Structural Imaging

Structural imaging continues to be the classical application of light microscopy. Fluorescence techniques are routinely used to explore the microscopic structure of biological specimen labeled with fluorescent markers, including living nerve cells. In order to improve the spatial resolution, particularly the optical sectioning performance, advanced techniques such as confocal microscopy and multi-photon microscopy have become popular. Specifically in lightscattering specimen, the result of these advanced techniques can be stunning. Confocal microscopy employs visible laser light for exciting a fluorescent indicator and obstructs out-of-focus and scattered light by spatial filtering of the resulting image with a pinhole that is confocal with the illumination spot. Multi-photon microscopy uses infrared (IR) laser light, which is inherently less scattered due to its longer wavelength. Multiple photons of lower energy have to be quasisimultaneously absorbed to excite a single fluorescent molecule. This non-linear mechanism results in significantly smaller excitation volumes compared to single-photon techniques, and, thus, clearly improved sectioning and reduced photo-damage of cells. The small excitation volume also allows for efficient pinhole-free wide-field detection. State-of-the-art structural imaging can document sub-micrometer compartments in living cells such as dendrites and dendritic spines.

To reduce photo-damage and allow for detailed structural imaging, we have recently developed means to upgrade this confocal microscope for multiphoton excitation. A novel ultrafast IR laser (BioLight 1000, on loan from Coherent) was fibercoupled to a commercial confocal scan-head (PCM 2000, Nikon). This scan-head is located on a research microscope, which can be positioned relative to the specimen. While this preferred configuration is routinely used with CW lasers employed in confocal microscopes, it has not been applied to common ultra-fast lasers since they are lacking the possibility of fiber coupling. The reduced light-scattering of IR light permitted efficient wide-field detection of emitted fluorescence, and the microscope was modified accordingly, bypassing the pinhole and connecting straight to the PMT detectors.