Fluorescent Imaging and Microscopy for Dynamic Processes in Rats.

The rat is a favored model organism to study physiological function in vivo. This is largely due to the fact that it has been used for decades and is often more comparable to corresponding human conditions (both normal and pathologic) than mice. Although the development of genetic manipulations in rats has been slower than in mice, recent advances of new genomic editing tools allow for the generation of targeted global and specific cell type mutations in different rat strains. The rat is an ideal model for advancing imaging techniques like intravital multi-photon microscopy or IVMPM. Multi-photon excitation microscopy can be applied to visualize real-time physiologic events in multiple organs including the kidney. This imaging modality can generate four-dimensional high resolution images that are inherently confocal due to the fact that the photon density needed to excite fluorescence only occurs at the objective focal plane, not above or below. Additionally, longer excitation wavelengths allow for deeper penetration into tissue, improved excitation, and are inherently less phototoxic than shorter excitation wavelengths. Applying imaging tools to study physiology in rats has become a valuable scientific technique due to the relatively simple surgical procedures, improved quality of reagents, and reproducibility of established assays. In this chapter, the authors provide an example of the application of fluorescent techniques to study cardio-renal functions in rat models. Use of experimental procedures described here, together with multiple available genetically modified animal models, provide new prospective for the further application of multi-photon microscopy in basic and translational research.

Non-linear optical microscopy of cartilage canals in the distal femur of young pigs may reveal the cause of articular osteochondrosis.

BACKGROUND: Articular osteochondrosis is a common cause of leg weakness in pigs and is defined as a focal delay in the endochondral ossification of the epiphysis. The first demonstrated steps in the pathogenesis consist of loss of blood supply and subsequent chondronecrosis in the epiphyseal growth cartilage. Blood vessels in cartilage are located in cartilage canals and become incorporated into the secondary ossification centre during growth. It has been hypothesized that vascular failure occurs during this incorporation process, but it is not known what predisposes a canal to fail. To obtain new information that may reveal the cause of vascular failure, the distal femur of 4 pigs aged 82-140 days was sampled and examined by non-linear optical microscopy. This novel technique was used for its ability to reveal information about collagen by second harmonic generation and cellular morphology by two-photon-excited fluorescence in thick sections without staining. The aims were to identify morphological variations between cartilage canal segments and to examine if failed cartilage canals could be followed back to the location where the blood supply ceased. RESULTS: The cartilage canals were shown to vary in their content of collagen fibres (112/412 segments), and the second harmonic and fluorescence signals indicated a variation in the bundling of collagen fibrils (245/412 segments) and in the calcification (30/412 segments) of the adjacent cartilage matrix. Failed cartilage canals associated with chondronecrosis were shown to enter the epiphyseal growth cartilage from not only the secondary ossification centre, but also the attachment site of the caudal cruciate ligament. CONCLUSION: The variations between cartilage canal segments could potentially explain why the blood supply fails at the osteochondral junction in only a subset of the canals. Proteins linked to these variations should be examined in future genomic studies. Although incorporation can still be a major cause, it could not account for all cases of vascular failure. The role of the caudal cruciate ligament in the cause of osteochondrosis should therefore be investigated further.

Motion correction for cellular-resolution multi-photon fluorescence microscopy imaging of awake head-restrained mice using speed embedded HMM.

Multi-photon fluorescence microscopy (MFM) captures high-resolution fluorescence image sequences and can be used for the intact brain imaging of small animals. Recently, it has been extended from anesthetized and head-stabilized mice to awake and head-restrained ones for in vivo neurological study. In these applications, motion correction is an important pre-processing step since brain pulsation and body movement can cause motion artifact and prevent stable serial image acquisition at such high spatial resolution. This paper proposes a speed embedded Hidden Markov model (SEHMM) for motion correction in MFM imaging of awake head-restrained mice. The algorithm extends the traditional Hidden Markov model (HMM) method by embedding a motion prediction model to better estimate the state transition probability. The novelty of the method lies in using adaptive probability to estimate the linear motion, while the state-of-the-art method assumes that the highest probability is assigned to the case with no motion. In experiments we demonstrated that SEHMM is more accurate than the traditional HMM using both simulated and real MFM image sequences.

Motion artifact correction of multi-photon imaging of awake mice models using speed embedded HMM.

Multi-photon fluorescence microscopy (MFM) captures high-resolution anatomical and functional fluorescence image sequences and can be used for the intact brain imaging of small animals. Recently, it has been extended from imaging anesthetized and head-stabilized animals to awake and head-restrained ones for in vivo neurological study. In these applications, motion correction is an important pre-processing step since brain pulsation and tiny body movement can cause motion artifacts and prevent stable serial image acquisition at such a high spatial resolution. This paper proposes a speed embedded hidden Markov model (SEHMM) for motion correction in MFM imaging of awake head-restrained mice. The algorithm extends the traditional HMM method by embedding a motion prediction model to better estimate the state transition probability. SEHMM is a line-by-line motion correction algorithm, which is implemented within the in-focal-plane 2-D videos and can operate directly on the motion-distorted imaging data without external signal measurements such as the movement, heartbeat, respiration, or muscular tension. In experiments, we demonstrat that SEHMM is more accurate than traditional HMM using both simulated and real MFM image sequences.

Extended-working-distance multiphoton micromanipulation microscope for deep-penetration imaging in live mice and tissue.

We constructed a multiphoton (2-P) microscope with space to mount and operate microphysiology hardware, and still acquire high quality 2-P images of tumor cells deep within tissues of live mice. We reconfigured for nondescanned 2-P imaging, a dedicated electrophysiology microscope, the Nikon FN1. This microscope is compact, with retractable objectives, allowing more stage space. The instrument is fitted with long-working-distance objectives (2.5- to 3.5-mm WD) with a narrow bore, high NA, and efficient UV and IR light transmission. The system is driven by a powerful 3.5-W peak power pulsed Ti-sapphire laser with a broad tuning range. This 2-P system images a fluorescent standard to a depth of 750 to 800 microm, acquires images of murine pancreatic tumors in vivo, and also images fluorescently labeled T-cells inside live, externalized mouse lymph nodes. Effective imaging depths range between 100 and 500 microm. This compares favorably with the 100- to 300 microm micron depth attained by many 2-P systems, especially descanned 2-P instruments, and 40-microm-deep imaging with confocal microscopes. The greater depth penetration is attributable to the use of high-NA long-working-distance water-dipping lenses incorporated into a nondescanned instrument with carefully configured laser beam introduction and image-acquisition optics. Thus the new system not only has improved imaging capabilities, but allows micromanipulation and maintenance of tissues and organs.

A three-dimensional multispectral fluorescence optical tomography imaging system for small animals based on a conical mirror design.

We have developed a three dimensional (3D) multispectral fluorescence optical tomography small animal imaging system with an innovative geometry using a truncated conical mirror, allowing simultaneous viewing of the entire surface of the animal by an EMCCD camera. A conical mirror collects photons approximately three times more efficiently than a flat mirror. An x-y mirror scanning system makes it possible to scan a collimated excitation laser beam to any location on the mouse surface. A pattern of structured light incident on the small animal surface is used to extract the surface geometry for reconstruction. A finite element based algorithm is applied to model photon propagation in the turbid media and a preconditioned conjugate gradient (PCG) method is used to solve the large linear system matrix. The reconstruction algorithm and the system feasibility are evaluated by phantom experiments. These experiments show that multispectral measurements improve the spatial resolution of reconstructed images. Finally, an in vivo imaging study of a xenograft tumor in a mouse shows good correlation of the reconstructed image with the location of the fluorescence probe as determined by subsequent optical imaging of cryosections of the mouse.

Modular design for in vivo optical imaging and ultrasound treatment in the murine brain.

Simultaneous optical imaging and ultrasound exposure to the brain is challenging due to geometric constraints. We demonstrate sonication through the ventral surface of the mouse, leaving the dorsal aspect free for imaging. Ultrasound components are integrated into a commercial multiphoton microscope, and animals are imaged during ultrasound-induced blood-brain barrier disruption.

Presence of cumulus cells during in vitro fertilization protects the bovine oocyte against oxidative stress and improves first cleavage but does not affect further development.

The present study was conducted to evaluate the function of cumulus cells during bovine IVF Oocytes within cumulus-oocyte complexes (COCs) or denuded oocytes (DOs) were inseminated in control medium, or DOs were inseminated in cumulus cell conditioned medium (CCCM). DOs exhibited reduced cleavage and blastocyst formation rates when compared with intact COCs. The reduced blastocyst formation rate of DOs resulted from reduced first cleavage but subsequent embryo development was not changed. Live-dead staining and staining for apoptotic cells revealed no differences in blastocysts from oocytes fertilized as COC or DO. Fertilization of DOs in CCCM partially restored the cleavage rate, suggesting that factors secreted by cumulus cells are important for fertilization but that physical contact between oocytes and cumulus cells is required for optimal fertilization and first cleavage. Exposure of COCs to hydrogen peroxide shortly before fertilization reduced the cleavage rate, but did not lead to enhanced death of cumulus cells or oocyte death. Exposure of DOs to hydrogen peroxide, however, resulted in oocyte death and a complete block of first cleavage, suggesting that cumulus cells protect the oocyte against oxidative stress during fertilization.