How to design a Microprobe Experiment

How to Design and Perform a X-ray Fluorescence Microscopy Experiment


In this section, you will find instructions for operating the X-ray Fluorescence microprobe once it has been setup by BioCAT staff members. If you find the microprobe is not working properly, there are a number of ways to determine which component is not working and how to fix it. If these instructions are not clear or you cannot solve the problem by your own, please call your scientific contact.

 

Please note: As of January 2012, the X-ray Fluorescence Microscopy is only available in collaboration with BioCAT staff and not via the APS General User Program.

 

The microprobe setup consists of the following components:

  1. Optics:
    KB mirrors, collimator slits, ion chambers, video camera 1, video camera 2.
  2. Sample Holder System:
    XYZ high precision positioner, rotary stage, Linkam cryostage, cryostream, sample holders
  3. Detector System:
    Ketek single element detector, Ketek 7 element detector, XIA Saturn electronics, XIA xMap electronics.
  4. Software:
    scanning software BioCAT-FMAP, on-the-fly-analysis software BioCAT-ROI and peak fitting and calibration software BioCAT-FIT.

This document is organized by microprobe components. First you need to know how to operate the microprobe under normal conditions. If any of these components fail then you will need to go to the troubleshooting section of this document.

How to run the microprobe

  1. Optics

When you arrive at the BioCAT beamline 18ID to perform a microprobe experiment, the main components of the microprobe will have been set up beforehand. The x-ray beam energy will be set to your requested value, the beam will be collimated, and the KB mirrors will be set to focus the beam at the sample position. Two ion chambers are in the beam path: one upstream of the mirrors to monitor the incoming beam and to provide a signal to the feedback control system and another ion chamber immediately downstream of the KB mirrors to monitor the focused beam intensity delivered to the sample. Finally, the Hitachi video camera will have been aligned and focused on the x-ray focal point. This is your reference point for sample alignment.

Please do not adjust the optical configuration.

Adjusting the optical configuration could result in losing the alignment and several hours of your beamtime. If you cannot find the beam or you suspect the system is not properly aligned and focused, please call your scientific contact.

  1. Sample holder system
    1. XYZ high precision positioner

The sample holder system is based on a high precision (0.1 micron step size) XYZ positioner. The positioners are driven by stepper motors controlled by the MEDM screens already opened in one of the workspaces of the main control computer. The positioners are named: <CENTERSample Vertical
Sample Horizontal
Sample Focus

Each motor is associated with a particular stepper motor channel. These have been already calibrated and preselected in the scanning software.

The video selector box above the computer monitor allows you to change the source of the video input. To align the sample, select VideoCamera 1 to view the focal spot and the sample in a close-up view. Move the sample focus positioner while following the video camera 1 image on the monitor on the left side of the main computer.

There is a second camera mounted on the table that allows you to visualize a larger area of the sample, although it is not intended to be used as a position reference. Use this camera to locate your sample near the focal point. The beam position has been marked on the screen to help you find the right spot for your samples. Once the sample is located near the focal spot you need to select the scanning range for the BioCAT-FMAP software.

Move the sample horizontally to define the borders of your sample. Always add a few microns more to the position of the edge of the sample. Repeat the same procedure in the vertical direction.

If you have more than one sample on the same sample holder, you can define multiple regions for the scanning software. Move your sample manually and repeat the previously described procedure to determine the limits of each region selected. Once you have the positions for each region selected you are ready to setup a mapping scan.

    1. Rotary Stage

There is a rotary stage available that can be easily mounted on the XYZ positioner. The rotary stage is driven by a high-precision stepper motor and the channel on the step pak is preselected. The stage allows the rotation of samples to perform tomography-like experiments.

    1. Linkam Cryostage

A Linkam cryostage is also available for users. The cryostage is attached to a kinematic mount allowing to mount it easily on the XYZ positioner. The cryostage can be programmed in several modes. In most of these experiments the samples are previously frozen, therefore the cryostage should be constantly running at low temperatures to keep the sample temperature constant. The cryostage cold finger has a hole at the center that allows the beam to pass through it without striking the metal. When using the cryostage the XYZ positioner cannot be use for scanning for that reason. Instead you have two additional motors mounted on the cryostage to perform the scans. These motors are already setup and their channels are preselected.

    1. Cryostream

If your sample holder does not allow you to use the Linkam cryostage for low temperature experiments, the option is to use a cryostream. Currently BioCAT has no cryostream available for users, although it is planned to incorporate one in the near future. A cryostream can be borrowed from the APS detector pool if scheduled in advance. Please contact your scientific contact in advance if you are planning to use a cryostream.

  1. Detector System

There are two detectors available at BioCAT,

    1. Ketek Single-Element Silicon Drift Detector

The Ketek single-element SDD is a 10 mm2 actve area detector. It has a thin polymer window that allows measurment of low atomic weight elements such as Aluminum or even Oxygen under vacuum conditions. Although we do not operate our microprobe under vacuum, it is possible to run an experiment in a He atmosphere (Aluminum is still practical). To avoid permanent damage, the detector must be operated under dark conditions as it is sensitive to visible light because of the thin polymer window. There is a protective aluminum cup with Be windows that can be used to run the detector under normal conditions where low Z element sensitivity is not needed. The Be windows limit the ability of low energy photons to reach the detector. Therefore, under these conditions, sensitivity to light elements is reduced. The detector can only be connected to the Saturn XIA digital spectrometer.

To start the Saturn follow these instructions:

      1. open a terminal window
      2. type: start_dxp <Enter>.

An MEDM screen "DXP Detector Control" will show up.

Do not change the DXP parameters, since they are already optimized for the Ketek detector.

      1. Click on "DXP & MCA Plots" and select MCA plots to open an MCA screen.
      2. In order to prepare the ROIs for the scanning software, you need to insert the following information in these fields:

Label:

Atomic Symbol

Low:

ROI lower channel number

High:

ROI higher channel number

                                         5.         Enter as many elements as you want to plot after the scan without performing any fitting on the data. The ROI data will be use by the Matlab code BioCAT-ROI to plot the image. If you need more elements click on "All ROIs" to open a new screen with more options.

                                         6.         Use the top buttons to start and stop counting manually. You don't need to start the dxp to run a mapping scan. The BioCAT-FMAP softwary controls the detector via EPICS.

                                         7.         You can also open a python program to visualize the mca traces and save spectra. This is very useful when measuring calibration standards and selected spot on a given sample.

                                         8.         To open the python MCA program follow these instructions:

        1. Open a terminal window and type:python2 <Enter>
        2. At the python ">>>" prompt, type: mcaDisplay.mcaDisplay() <Enter>

A new MEDM screen will appear:

    1. Ketek 7-Element Silicon Drift Detector

This device is currently under commissioning.

  1. Software

There are three programs that allow the user to perform mapping experiments with the BioCAT microprobe:

    1. BioCAT-FMAP

BioCAT-FMAP is scanning software written in python. To start the program follow these instructions:

      1. Open a terminal window and type: ./BioCAT-FMAP.py (case sensitive)
      2. Select the motors channels for X and Y directions.

By default, MOTOR X is set to channel 20 and MOTOR Y is set to channel 22. Motor channel 21 is dedicated for the sample focus motor.

      1. Define the X and Y scan ranges

You must choose the Initial point, Final point, and the Step size. All dimensions are in millimeters.

      1. Select Joerger channels 3 and 4

The Joerger scaler reads the two ion chambers on these channels. Please do not change these channels.

      1. Insert the output file name with extension *.stp, including the full path.
      2. Save the configuration file as *.par

This will help to re-run other scans without retyping all the numbers.
The ASCII configuration file can be edited to add more scanning regions. Simply open the file with any text editor of your choice. Add more regions by adding a second, third and so on columns with the new information separated by commas. Below is an example of a single scan region and a multiple scan regions.

    1. BioCAT-ROI

BioCAT-ROI is quick analysis software written in Matlab. This code reads the *.stp files and plots the images of the ROIs selected in the scanning software. This is the fastest way to get the image of the measured data. The program only plots the values stored on the ROI channels. There is no background removal, peak fitting, or calibration performed at this point. This program is only intended for quick analysis performed on the floor while you are measuring the next sample.

Typically the program takes less than a minute to read the data file and plot the images. You can change the color code or any other parameters of the plots using Matlab quite easily and the images can be saved as JPEG files.

Matlab

    1. BioCAT-FIT

This is Matlab software indended for full analysis of the retrieved data. This code reads the *.stp files and performs peak fitting of each measured spectra for each point of the scan. The peak fitting routine includes: gaussian shape peak fitting, escape peaks, both theoretical and experimental Ka/Kb ratios and background removal. The code plots the images of the ROIs selected in the scanning software. This is the fastest way to get the image of the measured data.

  1. Troubleshooting the Microprobe

If you experience difficulties during the operation of the microprobe, here are some hints that may help you fix the problem. If these actions do not solve the problem, please contact Raul Barrea for assistance.

    1. There is no signal on the MCA screen
      1. Ensure that there is current in the storage ring

If the current is zero, there is no beam in the storage ring. You will need to wait for the APS to refill the storage ring before x-rays will be avaliable. The Floor Coordinator may have information about why the beam is down and when the APS expects to return to operations.

The Ring Current is displayed at the lower right corner of the picture below. Typically, beam current is near 102 mA in top-up mode.

      1. Ensure that x-rays are being delivered to the experiment station

First, make sure that the "A," "D," and user shutters are open.

If x-rays are making it into the experimental station, they should be passing through the ion chamber "I0." The signal from this ion chamber is displayed on the voltmeter located in the upper left corner of the voltmeter array on the main control panel (see picture above). With no beam present, the DVM typically reads 0.02-0.03 V. If there is no beam you need to check whether the shutters are open and that the intensity feedback system is operating correctly.

The feedback system is located in the second rack on the right of the main console. All switches must be on. You can search for the beam by turning the offset knob while watching the I0 value until you see the intensity increase. Keep turning the knob until you get the maximum value.

      1. Ensure that x-rays are passing through the KB mirrors

The ion chamber "I1" monitors x-rays exiting the KB mirrors. Again, this DVM typically reads 0.02-0.03 V if there is no beam present. If this is true, call Raul Barrea for further assistance.

    1. The scanning software is frozen or the motors seem to be stopped.

If the scanning software is frozen and the motor positions are not changing, you must restart the program. Close the BioCAT-FMAP program. There should be a terminal window in the same working space that contains the BioCAT-FMAP GUI. If not, simply open a new terminal window and type: ./BioCAT-FMAP.py <Enter> A new instance of the program should show up immediately. Setup the parameters following the instructions given in previous sections for the scanning software. Check that the motors are now moving properly.

    1. The BioCAT-ROI routine does not read the *.stp files

The Matlab program BioCAT-ROI reads *.stp files only. If the file structure is not the one expected by the program, it will crash. Make sure you are trying to read *.stp files. Contact Raul Barrea to review the file structure and the routine that reads the file.

    1. The BioCAT-ROI graphs are distorted

The BioCAT-ROI program reads the header of the file to organize the data by horizontal and vertical steps. Sometimes there is a missing point at the end of the line because of rounding errors. The solution is to correct the file header and insert the proper numbers. Contact Raul Barrea for further assistance.

    1. The BioCAT-FIT routine does not read the *.stp files.

The BioCAT-FIT program is intended to perform peak fitting of the mca traces on every measured point of an *.stp file. The routine is expecting a specific file format. If it cannot read the file, there might be an additional line or structure in the file. Contact Raul Barrea to review the file structure and the routine that reads the file.

    1. The detector is not counting

In the ulikely event that the EPICS software which communicates with the detector via the Saturn electronics hangs, you must restart the EPICS DXP software:

      1. Close the MCA window
      2. Close the DXP Detector control window
      3. In the EPICS terminal window, type: exit at the epics> prompt.
      4. Once you return to a command prompt, type: start_dxp. The DXP Detector Control window should return. Check that the detector is now working.
      5. Contact your scientific contact for further assistance

FAQs

a) What is the BioCAT microprobe?

The BioCAT microprobe is a unique instrument dedicated to microfluorescence biological applications (Also called X-ray Florescence Microscopy). The microprobe program is available only in collaboration with BioCAT staff and not via the General User Program. Investigators wishing to use the microprobe capability should contact Tom Irving. The main goal of the microprobe program is to provide the experimenters with a very high efficiency tool for microfluorescence mapping with or without microdiffraction

bWhat kind of experiments are feasible with the BioCAT microprobe?

Microfluorescence experiment that require resolutions of 50 microns, 20 microns down to 5 microns are feasible with the  BioCAT microprobe.  Typically tissue sections of various kinds are prepared for scanning at the microprobe. The efficiency of the microprobe allows the experimenter to map a large section of tissue at low resolution and map of small selected spots at higher resolution. At this point only freeze and air dried samples are conveniently measure at BioCAT.  We will be develop ping cryogenic capabilities for studying frozen sections in the future.

c)  Which elements can be mapped with the microprobe?

Elemental mapping of elements from K up to U can be mapped with the microprobe. The detection limit for each element depends on the noise level , the sample substrate and detector's efficiency. Most of the experiment are performed at room temperature and normal atmorpheric conditions. There is an SDD detector for light element detection that is able to detect P and S under proper dark environment and He atmosphere. These kind of experiments require a special setup.


d)  How do I analyze the data?

There are two routines available for experimenters written in Matlab. One routine was  designed for quick  analysis of the experimental data to allow the users to visualize the results immediately after the scan is done and to help them to make decisions regarding the appropriate regions of interest and so on for their experiment. Another routine was designed for a complete peak fitting analysis including background removal, peak overlap removal and normalization by using standards. Both routines are available for the experimenters.

Updated 7/14/15 T. Irving.