하나 나노 미터의 정확도와 형광 이미징 (피오나)

The overall goal of this procedure is to demonstrate how to carry out fluorescence imaging with one nanometer accuracy or Fiona experiments. This is accomplished by first setting up the required equipment and aligning the optics for total internal reflection, fluorescence, microscopy, or turf. The next step is to immobilize CY 3D NA on the inner surface of a sample chamber and to localize CY 3D NA single molecules with nanometer precision.

Subsequently, Fiona is applied to study the movement of a single truncated myosin five a motor labeled with a quantum dot. Ultimately, the step size of myosin as it walks on, Acton was measured as 36 nanometers by Fiona analysis. Though this method can provide insight into molecular motors, it can also be applied to other systems such as the tracking of receptors on the membrane of cells.

Visual demonstration of this method is critical as the experimental details are difficult to learn. It is important to wear laser safety goggles at all times during the setup. For total internal reflection, fluorescence, microscopy, or turf.

To begin set the heights of all the optical components to the height of the center of the microscope, back port, mount the laser, laser shutter and ND filters. Use ND filters to attenuate the laser power down as low as possible while keeping the beam visible. Tighten screws with the appropriate hex keys.

Plan a beam path as illustrated by the dotted blue lines. In this schematic, mark the beam path with tape or marker on the optical table. Place mirror M1 at the first right angle turn, place the two irises along the second straight section of the planned beam path.

Adjust both the position and the tilt of M1 such that the laser goes through the irises place mirror M two. At the second right angle turn place the 10 x beam expander along the third straight section of the path. Adjust its tilt such that the beam expander is parallel to both the optical table and the planned beam path.

The next step is to adjust M1 and M two iteratively such that the laser goes straight through the centers of both lenses, L one and L two of the beam expander. Use a piece of white paper to block the beam after the beam expander to check the beam profile by eye. Adjust until the beam profile of the expanded beam is non-ED.

Gaussian adjust the distance between L one and L two such that the beam is collated shutter. The laser unscrew the microscope objective and screw in a fluorescent alignment. Target place mirrors M three and M four to direct the expanded beam into the microscope port and onto the dichroic mirror.

Inside the turret. Adjust M three to center the brightest part of the beam on the fluorescent target and M four to make the beam tilt vertical, shutter the laser and screw the objective back in.Fine. Tune the tilts of M three and M four to optimize the laser power and beam profile out of the objective.

Mount an E-M-C-C-D camera to the microscope and connect the camera to a computer. Start the software for the camera. Mount a fluorescent sample on the microscope.

Look at the bright fluorescent spot on the camera. Check that the spot does not shift on the screen as the focus has changed. Place the TIR lens on the XY, Z translation stage at a distance from the back focal plane of the objective which is equal to the focal length, the L three, which is about 30 centimeters.

Adjust the position of L three such that the laser goes through the center of the lens. Translate L three along the beam path to adjust the beam collation. Make sure that the beam is still centered on the monitor and symmetrical in shape.

Translate L three perpendicular to the beam path to tilt the beam out of the objective. Keep translating the TIR lens such that TIR is achieved prior to carrying out a Fiona. Experiment on localizing and mobilized CY 3D NA.Single molecules construct sample chambers as detailed in the protocol text.

Two strips of double-sided tape are applied onto the slide along the long edges, leaving a gap of three to five millimeters at the center and then a cleaned cover slip is placed on top of the slide side views of the chamber from the right and from the front are shown. The open ends of the chamber are left open and serve as the inlet and outlet to immobilize SI 3D NA on the inner surfaces of the sample chamber. Pipette 10 microliters of BSA biotin into the chamber.

Wait for five minutes. Wash the sample chamber by pipetting 40 microliters of T 50 BSA into the chamber. Then pipette 10 microliters of neut travain into the chamber and incubate for five minutes.

Wash the chamber again with 40 microliters of T 50 BSA pipette, 20 microliters of SI 3D NA into the sample chamber and incubate for five minutes. Lastly, wash the chamber with 80 microliters of T 50 BSA. To begin the procedure for imaging SI 3D NA single molecules under turf M pipette 30 microliters of imaging buffer into the sample chamber and wait for eight to 10 minutes.

Mount the sample for imaging on a turf microscope that is equipped with a green laser, a 100 X oil immersion objective, and an E-M-C-C-D camera. Set the exposure time and the EM gain. Acquire a movie of the sample for 1000 frames.

Compile and run Fiona Pro for Fiona analysis. Use this IDL program to import the acquired image to input the effective pixel size and the conversion factor from intensity to photon number and to choose spots for Fiona analysis. At the end, the program will output the fitting results with 2D Gaussian functions as well as total photon numbers and localization precision compile and run pH count pro.

To characterize photon count, use this DL program to measure the average number of photons emitted by a flora. Four before photo bleaching to import the acquired image and to input the conversion factor from intensity to photon number. The program will detect fluorescent spots, automatically calculate photon counts as a function of frame number and output the traces of photon counts.

Prepare the sample for this experiment by pipetting 20 microliters of biotinylated BSA at one milligram per milliliter in DD H2O into a sample chamber. Incubate for 10 minutes. Rinse with 30 microliters of D ddh, two O pipette in 0.5 milligrams per milliliter of neut travain and incubate for two minutes.

Wash the chamber with 30 microliters of M five PSA. The acton for this experiment must be prepared on the previous day as described in the protocol text. Dilute the prepared f actin 25 times in general, actin buffer to a final concentration of about 0.004 milligrams per milliliter.

Pipette the actin solution into the chamber and wait for 10 minutes. Rinse the chamber with 30 microliters of buffer dilute myosin five A with flag tags by 30. Fold an M five buffer to a final concentration of 250 nano molar.

Mix one microliter of the diluted myosin with one microliter of Anti-Flag Q.Do 7 0 5. Add in eight microliters of M five to fill to 10 microliters and pipette up and down to mix. Well incubate for 10 minutes on ice.

Pipette 20 microliters of imaging buffer containing myosin Q dot into the sample chamber. Incubate for eight to 10 minutes. Image the sample on the turf microscope at an exposure of 30 milliseconds.

Acquire at least 1000 frames to begin data analysis. Open the video file in image J and crop the video around a moving spot. Track the spot through the video to generate X and Y coordinates through time in pixels by applying Fiona analysis to each and every frame of the video.

Convert pixels to nanometers as described in the protocol text. Calculate displacement from initial position as a function of time. Run a T-test on the displacement to obtain the steps of myosin walking.

Delete all zero values from the step size column. Plot the distribution of the step sizes using origin or matlab. Fit a Gaussian to the histogram.

In this typical image of surface immobilize SI 3D NA.The yellow arrow points to a single SI 3D NA molecule whose point spread function or PSF is shown here. The PSF is fitted with a two dimensional Gaussian function, and fitting residuals are also shown. This plot is a typical trace of photon count versus frame number and exponential fitting gives the average photon number of about 1.4 times 10 to the sixth to measure myosin step size a video file with good signal to noise of a single myosin.

As it walks along, an actin filament is captured. Three example frames are shown. Application of Fiona to each frame yields a distance versus time trace plotted in red.

A step finding algorithm based on the T-test is used to extract individual steps and the output is overlaid in white Step sizes and nanometers are labeled in white. When steps from multiple traces are combined in a histogram, the measured step sizes are Gaussian distributed about 35.8 plus or minus 0.4 nanometers. After watching this video, you should have a good understanding of how to carry out Fiona experiments, including setting up a turf microscope To achieve the best results With these procedures, it is important to minimize photo damage to floor force use and experiments.

Don't forget that working with turf microscopy and Fiona can be extremely hazardous and precautions such as safety goggles should be worn while performing this procedure.