Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt

Abstract

This protocol describes procedures for performing fluorescence resonance energy transfer (FRET) microscopy analysis by three different methods: acceptor photobleaching, sensitized emission and spectral imaging. We also discuss anisotropy and fluorescence lifetime imaging microscopy-based FRET techniques. By using the specific example of the FRET probe Akind (Akt indicator), which is a version of Akt modified such that FRET occurs when the probe is activated by phosphorylation, indicating Akt activation. The protocol provides a detailed step-by-step description of sample preparation, image acquisition and analysis, including control samples, image corrections and the generation of quantitative FRET/CFP ratio images for both sensitized emission and spectral imaging. The sample preparation takes 2 d, equipment setup takes 2-3 h and image acquisition and analysis take 6-8 h.

Figure 1

Schematic diagrams depicting the three conditions that must be met for efficient FRET. (a) The energy of donor emission must be an energy that the acceptor can absorb. In other words, the emission spectrum from the donor fluorophore must overlap with the excitation spectrum of the acceptor fluorophore. (b) If the FRET donor and acceptor are more than 10 nm apart, then no FRET occurs and the donor emits fluorescence. If the donor and acceptor are within ~10 nm of one another, then energy transfer can occur from the donor (CFP) to the acceptor (Venus). (c) If the donor and acceptor fluorophore dipoles are perpendicular to one another, then the donor molecule will emit fluorescence. However, if the dipoles are parallel to each other, FRET will occur.

Figure 2

Schematic diagram of the intramolecular Akind FRET probe. This figure was adapted with permission from ref. . Akind consists of an Akt pleckstrin homology (PH) domain, followed by Venus FP, an Akt catalytic domain (CD) and finally CFP. (a) In its basal state, there is minimal FRET, as the CFP and Venus fluorophores are not within 10 nm of one another. Thus, if CFP is excited, cyan fluorescence will be emitted. (b) When Akind is recruited to the plasma membrane via an interaction between its PH domain and PIP₃, it is activated by two phosphorylation events (P). These phosphorylation events cause a conformational change in the Akind probe, bringing the CFP and Venus fluorophores in close-enough proximity to allow FRET to occur.

Figure 3

Acceptor photobleaching within regions of interest (ROI). HT-1080 cells expressing Akind are shown. (a) Images of CFP and Venus FP before (left) and after photobleaching Venus with the 514-nm laser line (right). Red circles denote the ROI where Venus was bleached with the 514-nm laser line, and white circles represent the unbleached control region. (b) Quantification of the change in CFP fluorescent intensity after photobleaching in both an unbleached control region (CFP control region) and a region in which Venus has been photobleached with the 514-nm laser line (CFP bleached region). Error bars represent the s.e.m. for 16 cells (*P = 0.0022). Scale bar, 10 µm. a.u., arbitrary units.

Figure 4

Crucial excitation and emission cross talk corrections. (a) Excitation curves for CFP and Venus showing excitation cross talk (blue region) from the 405-, 440- or 458-nm laser light. In the example shown, when a 440-nm laser is used to excite CFP, a large fraction of the Venus fluorescence (Venus image, based on direct excitation of Venus with 514 nm light) is also excited directly, and the emission enters the FRET image channel (FRET image). Venus excitation cross talk can be corrected for by calculating the ratio between the Venus image when the fluorophore is directly excited (514 nm) and when the fluorophore is excited by the CFP incident light (440 nm) in control cells only expressing Venus. See equation (1) for more details. When performing FRET measurements, a direct image of Venus expression excited at 514 nm is used so that the proportion of Venus direct excitation cross talk can be subtracted from the FRET image. (b) CFP emission is highly overlapping with the emission of Venus, and thus some emission from the CFP fluorophore is transmitted and collected within the FRET image channel (blue region of curve). This emission cross talk will be extensive regardless of which light source is used to excite CFP. A ratio of the CFP signal in the CFP channel and the FRET channel from a sample of cells only expressing cytosolic CFP can be used to correct FRET images for emission cross talk using equation (1). Adapted from http://www.microscopyu.com/articles/fluorescence/fret/fretintro.html with permission.

Figure 5

Spectral imaging of autofluorescence and the CFP-Venus Akind probe. (a) Spectral images of the Akind probe expressed in HT-1080 cells. The spectrum was measured from 421 nm to 645 nm in 10-nm increments using a ×20/0.8-NA objective lens and excitation from a 405-nm laser. Scale bar, 100 µm. (b) Reference spectra that were collected for cellular autofluorescence (green line), CFP (cyan line) and Venus (yellow line) and used for spectral unmixing of lambda image stacks such as the one shown in a. (c) Unmixed images from a showing autofluorescent cells, with a subset expressing the Akind FRET probe. As excitation was only from the 405-nm laser, the Venus image is a result of FRET from CFP to Venus fluorophores. Scale bar, 100 µm.

Figure 6

FRET-FLIM analysis of cytosolic GFP and a FRET-positive GFP-mCherry probe, as well as the effects of the local fluorophore environment. (a) Representative TCSPC decay curves for cytosolic GFP (green) or the FRET-positive GFP-mCherry probe (red), together with the instrumental response function (IRF; black). Note the steeper slope, or shorter GFP lifetime, of GFP-mCherry, indicating that FRET is occurring. (b) FLIM images (spatial distribution of lifetime value) of GFP fluorescence lifetime in HEK-293 cells expressing cytosolic GFP or the FRET-positive GFP-mCherry probe. Yellow and red represent a long lifetime for cytosolic GFP, whereas green and blue show the reduced lifetime due to FRET between GFP and mCherry. Scale bar, 10 µm. (c) Mean lifetime values for cells expressing either cytosolic GFP or the FRET-positive GFP-mCherry are shown for the indicated environmental conditions: live cells in their appropriate growth medium (live), fixed cells mounted with PBS (fixed), ProLong Gold (ProLongGold) or Fluoro-Gel (Fluoro-Gel) mounting medium. (d) Respective FRET efficiencies for cells expressing the FRET-positive GFP-mCherry probe calculated using equation (4). Fixed samples have a similar lifetime and FRET efficiency compared with live samples, whereas the same parameters measured in samples mounted in ProLong Gold or Fluoro-Gel are strongly affected by the mounting procedure. Error bars represent the s.e.m. for 5–7 cells for each condition.

Figure 7

Calculation of FRET/CFP ratio images. (a) Background-subtracted CFP, Venus and FRET_Raw images (first three images) are used with equation (1) (where Venus is the acceptor and CFP is the donor) to create a FRET_Corr image (far right image). (b) Upper images, the newly created FRET_Corr image and the CFP image from a are shown with a rainbow or pseudocolor-coding lookup table to emphasize subtle changes in intensity across the cells. These images are then normalized using the indicated equations to produce images with similar maximum intensity values (lower images). (c) The normalized images from b are then used to create a FRET/CFP ratio image by dividing the FRET_Corr image by the CFP image and multiplying the result by 1,000 (left panel). A median filter was used to reduce the image noise (right panel). Images are shown in pseudocolor coding. Scale bars, 10 µm.

Figure 8

Comparison of the FRET ratio images for wild-type and mutant Akind probes. (a) Representative FRET/CFP ratio images collected with a ×63/1.4 NA objective lens for HT-1080 cells expressing either wild-type Akind (WT-Akind) or a nonactivatable Akind mutant in which three key residues that have been mutated to alanine (3A-Akind). Scale bar, 10 µm. (b) Quantification of the average FRET/CFP ratio in HT-1080 cells expressing either WT-Akind or 3A-Akind. Error bars represent the s.e.m. for at least seven cells (*P = 0.035). The asterisk indicates a statistically significant difference compared with WT-Akind. FRET/CFP ratio images were calculated using equations (1) and (2).

Figure 9

Spectral imaging FRET of WT-Akind and 3A-Akind. (a) Spectrally unmixed images of autofluorescence, CFP and Venus (FRET) for HT-1080 cells expressing either WT-Akind, 3A-Akind or unlinked CFP and Venus collected with an ×63/1.4-NA objective lens. Untransfected HT-1080 cells are also shown for comparison. Note that essentially the entire florescence signal is due to autofluorescence. Scale bar, 10 µm. (b) Quantification of the average FRET/CFP ratio from images collected at ×20/0.8 NA in HT-1080 cells expressing either Wt-Akind or 3A-Akind. Error bars represent the s.e.m. for 10–21 cells (P = 0.0014). The asterisk (*) indicates a statistically significant difference compared with Wt-Akind. FRET/CFP ratio images were calculated using equation (2), where FRET_Corr = FRET_Spec and CFP = CFP_Spec.