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. 1992 Mar;13(3):131-47.
doi: 10.1016/0143-4160(92)90041-p.

Fluorescence lifetime imaging of calcium using Quin-2

J R Lakowicz  1 , H Szmacinski, K Nowaczyk, M L Johnson
Affiliations

Fluorescence lifetime imaging of calcium using Quin-2

J R Lakowicz et al. Cell Calcium. 1992 Mar.

Abstract

We describe the use of a new imaging technology, fluorescence lifetime imaging (FLIM), for the imaging of the calcium concentrations based on the fluorescence lifetime of a calcium indicator. The fluorescence lifetime of Quin-2 is shown to be highly sensitive to [Ca2+]. We create two-dimensional lifetime images using the phase shift and modulation of the Quin-2 in response to intensity-modulated light. The two-dimensional phase and modulation values are obtained using a gain-modulated image intensifier and a slow-scan CCD camera. The lifetime values in the 2D image were verified using standard frequency-domain measurements. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which may allow Ca2+ imaging using other Ca2+ probes not in current widespread use due to the lack of spectral shifts. Fluorescence lifetime imaging can be superior to stationary (steady-state) imaging because lifetimes are independent of the local probe concentration and/or intensity, and should thus be widely applicable to chemical imaging using fluorescence microscopy.

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Figures

Fig. 1
Fig. 1
Intuitive presentation of the concept of Fluorescence Lifetime Imaging (FLIM). It is assumed that the object has two regions which display the same fluorescence intensity (I1 = I2) but different decay times, τB > τF; a – object; b – color or grey-scale Ca2+ image; c – lifetime contour Ca2+ image
Fig. 2
Fig. 2
Schematic diagram of a FLIM experiment. The ‘object’ consists of a row of cuvettes, each with a different [Ca2+] and lifetime. This ‘object’ is illuminated with intensity modulated light. The spatially and temporarily-varying emission is detected with a gain-modulated image intensifier, which acts like a phase-sensitive detector and is imaged onto a CCD camera. A series of phase-sensitive images are used to compute the phase angle, modulation and/or lifetime images. The light source is a cavity-dumped dye laser
Fig. 3
Fig. 3
Absorption (A) and emission spectra of Quin–2 (B) in the presence of increasing amounts of Ca+. For the emission spectra the excitation wavelength was 342 nm. The dashed line (lower panel) shows the transmission of the Corning 3-72 emission filter used to isolate the emission during the FLIM or FD measurements
Fig. 3
Fig. 3
Absorption (A) and emission spectra of Quin–2 (B) in the presence of increasing amounts of Ca+. For the emission spectra the excitation wavelength was 342 nm. The dashed line (lower panel) shows the transmission of the Corning 3-72 emission filter used to isolate the emission during the FLIM or FD measurements
Fig. 4
Fig. 4
Frequency-response of Quin–2 in the presence of increasing amounts of Ca+. See Table 1 for additional detail and data
Fig. 5
Fig. 5
Above: Ca2+-dependent lifetime, intensity and fractional saturation of Quin–2. The fractional saturation was obtained from α2/(α1 + α2) Right: The dissociation constants (KD) were obtained from plots at log {(F − Fmin)/(Fmax − F)}, log{(τ¯−τ¯min)∕(τ¯max−τ¯)} or log {(α2 − α2 min)/(α2 max − α2)} versus log [Ca2+] where τ¯−f1τ1+f2τ2 from the double exponential fits (Table 2)
Fig. 5
Fig. 5
Above: Ca2+-dependent lifetime, intensity and fractional saturation of Quin–2. The fractional saturation was obtained from α2/(α1 + α2) Right: The dissociation constants (KD) were obtained from plots at log {(F − Fmin)/(Fmax − F)}, log{(τ¯−τ¯min)∕(τ¯max−τ¯)} or log {(α2 − α2 min)/(α2 max − α2)} versus log [Ca2+] where τ¯−f1τ1+f2τ2 from the double exponential fits (Table 2)
Fig. 6
Fig. 6
Ca2+-dependent phase and modulation values for Quin–2 at 34.155, 49.335 and 72.105 MHz
Fig. 7
Fig. 7
Phase-sensitive intensities of Quin–2 collected with the FLIM apparatus. θI is the instrumental phase shift between the modulated excitation and the intensifier gain modulation. The value of θI was determined from the known phase of the reference sample (θR = 72.3° for 602 nM Ca2+) using θi = θ′R − θR, where θ′R = 78.7° is the observed phase of the reference
Fig. 8
Fig. 8
Phase-sensitive images of Quin–2 measured at various detector phase angles
Fig. 9
Fig. 9
FLIM images of Ca2+ obtained from the phase angle image. The phase lifetimes were obtained from the calibration curve in Figure 6
Fig. 10
Fig. 10
FLIM images of Ca2+ obtained from the modulation image. The modulation lifetimes were obtained from the calibration curve in Figure 6
Fig. 11
Fig. 11
Ca2+ imaging using phase and modulation color scales. The color changes in the cuvettes from left to right indicate increasing phase angles and decreasing modulation. The lowest row of images shows the intensity images
Fig. 12
Fig. 12
Phase and modulation lifetime images of Ca2+. The color scale represents the apparent lifetimes
Fig. 13
Fig. 13
Intuitive descriptions of phase suppression. In a difference image with ΔI = I (θD + 180) − I(θD) a component with θ = θD is completely suppressed. Components with longer lifetimes (phase angles) appear to be negative, and those with shorter lifetimes (phase angles) appear to be positive
Fig. 14
Fig. 14
Ca2+ images with suppression of regions with [Ca2+] ≥ 80 nM (top) and Ca2+ ≤ 17 nM (bottom). The upper and lower suppression images were calculated from the phase sensitive images I(θ′D) using I(348.2°) − I(152.4°) and I(152.4°) − I(304.6°).
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