Digital Illumination

Technology

The core of Digital Illumination is the Digital Micromirror Device (DMD), a high speed and highly efficient semiconductor-based "light switch" array of up to 2 million hinge-mounted addressable, tiltable, microscopic mirrors. It is a mass produced spatial light modulator based on mature semiconductor material systems and processing.  With more than eight million in the field, the DMD is also the core of consumer electronics products such as HDTVs and projectors.

When a DMD chip is coordinated with a digital video or graphic signal, a light source, and beam delivery optics, its mirrors reflect a digital image onto the sample.  Infinitely complex geometries of excitation patterns are simultaneously mapped on to the sample.  In combination with continuous wave (CW) or arc lamp light fluorescence excitation sources and laser scanning confocal, spinning disk confocal or wide field microscopes, the computer controlled DMD spatial light modulator produces a diffraction limited mask at the specimen plane, at the image detector, and within the microscope eyepiece field of view. 

Like traditional galvo mirror scanning systems, Digital Illumination allows the user to apply illumination pixel-by-pixel where needed and for as long as necessary.  However, the inherent advantages of the DMD technology uniquely enable simultaneous pixel-by-pixel illumination of multiple regions of interest and infinitely complex sample geometries with zero delta image acquisition time.  There is no scanning of the sample and thus no time lapse between illuminating pixels in the mask.  This is not possible with traditional galvo mirror technology that scans the regions of interest over a period of time.

Live Cell Imaging

Live cells represent vital model systems for the study of organism development and human disease.  They are increasingly recognized as powerful discovery tools in genetic, proteomic and cytology research and have vast utility in drug discovery and studying efficacy and toxicity of novel therapeutics.

Functional, molecular, and morphologic quantitative fluorescence imaging techniques are important tools for providing data about the biochemical, genetic or pharmacological processes – non-invasively, real time, and repeatably.  The enormous potential of the fluorescence imaging modality is yet untapped due to limitations that include temporal based processes inherent to living cells, autofluorescence, and sample integrity.  In combination with confocal and wide field microscopy, Mosaic digital illumination, promises to overcome these limitations in live cell imaging.

Temporal Based Studies: Photoactivation and FRAP

Critical to maintaining data integrity in temporal based live cell studies is the ability to simultaneously excite entire and multiple regions of interest in real time with zero delta time.

For example, photoactivation experiments targeting multiple Regions of Interest (ROI) are often conducted to analyze the mobility of proteins within a living cell.  If the observed protein is mobile, eventually the signals in multiple ROI’s will become equal; conversely, if signals do not reach a point of equilibrium, this suggests that a significant portion of the protein is immobile.  Critical to the design of photoactivation experiments are the ability to simultaneously photoactivate multiple regions of interest within a living cell. Traditional galvo-mirror based laser scanning illumination cannot accommodate such temporal based studies involving multiple regions of interest.

Similarly, FRAP experiments may include both fast and slow recovery components.  Digital illumination uniquely can capture the fast component in an area larger than the diffraction limit whereas the intrinsic delta time of a point scanner can miss or obscure this event. 

Digital illumination provides FRAP and photoactivation data without recovery or diffusion gradients.  It is enabling researchers to now observe and validate time lapse data that was missed during the acquisition time associated with galvo mirror scanning. 

Phototoxicity and Photobleaching

An inherent limitation in fluorescence imaging is achieving sufficient excitation energy while maintaining the integrity of the sample and fluorophore.  When every pixel is illuminated evenly, as with conventional confocal microscopy, pixels without fluorophore molecules present are illuminated, compromising the integrity of the sample.  Likewise, when pixels with high fluorophore concentrations are illuminated with higher intensity than necessary, the image saturates with high signal-to-noise ratio – damaging the sample and reducing the dynamic range.

With Digital Illumination, precision in exciting complex sample geometries with zero-delta acquisition time, minimize and eliminate damage and modification the sample and the effects of inadvertent photobleaching.

Autofluorescence

Another inherent limitation in fluorescence imaging is auto-fluorescence from local non-targeted, but fluorescing biological compounds.  This leads to background noise and reduced sensitivity. 

Digitally illuminating complex sample geometries allows for greater precision and minimizes the effects of auto-fluorescence to achieve higher sensitivities and improved levels of quantitation. 

Multiplexing

Multiplexing enables multiple assays to be performed simultaneously on a single sample.  The challenges of achieving high sensitivity and dynamic range while maintaining sample integrity over time are amplified with complex, multiplexed cell assays.  The precision and accuracy in digitally exciting multiple, complex sample geometries with zero-delta acquisition time minimize and eliminate the effects of excitation leak through associated with multiple excitation wavelengths and make multiplexed assays possible.

Mosaic Digital Illumination Systems

Photonic Instruments’ proprietary DMD technology is the core of the Mosaic Digital Illumination System, a flexible and modular research tool that is readily integrated with off-the-shelf microscopes and imaging acquisition and analysis software.  The technology is also a robust and flexible platform amenable for OEMs to develop a plethora of  fluorescence imaging solutions.