Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models

Abstract

Bioluminescent and fluorescent reporter systems have enabled the rapid and continued growth of the optical imaging field over the last two decades. Of particular interest has been noninvasive signal detection from mammalian tissues under both cell culture and whole animal settings. Here we report on the advantages and limitations of imaging using a recently introduced bacterial luciferase (lux) reporter system engineered for increased bioluminescent expression in the mammalian cellular environment. Comparison with the bioluminescent firefly luciferase (Luc) system and green fluorescent protein system under cell culture conditions demonstrated a reduced average radiance, but maintained a more constant level of bioluminescent output without the need for substrate addition or exogenous excitation to elicit the production of signal. Comparison with the Luc system following subcutaneous and intraperitoneal injection into nude mice hosts demonstrated the ability to obtain similar detection patterns with in vitro experiments at cell population sizes above 2.5 × 10(4) cells but at the cost of increasing overall image integration time.

Figure 1

Pseudocolor representation of the bioluminescent or fluorescent flux from cell concentrations ranging from 1 million (1M) to several thousand (K) to approximately single cell levels (NEG = negative control wells) stably transfected with (a) holux, (c) Luc, or (e) GFP. Red lines indicate the combination of two separate runs, each represented by the corresponding color scale on the right or left side of the figure. The yellow box in (e) indicates wells containing equal numbers of untransfected HEK293 cells to determine levels of background autofluorescence. Note that autoscaling of the pseudocolor image assigns brighter colors and larger areas to the larger population sizes of low level detection experiments although their scale indicates overall lower levels of flux compared to larger population sizes. Average bioluminescent or fluorescent flux dynamics for the (b) holux, (d) Luc, and (f) GFP-containing cell populations of ∼1 × 10⁶ cells over a 24 hr period demonstrate the differences in signal intensity over time.

Figure 2

Despite presenting an intermittently detectable pseudocolor image, a population of ∼10000 holux-expressing cells could not be statistically differentiated from background light detection. Boxes represent the mean values of three trials, reported with overlapping standard error of the means.

Figure 3

Short integration times (∼1 s) are required to prevent saturation of the CCD camera when using a Luc-based reporter system due to its high levels of bioluminescent flux following D-luciferin amendment. (a) However, at integration times of 1 s it is not possible to differentiate Luc-expressing cell populations below ∼250 cells from background light detection. (b) Increasing the integration time to ∼10 s in the absence of larger population sizes to prevent camera saturation allows for detection down to ∼50 cells. Boxes represent the mean values of three trials, reported with the standard error of the mean.

Figure 4

Cells expressing GFP were visible down to population sizes of ∼5 × 10⁵ cells. Boxes represent the mean values of three trials, reported with the standard error of the mean.

Figure 5

Comparison of in vivo bioluminescence for holux and Luc cells. (a) The bioluminescent signal following subcutaneous injection of holux-expressing cells remains relatively stable following injection and (b) is detectable down to a minimum of ∼25000 cells. (c) Signal dynamics are significantly altered, but of approximately the same strength following intraperitoneal injection. (d) Total flux from subcutaneous injection of Luc-expressing cells is significantly higher, and (e) as such is detectable down to ∼2500 cells. (f) Bioluminescent output from intraperitoneal injected Luc-expressing cells expressed peak flux immediately following D-luciferin injection, but then quickly diminished over the remainder of the assay.

Figure 6

Comparison of pseudocolor images of subcutaneously and intraperitonealy injected holux and Luc Cells. Subcutaneously injected (a) holux- or (b) Luc-expressing cells are capable of presenting relatively similar images despite the large differences in total flux from each reporter system if the integration time is increased from 1 s (Luc) to 60 s (holux). Similar increases must be made to maintain uniform representative detection following intraperitoneal injection of the (c) holux and (d) Luc cells as well, with the holux system requiring a 60 s integration time to achieve similar pseudocolor patterning as a 10 s integration of the Luc system.