Green Wavelength Technology
Table 1: Excitation and emission peaks of some common fluorophores and genetically expressed fluorescent proteins.
Fluorophore name Excitation (nm) Emission (nm) DAPI BFP GFP FITC
eGFP
Alexa Fluor 488 YFP
TRITC RFP
MitoTracker™ Red mCherry
Texas Red Cy 5 Cy 7
360 380 395 475 488 499 514 554 555 577 585 589 647 753
460 440 509 520 509 520 530 578 585 600 610 615 665 775
achieve maximum excitation efficiency of their fluorophores. Researchers are now requesting LED replacements equiva- lent to the full range of the mercury spectrum to be able to enjoy the technological advantages of LEDs, including longer life, lower environmental footprint, smaller size, and reduced price/Watt power benefits. To meet this demand, light source manufacturers must ensure that LEDs used for microscopy illumination can cover the key components of the spectrum including the challenging “green gap” at sufficient power levels.
The Green Gap Challenge Te challenge faced by manufacturers is to design systems
that cover the same portion of the spectrum as the mercury arc lamp in order to efficiently excite common fluorophores used in fluorescence studies. Te most challenging wavelength band for LED manufacturers to match has been the 540 to 590 nm range, known in the solid-state lighting industry as the green gap (Figure 3). Emission in this region of the spectrum is fun- damentally limited by the lack of semiconductor materials to efficiently emit light in this range of wavelengths. LED manu- facturers for microscopy and fluorescence excitation have struggled with this for many years, and some have generated innovative solutions to bridge the gap. A range of solutions with varying degrees of success are now available, includ- ing LED arrays and wavelength conversion technology such as phosphor materials combined with LEDs. However, many microscopy systems continue to rely on lasers or even mercury arc lamps to efficiently excite fluorophores at these challenging wavelengths.
The Solution Wavelength conversion with phosphor materials has
Figure 3: Mercury lamp spectrum with the LED green gap excitation range highlighted in green.
YFP (yellow fluorescent protein), mCherry, and RFP (red fluo- rescent protein) have gained popularity (Table 1). Unlike a fluorophore that is intro-
duced into cells from an external source, the organism of interest intrinsically manufactures genetically expressed fluorescent proteins, thus eliminating stress on the cell and providing a less invasive source of fluorescence label- ing. Excitation of fluorophores like TRITC and fluorescence proteins like YFP, mCherry, and RFP require green light for excitation, and subsequently emit red fluorescence. With LEDs becoming more prev-
alent as an illumination source, inves- tigators must plan their experiments according to the difference in peak optical power between traditional lamps and LEDs in order to ensure optimization of their filters and to
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been used in the lighting and projection industries for several decades. Shorter wavelength light (blue, violet, UV) is absorbed by a material that re-emits light of a longer wavelength via phosphor conversion [3]. Excelitas Technologies’ LaserLED Hybrid Drive uses patented laser phosphor conversion tech- nology to generate high-power light to fill in the green gap region (Figure 4, Patent US #9,239,133). Tis system uses high- efficiency blue lasers to excite a phosphor layer, generating a
Figure 4: Comparison of LED power in the green gap without LaserLED Hybrid Drive (blue line) vs. with Laser- LED Hybrid Drive (green line).
www.microscopy-today.com • 2020 July
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