Nikon Confocal Laser & Epifluorescence Inverted Microscope

Confocal Laser & Epifluorescence Inverted Microscope

Nikon Eclipse Ti2-E Inverted Optical Microscope

Best for

Differential interference contrast (DIC) imaging
Rapid or weak epifluorescence imaging
Scanning over large areas or at multiple locations
Microscopic 3D imaging (Z-series)

This system can perform brightfield, differential-interference contrast (DIC) imaging and epifluorescence imaging with an inverted configuration (objectives below the sample). The microscope’s plan apochromatic objectives with 25 mm field of view include

10X magnification, 0.45 numerical aperture, 4.2 mm working distance,
20X magnification, 0.75 numerical aperture, 1.0 mm working distance,
60X magnification, 1.4 numerical aperture, 0.13 mm working distance, oil immersion.

Plan objectives are corrected to provide a flat image. Apochromatic objectives are the most highly corrected objectives, typically correcting for chromatic aberrations at 4‒5 colors and spherical aberration at 3‒4 colors, and are thus recommended for the highest quality color photomicroscopy. Oil immersion objectives require special oil between the objective and the sample’s coverslip to achieve the highest magnifications. The DIC (Nomarski) technique can provide enhanced contrast for nearly transparent specimens (e.g., unstained cells)

The system is fully motorized and computer-controlled including control of the horizontal and vertical motion of the stage, illumination devices, and imaging systems. A joystick is also available for stage control. The system also uses an infrared laser beam to continuously maintain a stable vertical focus via the Nikon Perfect Focus system.

Epifluorescence Microscopy

The system can perform widefield epifluorescence imaging by exciting the sample with one wavelength and then detecting the emitted fluorescence light at a different wavelength. Dichroic filter cubes are used to pass the light from the source to excite fluorescence in the sample, but then separate out the emitted fluorescence to pass to the binocular or camera. For this system, brightfield, DIC, and widefield epifluorescence illumination will occur via a SOLA SE II LED white-light engine connected via a rear port. For epifluorescence imaging, three dichroic filter cubes for TexasRed (red), GFP (green), and DAPI (blue) dyes are available. The user can view these images via the binocular port or on a computer monitor via the attached cameras. The system will have two cameras available:

Basic color microscope CMOS camera (Motic 10, 10 Mpixel)
High-sensitivity monochrome scientific EMCCD camera (Andor iXon Life 897, 0.26 Mpixel, 16 x 16 µm pixel size, 56 frames per second at full frame size, peak quantum efficiency of >95%).

The user can manually switch between cameras as needed. The CMOS camera is best for real-color static imaging of samples, while the EMCCD camera is best for low-light (e.g., epifluorescence) or real-time dynamic monochrome imaging. The EMCCD camera can also be used with static samples containing multiple dyes by taking multiple images with different dichroic filters and then combining them to create superimposed false-color images.

Confocal Laser Scanning Microscopy

The system can also perform confocal laser scanning microscopy. In this imaging mode, a laser beam excites fluorescence in a small volume on the surface or interior of the sample. The emitted fluorescence light is then collected, filtered, sent through a pinhole to eliminate out-of-focus light, and then collected by a detector. By raster-scanning the beam over the sample, an fluorescence image can be created.

This method, while sometimes slower than widefield epifluorescence imaging, can perform imaging that has shallow enough depth of field to allow for optical sectioning. By shifting the focus vertically, these virtual slices of the specimen can be assembled to form a Z-series or three-dimensional image of the sample . The maximum resolution of the confocal unit is 512 x 512 pixels at 8 frames per second, but frame rate can be increased with a smaller scan range. If larger regions need to images, then multiple scans can be taken and then digitally “stitched” together to form a composite image.

3D image of a brine shrimp taken using the 60X objective over a depth of 46 µm in 0.5 µm steps.

The system has four excitation lasers that can excite in the violet (405 nm), blue (488 nm), green (561 nm), and red (640 nm). The light is sent into scanhead unit on the left side port of the microscope which performs the laser scanning, and the resulting light is then sent back to the detector unit which contains a beam-splitter and a high-sensitivity, dual-channel GaAsP-based spectral detector. Using the provided software, this detector allows the emission range to be tuned and thus allows for spectral “unmixing” of closely-spaced spectral components. The second channel also enables other techniques like FRET, emission ratiometric imaging, and other applications that require multiple simultaneous channels. The confocal unit also has a transmitted light detector that will allow transmitted light images to be collected simultaneously with epifluorescence images.

Funding for the confocal laser scanning microscopy system with EMCCD camera was provided by National Science Foundation Major Research Instrumentation Grant Number 1828246 to Kettering University (PIs: R. Kumon, C. Rablau, U. Ramabadran, G. Ryan, and L. Wang). Funding for the color CMOS camera was provided by the Physics Department and a Kettering University Faculty Research Grant-in-Aid to R. Kumon.

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