In the Nikon A1si confocal microscope, fluorescence emission light entering the detector is first passed through a proprietary Diffraction Efficiency Enhancement System (abbreviated DEES) that separates incoming non-polarized emission light into two orthogonal polarized light wavefronts (termed p and s) using a polarizing beamsplitter. The purpose of the DEES system is to increase the efficiency of light diffraction by the gratings used to separate fluorescence emission into component wavelengths. After leaving the beamsplitter, the p-wavefront is rotated by 90 degrees (into an s-polarized wave) using a prism system and both beams are then diffracted by one of three interchangeable gratings. The diffraction gratings, which can be precisely controlled to ensure a high level of reproducibility, have wavelength resolutions of 2.5, 6, and 10 nanometers.
The tutorial initializes with a 2.5-nanometer diffraction grating installed in the grating wheel, and incoming non-polarized light passing through the DEES polarization unit. Polarized light is diffracted by the grating and directed to the 32-channel photomultiplier by twin mirrors that operate on each beam independently, but converge on the photocathode of the detector. Directly above the DEES configuration schematic drawing are spectra of enhanced yellow fluorescent protein (EYFP) at full resolution and as detected by the instrument in wavelength increments dictated by the diffraction grating. In order to operate the tutorial, use the buttons to select a diffraction grating and observe changes to the EYFP spectrum and the detection bandwidth. The Zoom button produces a close-up view of wavefronts moving through the beamsplitter and polarization rotator.
The most versatile confocal microscope configuration for spectral imaging can dramatically enhance the acquisition speed of gathering lambda stacks by utilizing a multiple-channel photomultiplier to gather limited-size wavelength bands of fluorescence emission after it has been dispersed using a diffraction grating. This acquisition strategy is available for the Nikon C2+ and A1 HD25/A1R HD25 confocal instruments, each of which are capable of high-speed spectral acquisition with only a single scan. The multi-channel photomultiplier (often termed a multianode photomultiplier) in these instruments contains a linear array of individual 10-nanometer detection channels built into a single unit, which enables multiple emission bands to be imaged in parallel, thus severely limiting specimen photobleaching and phototoxicity. The Nikon spectral detection units feature several diffraction gratings with sampling increments of 2.5, 5 (or 6), and 10 nanometers that can be individually rotated into the optical path to adjust the spectral bandwidth of lambda sections. The dispersed emission is then passed to precisely defined channels in a 32-channel multianode photomultiplier to generate a separate image from each channel. The total bandwidth of fluorescence emission is dictated by the diffraction grating sampling increment: 2.5-nanometer sampling produces an 80-nanometer bandwidth, a 5-nanometer grating produces a 160-nanometer bandwidth, while the 6-nanometer grating generates a 192-nanometer bandwidth, and the 10-nanometer grating yields a 320-nanometer band. In the Nikon instruments, the spectral imaging detector uses a laser shielding mechanism that eliminates reflected laser light from the excitation source, and the diffraction grating can be tilted to select any of the sub-sampled bandwidths.
Among the advanced features of high-performance spectral imaging confocal microscopes are Nikon's unique proprietary diffraction efficiency enhancement system (DEES), which is designed to eliminate polarization artifacts, decrease wavelength losses at the grating, and capture the maximum amount of fluorescence emission. The DEES system operates by passing non-polarized fluorescence emission through a polarized dual-beamsplitter optical element to generate two component wavefronts termed p and s that are oriented parallel and perpendicular to the plane of incidence, respectively. The most proficient diffraction efficiency is observed with s-polarized light, so a polarization rotator is positioned in the pathway of thep-polarized light to generate s-polarized light, dramatically improving the efficiency of the grating system. As illustrated in Figure 1(a), the diffraction efficiency of p-polarized light is above 90 percent over the wavelength range of 450 to 675 nanometers. In contrast, the efficiency of s-polarized light is 80 percent at 450 nanometers and drops almost linearly to approximately 45 percent at 675 nanometers. Therefore, the Nikon DEES system can significantly improve light throughput, and therefore sensitivity, in the spectral detection unit. In cases where the spectral width must be adjusted, additional specimen scans can be conducted or adjacent detector channels can be combined (termed binning) to double, triple, or quadruple the width of the detection band.
Although slit-based spectral imaging confocal instruments are capable of imaging emission spectra at high resolution, they are relatively slow when compared to microscopes equipped with multianode photomultipliers. Even those instruments that feature mirrored slits to reflect a portion of the bandwidth to a second or third photomultiplier still suffer from a lack of imaging speed on the timescales necessary for live-cell imaging. In many cases, measuring a spectrum over 200+ nanometers in a slit-based system can take several minutes or more, thus hampering spectral imaging of specimens that undergo temporal motion throughout the imaging period. Among the advanced features that improve the performance of spectral imaging microscopes are sensitivity correction in multianode-based microscopes (see Figure 1(b)). These instruments are corrected for wavelength accuracy for each individual channel using emission lines and luminosity adjustment based on a traceable light source. Additionally, the ends of fiber optic elements and the detector surfaces are coated with proprietary anti-reflection agents to reduce signal loss and to achieve high optical transmission. Finally, advanced dual integration signal processing (DISP) technology has been added to the image processing circuitry to improve electrical efficiency, preventing signal loss while the digitizer processes pixel data and resets. As a result, the signal is monitored for the entire pixel dwell time, resulting in a dramatically improved signal-to-noise ratio. In fact, these combined technologies enable 32-channel spectral imaging (512 x 512 pixels) at speeds of 24 frames per second, fast enough for a wide variety of live-cell imaging applications.
Jeffrey M. Larson and Stanley A. Schwartz - Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York, 11747.
Tony B. Gines, Alex B. Coker, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.