available laser range. Diode lasers use conventional la- ing fly-back in scanning systems to re- G.I.T. Imaging & Microscopy 2/2011, pp 41–42, GIT VERLAG GmbH & Co. KG, Darmstadt, Germany www.gitverlag.com www.imaging-git.com duce photobleaching. Furthermore, this feature is advantageous for microscopy techniques such as FRAP (Fluorescence Recovery After Photobleaching) or CLEM (Controlled Light Exposure Microscopy), techniques that require rapid switching of wavelengths and intensity modulation. Multicolor Applications For many applications more than one excitation wavelength is required in or- der to localize different structures with respect to each other. Usually, not more than two to four stains are used in one sample. An example to extend four colors Fig. 1: Left: Color-coded z-projection of axons in rat brain, acquired with the iMIC Andromeda (Till in order to discriminate up to 90 colors Photonics) and the iBeam smart 488 nm. Right: iBeam smart at several wavelengths. is the so-called “Brainbow”-technique where individual neurons express sto- chastic combinations of three fluorescent proteins. For multicolor experiments, two different approaches are available: first, the use of white-light lasers and tuna- ble lasers, respectively. In the second ap- proach several lasers are combined in one multi-laser system, whose output is then coupled into the microscope. Multi-laser Engines Fig. 2: Multi-laser engine iChrome MLE As for the latter, stable alignment of mul- tiple laser sources is still an issue which is crucial for a convenient use of such systems. Quite often “breadboard ap- Fig. 4: FLIM measurement of mitochondria proaches” are employed, where lasers (dsRed) in Cos7 cells acquired with the iChrome are mounted on an optical table and TVIS at 550 nm excitation (Courtesy of Dr. Ro- combined via dichroic mirrors. These land Nitschke, Life Imaging Center, Freiburg). approaches, however, are prone to mis- alignment due to environmental in- wavelength range, leading to powers in fluences. An alternative to that is the the range of several mW/nm. The third iChrome MLE which combines up to four concept is a tunable light source which individual lasers (wavelengths from ­­ allows setting all wavelengths in the visi- 405 nm to 640 nm can be selected) within ble range. In this approach narrow band- one box (fig. 2). The individual lasers can width pulses are not filtered from a su- be switched on and off independently via percontinuum; instead ultrashort laser TTL or using an integrated acousto-opti- pulses generate the new colors by four Fig. 3: Maximum projection of a z-stack of a cal modulator (AOM) for versions includ- mouse kidney section excited at 405 nm (DAPI), wave mixing in a highly nonlinear fiber. ing a DPSS laser. The system overcomes 488 nm (Alexa 488) and 561 nm (Alexa 568). SHG converts the output of the fiber, gen- the aforementioned stability issues by an erating laser pulses tunable in the visi- auto-calibration technology, which guar- ble range (from 488 to 640 nm). This ap- antees high and constant optical output (OPO), super continuum light sources proach is realized in the iChrome TVIS. levels. Depending on the lasers included, (also referred to as white light sources) The pulsed operation of the system those systems emit single-line power lev- or tunable visible laser sources. OPOs (3.5 ps) makes it ideally suited for fluo- els of up to 50 mW after fiber delivery have a broad tuning range (~300 nm) rescence lifetime measurements (FLIM). and can be easily integrated into exist- and high maximum output powers A FLIM image of dsRed stained mito- ing setups. Figure 3 shows a maximum (~ 1 W) but are rather complex systems, chondria is shown in figure 4. projection of a mouse kidney section ac- frequently pumped by Titanium:Sapphire quired with the iChrome MLE. lasers. White light sources on the other hand generate a supercontinuum, typi- Contact cally from 460 nm to 2 µm, by using a Dr. Marion Lang Tunable Light Sources Toptica Photonics AG photonic crystal fiber (PCF) and emit all Gräfelfing, Germany The second possibility for multi-color ap- colors simultaneously. Narrow band fil- Tel.: +49 89 85837 123 plications is to use tunable light sources, ters or an AOTF are required for wave- Fax: +49 89 85837 200 which cover the complete visible range length selection. The several Watt output [email protected] such as Optical Parametric Oscillators powers are distributed over the complete www.toptica.com.
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