Ever growing complexity in fluorescence microscopy-based investigations has prompted Aurora Spectral Technologies to develop OptiMiS d-Lux, a laser scanning fluorescence microscope specifically designed to deliver an unsurpassed combination of image acquisition speed, fluorescence sensitivity, and spectral resolution. OptiMiS d-Lux utilizes Aurora Spectral’s patented OptiMis technology , where fluorescence emission from a sample is projected through a transmission grating onto an electron-multiplying CCD (EM-CCD) camera with single-photon sensitivity. Because the detector is a 2D array, multiple points in a sample can be excited and imaged simultaneously, as in OptiMis TruLine , allowing for a two order of magnitude increase in speed and/or sensitivity. At the same time, the spectral resolution is only limited by the number of rows of the EMCCD, which serve as different spectral channels. The following principal components of OptiMiS d-Lux provide investigators with a powerful yet flexible tool for experiments in need of a hyperspectral fluorescence microscope.
The excitation laser is fed into the scanning module which contains the necessary optics for scanning the laser beam in the sample plane. A periscope raises the excitation beam to the height of the back port of the inverted microscope. After passing through a tube lens (TL-O), the beam is directed into the microscope objective by reflection from a dichroic beam splitter (DBS) which is mounted in the filter cube turret of the inverted microscope (see inset). The emission emanating from the excited sample passes back through the objective, DBS, and the inverted microscope tube lens (TL-M) to a side port image plane, where it is delivered by the detection module to the EMCCD detector
Separate Excitation and Emission Modules
In OptiMiS d-Lux, the excitation light is fed into the back port of the microscope, via a scanning module, while the signal is detected from a side port and analyzed spectrally by a detection module, connected to the EMCCD. This provides the customer with advanced features while preserving the convenience of being able to switch between the appropriate CW or ultrashort pulse laser line by simply rotating the filter cube turret, as done in standard microscopy setups.
The emission signal is descanned in the emission pathway, separate from the excitation path, using proprietary technology. Descanning compensates for motion of the signal on the EMCCD detector arising from exciting different regions in a sample. By actively descanning the emission signal, the size of the EMCCD array being read out can be cut down to the minimum needed for the spectral range of interest, leading to an increase in the potential frame rate of the camera and hence faster acquisition times.
Plot of the maximum number of maximum spectral acquisitions per second vs the number of spectral channels acquired. The acqusition size is 440 x 40 pixels. The EMCCD used was an Andor iXon Ultra.
Two sections from an HEK cell expressing a muscarinic M3 receptor tagged with one of two fluorescent proteins, i.e. either citrine or cerulean. The excitation wavelength used in this scan (405 nm) primarly excites the cerulean, so citrine signal arises due to FRET occurring between M3-cerulean and M3-citrine proteins.
A confocal slit can be added to the detection module to improve image sectioning in single photon excitation measurements. The excitation laser is fed through the back port of an inverted microscope equipped with a standard filter cube turret, allowing for multiple dichroic beamsplitter and emission filter combinations to be utilized. A simple rotation of the filter cube turret allows users to be able to switch to the laser line appropriate for their particular measurements. Therefore, OptiMiS d-Lux opens the door to additional excitation choices while still providing access to the increased levels of sensitivity achieved using line-scan excitation technology and the hyperspectral resolution inherent to all OptiMiS systems.
Key Features of OptiMiS d-Lux
Faster potential frame rates are achieved by reducing the active readout area of the EMCCD chip
Excitation light is fed through the back port of the corresponding inverted microscope, allowing the system to be configured for single photon excitation, two photon excitation, or both
A confocal slit can be added to improve image sectioning capability
Can be configured using either point- or line-scan excitation (as in the TruLine configuration)
Compatible with standard filter cubes and dichroic mirrors
Highly flexible spectral resolution range and bandwidth size. If spectral resolution is not needed, have the ability to visualize 1, 2, or 3 bandwidths