INVESTIGATIONS OF THE RECOVERY PHENOMENON AFTER LASER EXCITATION IN IMMUNOFLUORESCENCE G . Wick, K. Schauenstein, F. Herzog, and A. Steinbatz
Department of General and Experimental Pathology University of Vienna and C. Reichert Optical Co. Vienna, Austria Our interest in the application of laser excitation for immunofluorescence was triggered b y two facts: the poor performance of conventional light sources and the interesting data of Kaufman e t al.* and Bergquist.2 Kaufman first demonstrated recovery of FITC fluorescence after laser excitation in a sample (Escherichia coli treated with a specific conjugate in a direct immunofluorescence test) subjected t o periods of darkness u p t o 48 hr. Bergquist took advantage of this phenomenon by using a tunable pulsed laser system that allowed extremely short excitation times (0.4-50 psec). Laser pulses were fired manually every 20 sec, and under these conditions, no bleaching was observed with glutaraldehyde-polymerized microspheres of purified human IgG and the appropriate FITC and TRITC conjugates used in a direct test. Our, still preliminary, experiments were performed t o further investigate this interesting recovery phenomenon by varying b o t h illumination time and darkness rest periods, which thus would “spread” the underlying events and hopefully yield further insight into the mechanisms involved. This study was performed with standard immunopathologic systems, namely, indirect immunofluorescence tests for human antinuclear factors assayed o n acetone-fixed 4 pm-thick sections of rat liver and for human antibodies to colloid on buffered formaldehyde-fixed 4 pm-thick sections of a colloid stmma. Direct immunofluorescence tests were performed on myelomatous acetic acid-ethanol-fixed plasma cells. All experiments employed an antihuman IgG FITC conjugate of rabbit origin. We shall now discuss only the experiments with antinuclear factors. Our optical system consisted of a continuously working argon laser (type 162.2, Spectra Physics, Mountain View, Calif.) appropriately fitted t o a Reichert-Zetopan microscope. This laser has an output of 10 mW and affords t w o monochromatic spectral lines, at 488 and 5 1 4 nm. Light of the former line was employed for the present experiments, which involved excitation of FITC only. Advantages of our laser are a long lifetime and relative inexpensiveness. It can, however, only be used for excitation of compounds responsive to light of the two wavelengths mentioned above (e.g., FITC and TRITC), which are, however, the most important for immunofluorescence work. All tests were performed with vertical illumination (wedge of dichroic mirror 518 nm), a 40/0.90 dry objective, and a 4x eyepiece. With the proper optical alignment, the laser beam was collected to illuminate a round area of 250 p m 2 , t o which a total energy of 2.7 x l o 3 W/cm2 was delivered. The illumination time was regulated by a manually operated concentric diaphragm, and the emitted fluorescence was measured b y a type-8LC PIN diode and recorded with a storage oscilloscope. FIGURE 1 is an oscillogram of the fluorescence from a liver cell nucleus after
172
Wick et al. : Recovery Phenomenon
173
FIGURE 1. Fluorescence oscillogram from a rat liver cell nucleus treated with human serum that contained antinuclear factors and an FITC antihuman IgC conjugate. Three fading curves were obtained after repeated laser excitation of 1/50-sec duration interspersed with dark intervals of 20 sec. The lower curves reflect liver cell plasma background fluorescence. Abscissa, 1 msec/cm; ordinate, 200 mV/cm.
laser excitation. The lowest recording represents background fluorescence of the liver cell plasma; the three upper lines are fading patterns of the specific nuclear fluorescence of the same area obtained upon repeated excitation for 1/50-sec durations interspersed with darkness periods of 20 sec. The degree of recovery of initial fluorescence and its decrease after each further exposure t o the laser beam are evident. When darkness periods of less than 2 sec are spaced between the repetitive exposures, no recovery is observed (FIGURE 2). TABLE 1 summarizes the results of an experiment in which both exposure times and darkness periods were varied. The dependence of percentage recovery of initial fluorescence o n laser excitation time and duration of darkness periods is documented. Although the exact mechanism of the recovery phenomenon remains to be determined, it may be suggested that electron transitions of different types can be expected after laser irradiation, and the relaxation of triplet states could elicit the formation of singlet states, which thus would reinforce fluorescence emission. In view of the present data, the shape of the fading curves for FITC conjugates seems to result from several processes that occur in parallel, namely, electron relaxations t o the ground state and irreversible dye decomposition.
174
Annals New York Academy of Sciences
FIGURE 2. Oscillogram from same preparation, obtained with identical laser excitation conditions but with dark intervals of 1-sec duration only.
TABLE I INITIAL FLUORESCENCE RECOVERY (%) AFTER LASER IMPULSE EXCITATION INTERSPERSED WITH DARK INTERVALS
0 0 23.0 Dark intervals (sec)
1
7.1 30.0
54.5 5
66.6 77.7 70.0
84.6
15
60
66.6 72.7
1/10 1/25 1/50
REFERENCES 1. KAUFMAN, G. I., J. F. NESTER & D. E. WASSERMAN. 1971. An experimental study of lasers as excitation sources for automated fluorescent antibody instrumentation. J. Histochem. Cytochem. 19: 469. 2. BERGQUIST, N. R. 1973. The pulsed dye laser as a light source for the fluorescent antibody technique. Scand. J. Immunol. 2: 37.
Department of General and Experimental Pathology University of Vienna and C. Reichert Optical Co. Vienna, Austria Our interest in the application of laser excitation for immunofluorescence was triggered b y two facts: the poor performance of conventional light sources and the interesting data of Kaufman e t al.* and Bergquist.2 Kaufman first demonstrated recovery of FITC fluorescence after laser excitation in a sample (Escherichia coli treated with a specific conjugate in a direct immunofluorescence test) subjected t o periods of darkness u p t o 48 hr. Bergquist took advantage of this phenomenon by using a tunable pulsed laser system that allowed extremely short excitation times (0.4-50 psec). Laser pulses were fired manually every 20 sec, and under these conditions, no bleaching was observed with glutaraldehyde-polymerized microspheres of purified human IgG and the appropriate FITC and TRITC conjugates used in a direct test. Our, still preliminary, experiments were performed t o further investigate this interesting recovery phenomenon by varying b o t h illumination time and darkness rest periods, which thus would “spread” the underlying events and hopefully yield further insight into the mechanisms involved. This study was performed with standard immunopathologic systems, namely, indirect immunofluorescence tests for human antinuclear factors assayed o n acetone-fixed 4 pm-thick sections of rat liver and for human antibodies to colloid on buffered formaldehyde-fixed 4 pm-thick sections of a colloid stmma. Direct immunofluorescence tests were performed on myelomatous acetic acid-ethanol-fixed plasma cells. All experiments employed an antihuman IgG FITC conjugate of rabbit origin. We shall now discuss only the experiments with antinuclear factors. Our optical system consisted of a continuously working argon laser (type 162.2, Spectra Physics, Mountain View, Calif.) appropriately fitted t o a Reichert-Zetopan microscope. This laser has an output of 10 mW and affords t w o monochromatic spectral lines, at 488 and 5 1 4 nm. Light of the former line was employed for the present experiments, which involved excitation of FITC only. Advantages of our laser are a long lifetime and relative inexpensiveness. It can, however, only be used for excitation of compounds responsive to light of the two wavelengths mentioned above (e.g., FITC and TRITC), which are, however, the most important for immunofluorescence work. All tests were performed with vertical illumination (wedge of dichroic mirror 518 nm), a 40/0.90 dry objective, and a 4x eyepiece. With the proper optical alignment, the laser beam was collected to illuminate a round area of 250 p m 2 , t o which a total energy of 2.7 x l o 3 W/cm2 was delivered. The illumination time was regulated by a manually operated concentric diaphragm, and the emitted fluorescence was measured b y a type-8LC PIN diode and recorded with a storage oscilloscope. FIGURE 1 is an oscillogram of the fluorescence from a liver cell nucleus after
172
Wick et al. : Recovery Phenomenon
173
FIGURE 1. Fluorescence oscillogram from a rat liver cell nucleus treated with human serum that contained antinuclear factors and an FITC antihuman IgC conjugate. Three fading curves were obtained after repeated laser excitation of 1/50-sec duration interspersed with dark intervals of 20 sec. The lower curves reflect liver cell plasma background fluorescence. Abscissa, 1 msec/cm; ordinate, 200 mV/cm.
laser excitation. The lowest recording represents background fluorescence of the liver cell plasma; the three upper lines are fading patterns of the specific nuclear fluorescence of the same area obtained upon repeated excitation for 1/50-sec durations interspersed with darkness periods of 20 sec. The degree of recovery of initial fluorescence and its decrease after each further exposure t o the laser beam are evident. When darkness periods of less than 2 sec are spaced between the repetitive exposures, no recovery is observed (FIGURE 2). TABLE 1 summarizes the results of an experiment in which both exposure times and darkness periods were varied. The dependence of percentage recovery of initial fluorescence o n laser excitation time and duration of darkness periods is documented. Although the exact mechanism of the recovery phenomenon remains to be determined, it may be suggested that electron transitions of different types can be expected after laser irradiation, and the relaxation of triplet states could elicit the formation of singlet states, which thus would reinforce fluorescence emission. In view of the present data, the shape of the fading curves for FITC conjugates seems to result from several processes that occur in parallel, namely, electron relaxations t o the ground state and irreversible dye decomposition.
174
Annals New York Academy of Sciences
FIGURE 2. Oscillogram from same preparation, obtained with identical laser excitation conditions but with dark intervals of 1-sec duration only.
TABLE I INITIAL FLUORESCENCE RECOVERY (%) AFTER LASER IMPULSE EXCITATION INTERSPERSED WITH DARK INTERVALS
0 0 23.0 Dark intervals (sec)
1
7.1 30.0
54.5 5
66.6 77.7 70.0
84.6
15
60
66.6 72.7
1/10 1/25 1/50
REFERENCES 1. KAUFMAN, G. I., J. F. NESTER & D. E. WASSERMAN. 1971. An experimental study of lasers as excitation sources for automated fluorescent antibody instrumentation. J. Histochem. Cytochem. 19: 469. 2. BERGQUIST, N. R. 1973. The pulsed dye laser as a light source for the fluorescent antibody technique. Scand. J. Immunol. 2: 37.
