Video-rate Scanning Confocal Microscopy and Microendoscopy

This video demonstrates how to construct a video rate scanning confocal microscope using standard off the shelf components. A scanning confocal microscope relies on the coordinated movement of two scanning mirrors to translate the beam across the raster plane light from the first mirror is scanned horizontally across the first set of relay lenses, which focuses it back to a stationary point on the second scanning mirror. The same scanning motion is then translated through the scan and tube lenses, placing the now expanded light beam on the back aperture of the objective lens.

The second mirror whose motion is slow compared to the first mirror simultaneously scans the Y axis allowing for scanning action above and below the illustrated plane and creating a rectangular raster on the sample surface. The main advantage of this protocol over purchasing a commercially available microscope is the low cost and the ability to customize the instrument to the specific needs of an imaging project. When working with any laser, it is vital to take appropriate safety measures to prevent injury to the eye.

Be sure to wear the appropriate laser safety glasses whenever possible. Spectators and or researchers not actively aligning or working with the beam should always wear glasses. Remove all shiny and reflective watches or jewelry.

Most importantly, be sure to follow and control the beam at all times by terminating its path using a beam block or mounted business card. This is especially important while lining mirrors as the beam's path can be difficult to predict. Also, never make your eyes level with the beam path.

Turn off the laser if you need to cross the plane of the beam before assembling the microscope. Ensure that all the equipment necessary for the construction is available, including the optical breadboard and 90 degree brackets, all mirrors, lenses and dichroic, along with the appropriate clamps and mounting hardware, both scanning mirrors and the photo detector. It is also useful to have a function generator handy to test the scanning action and manipulate the mirrors during alignment.

It is also highly suggested to bolt the breadboard down for stability. Begin the construction of the microscope by placing the pigtail fiber ator in the optical mount and placing this assembly in the middle of the optical breadboard. This corresponds to the origin of the blue beam as shown here.

Turn on the light source. Use the adjustment screws to roughly adjust the beam such that it travels in a straight line both horizontally and vertically. Place an iris in front of the fiber collator.

Next, adjust the height of the iris so that the beam passes through the center. Then move the iris away from the ator along the beam path. Check the alignment of the beam.

Use the two adjustment screws to reposition the beam on the iris if needed. Place the mounted diic mirror in the beam path with the laser beam positioned at the center of the mirror. Rotate the mirror holder to reflect the beam at approximately 90 degrees, ensuring that the height of the beam does not change.

Clamp the mirror to the table by tightening the screws on the mount. Next place the mounted resonant galvan metric mirror into the laser beam path. Taking care to ensure that the laser beam is positioned at the exact horizontal center of the mirror surface.

Rotate the mirror mount to reflect the laser beam at a 90 degree angle. Then roughly adjust the reflection off the mirror to maintain the same laser beam vertical height. Next, take two irises and place them directly in front of the resonant gal.

Then using the laser beam reflected off the resonant gal mirror as a reference, set their vertical height as before. Now using the screw holes on the optical breadboard as a guide to the eye clamp the two irises down in a straight line. Adjust the first mirror in the path, which is the dichroic to center the beam on the first iris.

Then adjust the second mirror in the path. The resonant gal to center the beam on the second iris. Iteratively adjust these two mirrors until the beam is aligned through both irises.

Ensuring that the laser beam reflected from the resonant gal mirror is still reflected from the approximate mirror center. If the beam is deviated, adjust the fiber collator mount and repeat the adjustments with the beam centered on both open irises. The next step is to place the two relay lenses that will image the first stationary tele centric plane onto the second stationary tele centric plane.

For this particular microscope, the selected lenses in the first relay have the same focal length F, so the distance between the two mirrors here is simply four F.For this setup, F equals 90 millimeters, so the four F position will be 360 millimeters away from the scanning mirror. To ensure that the lenses are precisely centered in the beam path, place the first lens in the beam path and look at the laser beam spot on the next iris in the beam path. Following the lens, adjust the lens height so that the vertical center of the beam is at the iris center.

Next, adjust the horizontal beam position to center the beam on the iris. Repeat this process with the second lens. Next, the scanning mirror will be set up and the microscope will be rotated to facilitate straightforward placement and alignment of parts that would otherwise need to be adjusted vertically.

To find the exact position of the second tele centric plane, plug the resonant gal O into its scanning unit and turn it on. Use a business card to follow the path of the scanning beam through the two lenses. The second tele centric plane will be located approximately four F from the resident gal O and can be identified as the location where the scanning beam appears stationary.

Mark the breadboard beneath this location with a pencil. Once the tele centric plane has been identified, set the scanning mirror input to zero volts and power up the mirror control hardware so that the mirror settles to its neutral position. Then position the standard scanning galil mirror at the exact location of the tele plane.

Adjust the mirror position such that the beam at the tele plane strikes the exact center of the scanning mirror and tighten the mirror mount. Now loosen the locking screw holding the gal mirror and rotate it, ensure the beam is propagating vertically. Now turn off the laser and scanning electronics.

Disconnect the fiber and disconnect the scanning mirrors. Then using 90 degree breadboard mounting brackets. Secure the second breadboard to the first.

Finally, carefully rotate the entire microscope so that the new breadboard is laying flat and use the clamp to secure the microscope in position. Reconnect the gal o and laser here and remember to return the gal O to zero volts. The remainder of the formerly vertical setup can be easily carried out on the flat breadboard.

Next, we'll set up the second set of relay lenses formally referred to as the scan lens and tube lens. Please see the accompanying written protocol for very important information on choosing the right combination of lenses. Start by setting up the lenses.

First, lay out the mirrors needed to steer the beam to the objective lens. Place the first large two inch mirror close to the edge of the breadboard and rotate the mirror mount to reflect the laser beam approximately 90 degrees roughly. Adjust the reflection off the mirror to maintain the same vertical beam height.

Then place the other two inch mirror at the opposite edge of the breadboard to direct the beam at a 90 degree angle and use the adjustment screws to fix into place. Next, set up two irises and adjust the two mirrors as before to center the beam. Then place the scan lens at its focal length from the gal in the beam path and adjust its horizontal and vertical position.

To center the laser spot on the first iris between the two mirrors. Carefully place the large two inch tube lens at the appropriate distance from the scan lens and adjust its horizontal and vertical position to center the beam on the first iris. In this setup, the tube lens is placed at a distance of 75 millimeters plus 300 millimeters With all components in place.

Attach the gal O to the output of a standard electrical function generator or control circuit, and set the peak to peak voltages to one to three volts. Then begin scanning the two mirrors using a business card. Locate the laser beam at the stationary plane, 300 millimeters after the tube lens.

Place the objective lens in the beam path, making sure to position the objective lens back aperture as close to the stationary plane as possible. Set up the sample stage, making sure that the translatable Z axis mount does not strike the objective lens mount as it is translated across its range. To set up the confocal, pinhole and detector begin by disconnecting all power supplies and fiber optics.

Rotate the microscope assembly such that it again rests on the breadboard, holding the resonant scanning mirror. After reconnecting the fiber to the collimator and the standard scanning mirror, power, supply and control cables, place zero volts on the control voltage driving the standard scanning gal as before on the stage, place a sample of a bright dye sandwich between two cover slips. Then turn on the laser source and bring the dye into focus.

Adjust the laser power to make the fluorescence as bright as possible using a business card. Trace the fluorescent emission from the sample through the objective lens through the scanning system to the dichroic mirror, which reflects the laser beam and will transmit the fluorescent emission. Find this fluorescent signal on the other side of the dichroic mirror.

Now dim the room lights. Place a mirror behind the dichroic mirror to reflect the emission at a 90 degree angle. Then using an iris with a mirror, direct the fluorescence beam as straight and parallel to the breadboard as possible.

Next, the confocal pinhole unit will be set up using the spatial filter cage mount assembly from Thor Labs. This diagram outlines the setup. The scanning mirror sit at stationary planes that are tele with a stationary objective.

Back aperture plane pairs of lenses between the stationary planes act to relay the scanned beams. The first pair of lenses have equal focal lens forming a one-to-one telescope. The second pair of lenses known formally as the scan lens and tube lens do not need to be equal and focal length and often serve as a beam expanding telescope to ensure the objective back aperture is overfilled.

Light emitted from the sample, travels back through the scanning system and is passed through the dichroic mirror. A short focus lens or an objective lens focuses the emission light through the confocal pinhole, which is then collated by a lens. A final lens focuses the confocal filtered emission onto a photo multiplier tube.

Place the spatial filter unit in line with the emission beam path taking care to center the first focusing lens mount on the emission beam. In this instance, a spatial filter unit with a pinhole size of 100 microns was selected. Next, mount a short focal length lens in the unit and slide the Z translation mount until a clear focus can be observed on the pinhole surface.

Ensure the entire unit is oriented along the straight line set by the fluorescent beam. Clamp the unit to the breadboard. Now using the adjustment knobs on the translation stage, adjust the two axes to perform a 2D search over the pinhole mount surface to find the point where the fluorescent signal through the pinhole is maximized.

Next, place the coating lens on the cage mount After the pinhole using a business card, find the fluorescent ion that goes through the confocal unit and slide the coating lens along the posts until the emitted fluorescence is as collated as possible. Set up the photo multiplier tube assembly. Place a 50 millimeter focal length lens in the fluorescent emission beam path and find its focal point.

Mark this position on the breadboard. Now turn off the laser completely and position the photo multiplier tube so that its window is located as close to the marked focal point as possible. Then using adjustable lens tubes, connect the assembly to the focusing lens.

Wrap dark tape around all exposed beam paths following the pinhole, turn on the laser, keeping its power extremely low. Turn on the photomultiplier tube carefully reading out its voltage on oscilloscope. As the laser intensity is increased, increase the voltage until a spike like readout and or a DC offset can be seen on the oscilloscope screen.

Confirm that this signal indeed arises from the fluorescence by turning the laser power off or blocking the beam path to observe a loss of signal. Finally, iteratively align the pinhole for maximum signal on the oscilloscope. By first manipulating the focusing lens Z position and then adjusting the YZ translation stage, the video rate microscope hardware is complete.

First, turn off the photo multiplier tube. Now hook up the mirrors custom control boards and computer as diagrammed in the accompanying written protocol. This system can be adapted for performing confocal scanning micro endoscopy.

By replacing the stage with a fiber holding stage, please see the accompanying text or details. The finished upright confocal scanning microscope and micro endoscope should appear as shown here. The laser and emission beams have been drawn as a guide to the eye.

During micro endoscopy operation, a fiber mount holds the image fiber in place for experiments. This fiber mount could be readily replaced with an XY stage for uses. An upright microscope platform shown here is a representative test image taken of a lowercase M printed on a white business card using a video rate frame grabber card to generate images from the incoming signal.Bleached.

White paper contains Fluor force that are easily excited by UV and blue light resulting in the bright background behind the dark. Letter M and emission filter centered at 515 nanometers was chosen to collect this fluorescent emission. A minor distortion of the image can be observed especially near the lateral edges of the image frame.

This distortion results from the sinusoidal scanning pattern of the eight kilohertz gvo mirror and is discussed in detail in the accompanying document. This movie shows sample video rate data obtained by scanning a business card. After watching this video, you should now have a good understanding how to build and align a video rate confocal scanning microscope.

Don't forget that working with laser light can be extremely hazardous and precautions such as removing all reflective jewelry as well as avoiding working with your eyes level with the beam line should always be taken while performing this procedure.