Biomedical research - an image of the future

James R. Joubert discusses applications of integrated microscopy methods including live cell mechanical stimulation in relation to medical developments

Researchers across life science disciplines use microscopy to characterise molecular interactions. Those molecules’ fluorescent signal can be faint, requiring advanced sensors for detection. The steady improvement in sensitivity of CCD cameras and the advent of EMCCD cameras, which offer optimal low-light-level sensitivity and boost signal-to-noise ratios by multiplying incoming light signals, have revolutionised the world of life science imaging in the past decade. Now, researchers have the technology needed to image samples not previously feasible, from whole animals in vivo to sub-cellular structures, to enhance the drug discovery process.

Understanding the molecular mechanisms behind how cells remodel in response to mechanical stimulation is essential to develop therapies for many vascular diseases. Studies of how cytoskeletal proteins respond to mechano-chemical stimulation have traditionally relied on post-stimulation, cell fixation, and staining. Because this method offers only static information, researchers began considering how to investigate the dynamics of fluorescently tagged proteins using the latest low-light cameras.

An ideal experiment would mechanically stimulate and image live cells in real time. However, the technical requirements of optical microscopy and mechanical stimulation techniques conflicted. Dr Andreea Trache at Texas A&M Health Science Centre tackled this issue and designed the first integrated microscopy system that can simultaneously stimulate and image live cell response in real-time.

Trache's integrated system uses an atomic force microscope (AFM) tip coated with fibronectin to mechanically stimulate the cortical actin fibres beneath the apical cell surface. Simultaneously, the mechanical-induced cytoskeletal reorganisation throughout the cell is captured at high spatial and temporal resolution by either total internal reflection fluorescence (TIRF) microscopy or fast spinning disk (FSD) confocal microscopy.

This setup is challenging because it's hyper-sensitive to noise and vibrations. The AFM can measure nanometre displacements at picoNewton force. At that level, the rotation of the spinning disk or the camera fan would disturb the experiment.

Because photobleaching would misrepresent the biological process they were recording, they needed a highly sensitive camera that would allow them to minimise the intensity of the laser excitation. Critical, too, was a high signal-to-noise ratio to record dim fluorescence images with a dark background, in order to minimise post-processing of hard data.

Trache also needed to synchronise the spinning disk of the confocal scanning head, which rotates at up to 5,000 rpm, with the low-light camera to obtain uniformly illuminated images. In turn, the field of view of the confocal camera needed precise alignment with the TIRF camera, the AFM tip, and the AFM video camera and eyepiece.

Trache methodically isolated all sources of vibration by mounting the confocal scanner on a silicone damper pad, and isolating it from the microscope body. Vibrating equipment, including external Photometrics camera fans, were mounted to adjacent structures. She also used a pair of Photometrics QuantEM EMCCD cameras for TIRF and FSD confocal microscopy because of the synchronisation between the spinning disk and the camera.

"Before, everyone was looking at mechanotransduction as a before and after event. Currently, we obtain consistent data from studying real-time live cell remodelling," said Trache. "We can image focal adhesion proteins recruitment at the basal cell surface as the cell remodels in response to the AFM's applied force. It's action and reaction."

The TIRF microscopy images of the cytoskeleton and focal adhesions proximal to the basal cell surface can be directly overlaid and analysed in conjunction with the FSD confocal microscopy data, which scans across the entire cell. The result is a 3-D capture of whole-cell adaptive responses to mechanical force at high spatial and temporal resolution.

Trache's research will open novel investigation pathways to analyse cellular adaptive responses to the local mechanical microenvironment. This research will yield a deeper understanding of the structure-function relationship between contractile proteins involved in force transmission. Her goal is to provide the knowledge base needed to develop better therapies for diseases such as hypertension, atherosclerosis, or tissue edema, for which cellular changes due to mechanical force are key.

Radiation therapy is one of the most successful treatments for malignant tumours. In addition to causing tumour death it also weakens and collapses the rapidly forming vasculature around the tumour. For tumours in the central nervous system (CNS), radiation-induced vascular weakening and activation of astrocytes can cause acute and long-term damage to normal brain tissue.

M. Waleed Gaber, an associate professor at Baylor's College of Medicine and co-director of the small animal imaging facility at Texas Children's Hospital, is investigating factors that influence the health of vasculature surrounding CNS tumours to optimise efficacy and safety of anti-cancer therapies. His team recently identified that tumour necrosis factor-alpha is linked to acute microvascular damage and astrocyte activation following radiotherapy.

The discovery stemmed from intravital fluorescence microscopy techniques that Gaber's team developed. Their goal was to visualise long-term changes in the health of the same section of cerebral microvasculature in live laboratory mice.

"By testing the same vessel or network for the duration of therapy, we can reduce variability and enable correlative studies of key markers of vascular health. This is not possible using ex-vivo sections," explained Gaber.

Gaber integrated Photometrics' CoolSNAP and Photometrics' Cascade II cameras for low-light fluorescence microscopy with his custom engineered stereomicroscope for intravital imaging. For maximum sensitivity and lowest noise, the cameras are thermoelectrically cooled – without the need for bulky circulators or cryogenics – and provided quantum efficiencies upwards of 60% and 90%, respectively.

By integrating a Photometrics high-quantum efficiency camera, he could visualise the weak fluorescence signals within the vascular bed without phototoxicity risk to perform the needed experiments. Using this approach, it delivered the final image that proves Gaber‘s concept. His team recently introduced fluorescent beads coated with specific leukocyte cell surface receptors to murine models. They imaged the interactions between those receptors and the vascular epithelium. Gaber has been recently funded to study the combined effect of irradiation and wound injury on vascular integrity.

Epilepsy that is untreatable by medication can be relieved by surgically removing excitable brain tissue. However, seizures originating in the cerebral cortex cannot be localised using non-invasive imaging techniques such as fMRI or PET. To localise the site of seizure onset, electrodes are surgically placed atop the cortex to monitor and triangulate epileptic seizure activity. The procedure poses significant discomfort and infection risk to the patient, as the boreholes through which electrodes are placed remain open for the seven-day monitoring period.

Daryl Hochman, Ph.D., and his colleagues at Duke University are developing a novel, nanoparticle-based imaging technique that has potential to quickly localise excitable cortical tissue and eliminate the electrode-based procedure.

The nanoparticles, developed in the lab of Cassandra L. Fraser, Ph.D. at the University of Virginia, exhibit oxygen-independent fluorescence at 450 nm and oxygen-dependent phosphorescence at 550 nm under UV illumination. Dual wavelength imaging and ratiometric calculations can reveal dynamic oxygen concentrations in the pial arterioles across the cortical surface and provide an indication of local neural activity. The project required a wide dynamic range to detect small oxygen-dependent changes in light absorption against the strong intrinsic signal, as well as exquisite sensitivity to capture the dim fluorescent signal. They also needed a camera that had the potential to do ultrafast imaging to capture bursts of neural activity.

To definitively quantify the nanoparticles' response to specific stimuli as well as compensate for fluctuations in dye loading and interference from intrinsic optical signal, Hochman had to economically image two wavelengths simultaneously.

Hochman placed Photometrics’ Evolve EMCCD camera directly into his surgical macroscope and began imaging his primate models. The camera's dual amplifiers met his demands for high sensitivity and wide dynamic range. The Evolve's deep-cooled engineering enabled Hochman to capture clear, usable images at ultrafast frame rates and thus resolve the nanoparticles' behaviour over the time course of bursts of neural activity.

“The Evolve can be used in traditional CCD mode, where it has dynamic range, and EM mode, where it is still very sensitive for fluorescence. Without the Evolve, we would need two expensive cameras that we would have to swap on and off the microscope. It wouldn't be a feasible thing to do," Hochman explained.

Hochman's lab has spent several weeks labelling primate brains with nanoparticles and measuring responses to electrical stimulation for epileptic activity. Hochman's team is on course for developing the nanoparticle model for measuring neocortical activity. The results prove that fluorescence increases as oxygen concentration decreases. The converse is also true.

With a better understanding of how the nanoparticles reflect cortical activity, Hochman plans to develop the biodegradable, non-toxic nanoparticles for use as a clinical diagnostic capable of quickly localising inter-epileptic activity. They plan to make quantitative measurements of blood oxygenation, based on ratios of two signals. Expressing those in physically meaningful units raises questions that wouldn't be obvious otherwise. With the Evolve, they gained the ability to collect quantitative data to better analyse accuracy, which he feels will be essential in developing the clinical application.

References
ResourcesTrache, Andreea and Lim, Soon-Mi. Integrated microscopy for real-time imaging of mechanotransduction studies in live cells. Journal of Biomedical Optics. 13(3), 034024 (May/June 2009).

Author
James R. Joubert, Applications Scientist, Photometrics