Page 204 - Fundamentals of Light Microscopy and Electronic Imaging

AUTOFLUORESCENCE OF ENDOGENOUS MOLECULES 187

1.0
Abs
Alexa Dye 495 519
Em
Alexa 488
Intensity Alexa 546 556 573
578
603
Alexa 568
Alexa 594
617
590
400 500 600 500 600 700
Wavelength (nm) Wavelength (nm)
1.0
Cyanine Abs Em
Dye
Intensity Cy2 492 510
570
550
Cy3
650
670
Cy5
400 500 600 700 400 500 600 700
Wavelength (nm) Wavelength (nm)
1.0 Fluoresc. Abs Em
Protein
EBFP 380 440
Intensity ECFP 433,453 475,501
509
EGFP
488
513
EYFP
527
DsRed 558 583
400 500 600 400 500 600 700
Wavelength (nm) Wavelength (nm)
Figure 11-5
Absorption and emission spectra of recently introduced dyes and proteins for fluorescence
microscopy. The Alexa series of dyes introduced by Molecular Probes, Inc. (Eugene,
Oregon), and the cyanine dyes of Amersham International, Inc. (available from Jackson
ImmunoResearch Laboratories, Inc, West Grove, Pennsylvania), are exceptionally
photostable, have high quantum efficiency, and are soluble in aqueous media. The GFP
series of proteins and DsRed protein, provided as DNA vectors by Clontech, Inc. (Palo Alto,
California), are used to construct fluorescent protein chimeras that can be observed in cells
after transfection with the engineered vectors. This technique avoids the problem of purifying,
tagging, and introducing labeled proteins into cells or having to produce specific antibodies.
Fluorescent protein chimeras are also suitable for studies of protein dynamics in living cells.
For examples and visualization methods, see Sullivan and Kay (1999).

(see Demonstration and Table 11-2). Background fluorescence emission is greatest
when live cells are examined with blue and UV excitation wavelengths. The strength of
endogenous autofluorescence depends on the particular metabolite and excitation wave-
length being employed and also on the cell type. Macrophages, neurons, and sperm cells
exhibit particularly strong autofluorescence. Fixation of cells with aldehydes in prepa-
ration for labeling with fluorescently tagged marker molecules may also induce