Accessible Micron Resolution Scanning Laser Microscopy

Sean Cheng, Donovan Gini, Vincent Chyn, Ritwik DanAEP/ENGRD 2640, Cornell University

Ithaca, NY 14853

Devices have been built to achieve nanometer resolution within images, but most are designed forexperts in the field. We present a scanning laser microscope design for less experienced users thatscans and interprets data at a micron resolution. It is accessible and a↵ordable for anyone with aninterest in the field of microscopy.

I. INTRODUCTION

The recent rise in microscopic research has come withthe necessity of understanding and imaging test subjectswith a high resolution. Various products have been re-leased to the market to address this need but often donot cater to the less experienced user or researchers newto microscopy, due to price and complexity This articlewill address this issue by presenting a scanning laser mi-croscope that is inexpensive, simple to use, and straight-forward to build.

A scanning laser microscope allows point by point datacapture. This is achieved through a system consisting ofmirrors and lenses, as well as a laser and photodiodedetector. By directing the laser beam through a beamsplitter and a motorized mirror, the beam can be sweptacross the surface of the object under inspection, andlight can be reflected onto a photodiode (shown in Fig.1).

To make the photodiode readings meaningful, we cre-ated an analog-to-digital converter virtual instrument(VI) in LabVIEW to convert the voltage from the pho-todiode detector into a 2-D array. This array is thenpresented as an image of the sample.

Using this system, described more thoroughly in Prin-ciples of Operation, our team was able to use one hori-zontal and one vertical stepper motor to control the mir-ror in our microscope, which in turn allowed the laserto illuminate the entire object under inspection point bypoint. A separate VI, built to control the moving of the

FIG. 1. Schematic diagram of an inexpensive scanning lasermicroscope as presented in the article [1].

laser across the surface of the object (through motorizedmirror control), read and organized the data into a two-dimensional image.This microscope has various benefits and capabilities

for viewing objects at a micron level. We tested on atransmission electron microscopy (TEM) grid to betterunderstand our microscope's functions and limitations.With this type of microscope, built with a simple and in-expensive design, both beginners and researchers withlimited resources can experience the benefits of laserscanning microscopy.

II. SAMPLE MOUNTING AND POSITIONING

Before the microscope can be used, a sample must beproperly mounted and positioned to be scanned. Samplesare mounted in front of the objective lens (see Figs. 2 and3). To begin mounting a sample, its square mount mustbe removed from the setup. Next, the sample is tapedto the center of a glass sample disk. The ring meantfor holding the glass sample disk is unscrewed with agraduated spanner wrench (see Fig. 4) and put in theglass sample disk. The ring is then screwed back ontothe square mount, and the square mount is screwed backonto the microscope so that the sample is almost touchingthe objective lens.To focus the microscope on the sample, the light re-

flected from the sample is observed directly in front of thephotodiode using a white piece of paper or similar opaque

FIG. 2. Sample mounted on the objective lens removed fromthe microscope setup.

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FIG. 3. Sample mounted on the microscope setup, ready forinspection.

FIG. 4. Thorlabs, Inc. graduated spanner wrench used toscrew in the sample-holder ring, so that the glass sample diskis held in place [2].

screen. The knob on the square mount is screwed in untilthe rough reflection pattern of the sample is seen. Onceit is visible, the knob is turned further clockwise untilthe back aperture of the objective lens, characterized bya sharp, uniformly colored circle, can be seen. Once thesesteps are complete, the microscope is ready to scan thesample.

III. PRINCIPLES OF OPERATION

The scanning laser microscope is comprised of the fol-lowing subsystems. The motorized mirror control subsys-tem is designed to redirect the laser beam such that thethe beam scans across the sample in a progressive man-ner. The laser originates from the laser diode, travels tothe sample, and is reflected into a photodiode. The sig-nal detection subsystem converts the light received by thephotodiode into a voltage that can be processed in Lab-VIEW. Finally, we describe the scanning method used todrive the motors.

FIG. 5. Motor submodule for scanning laser microscope. TheNI 622x is connected to two BigEasyDriver boards, each witha separate power supply. These drivers are connected to theirrespective step motors.

A. Motorized Mirror Control System

By using two stepper motors to move the mirror in re-spective directions, our microscope scans both horizon-tally and vertically. We measure a two dimensional im-age of the object under inspection. To achieve this, weuse a NI 622x with rmware written in LabVIEW to con-trol two units of BigEasyDriver, a stepper motor driverfrom Schmalz Haus LLC [3]. Each driver is connectedto a stepper motor [4]. A full system setup is shownin Fig. 5 and Tables I and II. The current setup cancontrol one motor at a time. For each motor,the NI 622xsends a direction signal followed by a pulse train (STEP).Each driver converts these signals to motor currents. Wecan control the rotation speed by the STEP frequencyand the rotation amount by the number of pulses sent.These settings are adjustable from the front panel of theLabVIEW VI as shown in Fig. 9 in Scanning and Syn-chronization.Our current setup controls each stepper motor in open

loop. Assuming no errors, this corresponds to 768 stepsper revolution. A reasonable step frequency for thesemotors is around 1 kHz a rotation speed of about 76rpm.When attached to the microscope, the maximumstep frequency is approximately 5 kHz. This correspondsto a maximum no-load rotation speed of 391 rpm.

B. Signal Detection

The signal detection subsystem works by converting aphotodiode short-circuit current (Isc) to voltage. As seen

TABLE I. Pin Mapping from LabVIEW to Driver [5]

NI 6221GPIO Name

GPIO No. BigEasyDriverPin

Motor Unit

CTR0 2 STEP H

P0.0 52 DIR H

CTR1 40 STEP V

P0.1 17 DIR V

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TABLE II. Pin Mapping from Driver to Motor

BigEasyDriver Output Pin Motor Lead

A+ Green

A- Yellow

B+ Red

B- Black

in Figs. 6 and 7, the output voltage is a linear function ofphotodiode illuminance, equal to Isc times the feedbackresistance Rf. We use an Intersil CA3140AEZ and 100kresistor [6]. The expected output voltage is between 0 to1V. Next, the voltage is fed into the ADC of the NI622x.The voltage data can then be processed in LABView.The signal detection subsystem is susceptible to one ma-jor source of error: temperature. As seen in Fig. 8,Isc (and the corresponding output voltage) varies greatlywith temperature. Therefore, regulating the photodiodetemperature is critical.

In addition, many other sources of noise may also existat magnitudes dependent on circuit implementation. Wediscuss only two major ones. First, parasitic capacitancescombined with high gain can cause frequency peaking.This translates to unstable time domain responses, sosampling deadtime becomes an issue. This phenomenonhappens at relatively high frequencies (dependent on im-plementation) and can be avoided by adding delay be-tween a motor step and sampling the photodiode. Sec-ond, we power the laser and photodiode using batteriesto avoid power supply noise.

FIG. 6. Photodiode short-circuit current vs. illuminance.The relationship is linear. Adapted from [7].

FIG. 7. Photodiode gain circuit. The output voltage is mul-tiplied by the feedback resistor value Rf. We choose a diodepolarity such that the output voltage is positive. Rf is 100k,and the output voltage ranges from 0 to 1 volt. The op-ampis powered by a 9 volt battery. Adapted from [7].

FIG. 8. Photodiode current vs. operating temperature. In-creasing the temperature of the photodiode from room tem-perature (25�C) to 60�C increases the current by an order ofmagnitude. Therefore, temperature regulation is critical toaccurate measurement. Adapted from [8].

C. Scanning and Synchronization

In order to integrate signal detection with mirror scan-ning, we used the aforementioned signal detection andmirror control LabVIEW VIs as subVIs in a larger VI(Fig. 9 includes the VI front panel and block diagram; alarger figure is available under request). The function ofthis VI is to scan a sample by moving the laser across,while simultaneously detecting its voltage signal from thelaser and storing it as data. This line-scanned data isthen converted into a 2D image of the scanned portionof the sample.To begin a scan, it is important that the laser is ini-

tially in the center of the region of the sample to beimaged. The VI first uses the mirror control subVI toshift the laser to the top left corner of the region to be

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FIG. 9. Full laser scanning microscope LabVIEW VI: frontpanel (top) and block diagram (bottom). For a larger image,see Appendix.

scanned. Then, the VI iterates a line scan using the scan-ning/signal detection subVI followed by a mirror motiondownward, before beginning the next iteration, whichagain starts with a line scan. The data from each of theline scans is compiled together to build a 2D array, whichis then plotted on an intensity graph on the front panel– this is the 2D image of the sample. Backlash causesthe line scans to be misaligned, and must be taken intoconsideration when setting up the microscope. Methodsof countering backlash, either during or after a scan, arediscussed in the Example Measurements section. Afterthe scan, the laser is re-centered and the VI terminates.

The VI is operated from the GUI, or front panel (shownin Fig. 9) of the VI. This consists of several controls, in-dicators and graphs. The X Range and Y Range controlsallow a user to set the horizontal and vertical lengths (inmicrons) of the rectangular portion of the sample to bescanned, while the Number of X Points and Number ofY Points determine how many data points are to be col-lected in each respective direction; thus, the total numberof data points taken at the end of a scan is the Numberof X Points multiplied by the Number of Y Points.

The last control on the front panel is the scan rate,which simply sets the speed at which line scans occur, inmirror steps per second. We found that the maximumscan rate that can be used, without malfunctions in thestepper motors that control the mirror motion, is 10 kHz,although this may vary by individual setup. All thesecontrols must be set before starting a scan.

Two graphs are also included on the front panel. Thesmaller one titled Latest Line Scan plots the voltage in-tensity as a function of X-position after each line scanwhile the VI runs. The data from each of these scans isstored and built upon by each successive scan. After thefull scan is complete, this data is plotted, as mentionedbefore, as the 2D image of the sample on the larger graphtitled Intensity Graph. There may be issues with the 2Dimage after a scan, however – in particular, a misalignedor blurred image due to backlash. Troubleshooting forbacklash is discussed further in the Example Measure-

ments section.The last parameter on the front panel is an indicator

which shows the calibration factor from mirror steps tomicrometers, corresponding to a constant value in theblock diagram. The VI uses this constant to convert itsX range and Y range from a distance to a number of stepsfor the mirror to move during a scan. This parameter,like the formers, must be set set before running a scan,and a method for finding its value for a microscope setupis discussed in the following section.

IV. CALIBRATION AND SPATIALRESOLUTION

To calibrate the microscope, we took a measurementof a TEM grid. By comparing one grid crossing to thenext grid crossing in the intensity chart, we found thenumber of steps per µm as the calibration factors for eachdirection (vertical and horizontal). The scales for theaxes are related to the number of steps by this relation:

D

Cx

= X Scale andD

Cy

= Y Scale (1)

where D is downsampling (input as steps per measur-ing points), and C

x

and Cy

are for calibration factorsin steps/µm of horizontal and vertical directions, respec-tively. We input 1 for each value, D, C

x

and Cy

so thatthe scale is equal to number of steps.Since there is a well defined distance between two grid

lines, 62 µm, the actual calibration can be found fromdistance measurements. This way, when we measuredthe distance between each grid crossing in the scale ofthe intensity chart, the calibration factors were foundthus:

Cx

=dx

62and C

y

=dy

62(2)

where Cx

and Cy

are the same calibration factors asin Equation 2, and d

x

and dy

are the distances betweengrid lines in the scale of each chart in horizontal andvertical directions, respectively. Using this method tofind calibration factors, we found that both the X and Ycalibration factors for our microscope were 14 µm.Once the calibration factors are known, the spatial res-

olution of the microscope can be calculated. This is thesmallest distance between which the microscope can dif-ferentiate between two objects, or in this case, two de-tected voltage signals. With a smaller spatial resolution,there are more well-defined and crisper images. The firststep in finding the spatial resolution is to measure thelength of an individual data point, or pixel, on the imageand scaling it to find its length in steps, as is done incalculating the calibration factor.Using the calibration factor for the direction in which

the data points length was measured, this length in steps

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FIG. 10. TEM Grid scan before backlash correction, imagedusing MATLAB.

FIG. 11. TEM Grid scan corrected for backlash, imaged usingMATLAB.

can directly be converted to a physical length by divid-ing by the calibration factor. This value represents thespatial resolution of the microscope in micrometers. Wefound the spatial resolution of our microscope to be 1µm. In the following section, we present a scan usingour microscope setup with these calibration and spatialresolution values.

V. EXAMPLE MEASUREMENTS

Figures 10 and 11 show a laser scanned image of a180-by-180 micron portion of a TEM grid plotted usingMATLAB. This is not necessary, but MATLAB allowsfor labeling and the inclusion of other features like thecolormaps next to the images.One issue during a scan that must be addressed is back-

lash. Backlash causes misalignment in alternating linescans, causing an image to appear as in Fig. 10. Twomethods will be outlined to fix this problem. The firstis to shift the laser in the direction it is about to per-form a line scan in (directly within the for-loop of theVI) by several steps before beginning the line scan itself.The second is to export the image data from the VI andmanipulate it after the scan is complete. This can bedone by importing the data as an array to a programthat allows for data manipulation, like MATLAB, anditeratively shift every other line scan, or row of the dataarray, by several data points.Trial and error must be used to determine by inspec-

tion how many steps or data points (depending on themethod) to shift by in order to get the best image. Fig.11 shows a properly aligned image of the same TEM gridscan as in Fig. 10, using the second of the aforementionedmethods for backlash correction.When performing a scan, one should keep in mind that

increasing the number of data points taken, or the defi-nition of the image, comes with the cost of a longer scantime. For example, for our TEM grid scans, 100 Y datapoints were taken and each horizontal line scan took ap-proximately 1 second, so the total scan only took abouta minute and a half. With this few data points, seeingeach data point is easy and gives a relatively low defini-tion. However, a scan with a definition ten times greater,containing 1000 rather than 100 data points, using thesame scan rate as we did for these measurements, wouldtake over 16 minutes.

VI. CONCLUSIONS

This method of laser scanning microscopy is not with-out limitations. However, it is an e↵ective alternativeto more expensive and complex devices that are meantfor experienced users in the field. We presented a de-sign capable of achieving micron resolution imaging whilestill being accessible. The microscope as described canachieve a scan rate of 5 kHz to generate an image withina matter of minutes. It is an e↵ective, simple, and in-expensive instrument ready for use by both novices andresearchers who are less experienced in microscopy.

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2015, pp. 40.

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