Introduction

Fluorescent probes are being used extensively in developmental biology for various imaging techniques, including nontoxic live staining and visualization of expressed fluorescent proteins. These techniques have enabled the identification and tracing of numerous cellular and subcellular structures and molecules. Nonetheless, a number of limitations are associated with the use of existing microscopy techniques. For instance, live cell fluorescence microscopy using laser-scanning confocal microscopy (LSCM) induces embryo damage, as the cells are exposed to high-intensity light (1). A couple of microscopic techniques that are less toxic to cells lend themselves to exploitation. Two-photon fluorescence microscopy has proven less toxic for live samples and was used by Squirrell and colleagues to image hamster preimplantation embryos with no signs of developmental damage (1). However, despite its lower phototoxicity, the use of two-photon fluorescence microscopy has been hindered by its high cost and demand for technical expertise. In contrast, spinning-disk confocal microscopy is significantly more affordable and causes no discernable cellular damage and, as a consequence, is becoming the method of choice for dynamic imaging of living cells (2,3).

The spinning-disk technology uses a Nipkow disk, which was invented in 1884 (4) and adapted for optical microscopy in 1968 (5). In essence, the spinning-disk apparatus uses the same principle as LSCM to obtain confocal images (i.e., the excitation light goes through a small pinhole and then the fluorescence emission passes back through a pinhole, thus eliminating out-of-focus light). The main difference of the spinning-disk systems is that they rely on several confocal pinholes mounted on a perforated disk that rotates at high speed, thus merging the fluorescence into one uniform two-dimensional image. As a result, photobleaching and damage to the living cells are minimal. This technology is particularly useful in biological studies that require a high rate of microscopic imaging (2). Applications of the technique include, among other things, monitoring cytokinesis regulation (6), microtubules dynamics (7), Ca^2 + and cAMP signaling (8), calcium dynamics (9), membrane receptor internalization (10), and microvasculature physiology (11).

We have extended the use of the spinning-disk confocal technology to image live mammalian embryos (mouse and bovine) without compromising their developmental potential. We hypothesize that by using fluorescent markers during preimplantation development, it should be possible to capture high-resolution images that can later be correlated with the developmental competence of the embryos. Such a procedure could be useful for monitoring embryos of certain species, particularly under experimental conditions in which few embryos develop into normal individuals. For instance, only about 20%-30% of bovine 2-cell embryos produced by in vitro fertilization (IVF) reach the blastocyst stage, and barely half of these generate a pregnancy when transferred into a synchronized recipient (12). In contrast, approximately 70% of in vivo produced bovine embryos, when transferred to the uterus of a recipient cow, give rise to healthy calves. Likewise, a dramatically lower developmental efficiency is seen in somatic cell nuclear transfer (SCNT)-derived embryos, in which only 1%–10% of the embryos transferred into the uterus develop into healthy offspring (13). This implies that most of the in vitro produced embryos are destined to die; yet it remains very difficult to identify the phenotype of a developmentally competent embryo. Therefore, there is an imminent need for an affordable and reliable method to assess preimplantation embryos before they are transferred into surrogate mothers.

Similarly, despite the fact that SCNT has been successfully implemented in several mammalian species, attempts to improve its efficiency are hindered by the lack of reliable markers to indicate when the somatic nucleus transferred into the oocyte has undergone complete genome reprogramming and when the resulting embryo is able to generate a live offspring once transferred into the uterus of a surrogate mother. To date, the only reliable marker for success of complete reprogramming is the birth of a healthy offspring. This implies a long waiting period—at least 9 months in cattle—to determine the health status of the cloned offspring. It is therefore of utmost importance to find alternative markers for determining the developmental competence of a cloned preimplantation embryo. Each marker should be easily detectable by noninvasive methods and must be nontoxic to the embryo.