A new class of fluorochromes, the carboxymethylindocyanine succinimide esters, has recently been introduced (Southwick et al., 1990). Of these, Cy3
Table 12.4 Typical filter combinations for epi-illumination
Zeiss Filter Set
FITC, DTAF TRITC, Texas red, phycoerythrin, Cy3 AMCA
BP 450-490 BP 546
FT 510 FT 580
LP 520 LP 590
FITC and DTAF give green fluorescence; phycoerythrin gives orange, and AMCA gives blue. Mercury lamps provide strong lines at 313,334,365,405,435, 546 and 578 nm. There is no discrete line in the optimal range for FITC excitation. The filters for TRITC may also be used for Texas red, XRITC and phycoerythrin.
The filter set for AMCA would be suitable for ELF (Enzyme Labeled Fluorescence) substrate for alkaline phosphatase (Molecular Probes).
Filter sets are also available from Omega Optical, distributed by Molecular Probes. The filter set 0-5857 is a high-efficiency red, green and blue triple-band filter set, which works well for multicolour experiments using Texas red, FITC or DTAF and AMCA or Cascade blue. More details on filter sets are given by Haugiand (1992).
and Cy5 are the most promising. They have high quantum yields, although they are prone to self-quenching if antibodies are over-conjugated (Southwick et ah, 1990, and see Section 12.8.5). Cy3 has an absorption maximum at 550 nm and an emission maximum at 565 nm; it can be excited at about 50% efficiency with the 514 nm line of the argon laser, or to about 75% by the 546 nm line of the mercury lamp. Cy3 has very similar spectral properties to TRITC, and so the filter combinations for TRITC are suitable (Table 12.4).
Cy5 has an absorption maximum at 652 nm and an emission maximum at 667 nm. It is suitable for specialized imaging systems, such as those using CCD cameras, which work in the long emission wavelength range. It can be excited efficiently by the krypton/argon laser at 647 nm or the He/Ne laser at 633 nm, or by mercury or tungsten-halogen lamps. Cy5 is sometimes used in conjunction with other fluorochromes in 'energy transfer' systems, where the emission of a shorter wavelength is converted to a longer wavelength by Cy5 (see Table 12.2).
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