The overall goal of this procedure is to control complex patterns of light in time and space so that it can be used to stimulate neurons. This is accomplished by first positioning A DMD chip at an appropriate conjugate image plane in the optical path of the microscope. The second step of the procedure is to properly illuminate the DMD with an intense light source such as a laser.
The third step of the procedure is to eliminate the speckling effect of coherent illumination. The final step of the procedure is to integrate a graphic user interface to manipulate the DMD chip. Ultimately, results can be obtained that show experiments that were previously difficult to accomplish can now be completed relatively easily through multi-site lysis of cage neurotransmitter employing the DMD system.
Hi, I'm Michael Mohammed and the laboratory of Dr.Chaman Tang at the University of Maryland Department of Neurology. Today we will show you a procedure for generating complex patterns of elimination in an optical microscope. We use this procedure in our lab to study dendritic integration.
So let's get started. This protocol will demonstrate the operation of a digital micro mirror device or DMD illumination system for experimental neuroscience. A DMD is an optical semiconductor chip with several hundred thousand microscopic movable mirrors arranged in a rectangular array On its surface, the position of each mirror corresponds to the pixel of an image.
Each mirror can be individually controlled to tilt between two orientations. In the off orientation, the light is directed away from the optical axis of specimen illumination. In the on orientation, the light is directed along the optical axis.
DMD is well suited for lysis because it tolerates very intense UV light generates high contrast ratios between the on and off pixels and can switch between the two states in tens of microseconds. The application of DMDs for biomedical research is aided by the wide use of image manipulation software and hardware, and by commercial interest in the image display field. Modular components are marketed by Texas Instruments as DLP discovery kits that provide the supporting electronic drivers to adapt the DMD to a microscopic system.
Basic concepts regarding the optics of microscopy need to be understood. The basic modern fluorescent microscope consists of an imaging path and an illumination path. It is comprised of an objective, a filter cube, and two distinct tube lenses for the imaging and illumination path.
The microscope is designed to have positions that are conjugate to the specimen image plane, where objects in focus at the specimen plane will also appear in focus at these conjugate image planes. A corollary to this concept is that any bright image created at the conjugate image plane would also be sharply projected onto the specimen image plane. This strategy is used to project a computer generated illumination pattern onto the specimen.
The laser beam serves as a very bright light source that will illuminate the pattern implemented by the micro mirrors on the DMD chips positioned at a conjugate image plane. The design of A DMD illumination system depends on the excitation lights used for optogenetics experiments, which utilize light in the visible wavelength. The illuminated pattern can be brought through the camera path.
Therefore, the DMD chip can simply be mounted on one of two camera port of a dual camera port module. Conversely, for experiments that use light in the UV range, such as lysis of caged compounds, the light must be brought through the epi illumination path and a more accessible conjugate image plane must be created. This is because the imaging tube lens is not designed for UV transmission.
Instead, the illumination tube lens is utilized for UV transmission and performs an adequate job in image formation. Each micro mirror on the DMD can switch between a positive and a negative 12 degree tilt relative to the plane of the chip. Depending on the digital input it receives from the computer, the axis of rotation is along the diagonal corners of each mirror, 45 degrees to the rectangular sides of the chip.
The orientation of the mirror tilt is designated by a gold colored triangle at one corner on the front surface of the chip. The tilt of the micro mirror dictates the alignment of the pitch and azimuth of the incoming illumination beam. To illuminate the DMD first configure the pitch of the illumination beam to be 24 degrees from the perpendicular axis of the DMD chip and configure the azimuth to be perpendicular to the axis of mirror rotation.
Precise beam alignment is critical for efficient operation. For lysis experiments. Use a laser source to generate the high intensity focusable beam necessary for rapid uncaging.
Here a quasi continuous diode pumped solid state frequency tripled ND ate laser is employed, use a relatively high power laser because only a small fraction of the laser output is actually delivered to the specimen. When using a DMD system, then launch the output of the laser into a multi-mode fiber so that it can be easily positioned and oriented along the correct axis for illuminating the DMD to provide a solution to the speckle. Problem generated from coherent illumination transmit the light through the optical fiber.
The fiber is wound around a pazo electric fiber stretcher that is driven to oscillate it around 40 kilohertz. The microscopic stretching of the fiber is sufficient to move the speckle pattern many times during the millisecond duration of each photo stimulation pulse, thus effectively eliminating the speckling. Finally, the output of the optical fiber is collated with a UV microscope objective to illuminate the DMDs to coregister the CCD pixel with the DMD image software that correlates individual DMD mirrors to specific pixels of the imaging CCD camera was written.
The graphical user interface within the software allows the user to assign the DMD mirror orientation that corresponds to the region on the CCD image with the computer mouse using the graphical user interface. Tag the location for photo stimulation by moving the cursor over the image displayed on the computer screen and click the tagged region of interest to program the light delivery patterns store. The pattern marked on the computer screen as a series of separate images program the timing of the laser pulses for each spatial pattern into the software.
This will integrate with the data acquisition program P clamp. Finally, use the data acquisition program to coordinate the timing of the DMD electronics, the gating of the laser, and the acquisition of the patch clamped electrical signal from the target neuron. Here, a calibration of optical system resolution is shown.
The minimal spot size is shown as measured from a fluorescent target. The effective physiological resolution is measured by the amplitude of current flow as a function of photo stimulation. Spot position here, caged glutamate was photoed adjacent to a two micrometer diameter dendritic branch.
As the spot was moved. Orthogonally across the dite electrical responses to photo stimulation of different intensities are illustrated. Voltage clamped responses as a function of lysis intensity are shown.
Distributed dendritic stimulation can be easily implemented using a DMD based system. Here, input intensity was varied by increasing the number of spots of photos stimulation marked as circles on the two dendritic branches. Spatial summation can be non-linear across branch points.
It becomes increasingly super linear with increasing input amplitudes arriving on the two door to branches. These results clearly illustrate the non-linear manner of summation across dendritic branch points. The super linearity is largely mediated by NMDA receptors and not by voltage-gated channels.
This can be demonstrated as application of two NMDA antagonists A PV and MK 8 0 1 largely eliminate the non-linear behavior. We've just shown you the basic design and operation of A DMD based system for pattern photo stimulation of neural tissue. The system can be adapted for stimulation with visible light, such as used for optogenetic experiments.
It can also be adaptive for near UV lights as used for single photon lysis. When doing this procedure, it's important to remember that if a light at wavelength less than 400 nanometers is to be used and most people out through the epi illumination path, that precise alignment of the illuminating beam within a few degrees is critical, and that if a coherent light is used, speckling must be effectively eliminated. So that's it.
Thanks for watching and good luck with your experiments.
