Optical molecular imaging for systems biology: from molecule to organism

Abstract

The development of highly efficient analytical methods capable of probing biological systems at system level is an important task that is required in order to meet the requirements of the emerging field of systems biology. Optical molecular imaging (OMI) is a very powerful tool for studying the temporal and spatial dynamics of specific biomolecules and their interactions in real time in vivo. In this article, recent advances in OMI are reviewed extensively, such as the development of molecular probes that make imaging brighter, more stable and more informative (e.g., FPs and semiconductor nanocrystals, also referred to as quantum dots), the development of imaging approaches that provide higher resolution and greater tissue penetration, and applications for measuring biological events from molecule to organism level, including gene expression, protein and subcellular compartment localization, protein activation and interaction, and low-mass molecule dynamics. These advances are of great significance in the field of biological science and could also be applied to disease diagnosis and pharmaceutical screening. Further developments in OMI for systems biology are also proposed.

Fig. 1

Utilization of OMI in life science research. OMI can investigate the dynamics of biological events in real time from molecules (left), cells, tissues and organisms (right) digitally and quantitatively. It can handle a wide range of intensities (about 12 orders of magnitude), times (femtoseconds to years) and spatial dimensions (nanometers to centimeters), and it gives high spatial and temporal resolution of the targeted cellular structures—better than any other method

Fig. 2a–c

Various FP mutants. a Introduction of the mutation of Thr203His in GFP results in significantly red-shifted maximum excitation and emission wavelengths; this mutant is named YFP. b By using mutagenesis, the original tetrameric DsRed is reconstructed into the monomeric DsRed variant. c Interchanging the amino and carboxyl portions of GFP and rejoining them with a short spacer generates cpGFP

Fig. 3

Method of conjugating QDs to target proteins. The pG-zb acts as a molecular adaptor, connecting the QDs with the target protein through interactions of its protein G portion with a specific antibody as well as interactions of its positively charged tail with QDs capped with a negatively charged dihydrolipoic acid surface

Fig. 4a–f

Fluorescent OMI approaches. a LSCM only collects in-focus emitted light. b The principle of multiple photon excitation is based on the use of pulsed long excitation wavelengths to excite fluorescence. c FRET occurs between a donor and an acceptor that are in molecular proximity if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. d FRAP can reveal the mobility of FP-labeling proteins. These images illustrate the change in fluorescence of cells expressed with YFP-hGR before and after photobleaching. Reproduced from [51] with permission. e FLIM can measure the time-dependent emission intensity. The histogram represents the fluorescence lifetime distributions for the donor in the presence of interactions (red) or not (yellow). Reproduced from [57] with permission. f FCS can monitor the fluorescence signals emitted from the ROI. The cross-correlation curve (black) indicates a higher level of dimer or oligomer formation in the R1- and R5-expressing cells. Reproduced from [59] with permission

Fig. 5a–d

General designs of FRET-based fluorescent probes. a An intermolecular probe consists of two interacting proteins that are labeled with CFP and YFP, respectively, which interact and result in FRET. b An intramolecular probe consists of CFP and YFP fused together with a cleavable linker or protein, which can be cleaved by proteolysis and disrupt FRET. c An intramolecular probe consists of sandwiching two domains between CFP and YFP, which can interact after phosphorylation or binding to calcium, resulting in a change in FRET. d An intramolecular probe consists of CFP, YFP and a protein/domain, which permits conformational change by binding to another biomolecule, leading to a change in FRET

Fig. 6a–d

Single FP-based fluorescent probes. a The probe consists of the fusion of two interacting proteins to two complementary fragments of one FP, respectively, which can interact and reinvoke the fluorescence. b Insertion of a conformationlly responsive domain/protein into cpYFP can lead to a change of fluorescence when its conformation is changed. c The probe consists of the fusion of two interacting proteins/domains to the amino and carboxyl termini of cpGFP, which can interact and change the cpGFP fluorescence. d By using mutagenesis, AFP can be engineered to be directly sensitive to a small molecule, such as Cl⁻, H⁺

Fig. 7a–k

Whole-body OMI using FPs and QDs. Imaging of Ca²⁺ signals in pharyngeal muscles under the conditions of noncontraction (a) and contraction (b) (red color indicates higher calcium) in transgenic C. elegans expressing cameleon. Reproduced from [130] with permission. c–f Ca²⁺ signals evoked by different odors in Drosophila brain expressing G-CaMP. Reproduced from [135] with permission. Imaging of a three-day-old transgenic fish (g) carrying the cameleon and its RB neurons (h) by confocal optical section, and the change in the fluorescence ratio (i) (representing the calcium concentration) in an RB neuron under electrical stimulation of the skin. Reproduced from [136] with permission. j Simultaneous multicolor imaging in a mouse injected with QD-encoded microbeads; k QD imaging of a prostate tumor in the mouse. Reproduced from [33] with permission