Senior Projects

Senior Projects and Senior Theses

2019 – 2020

Nelson, Emma. “Understanding Supercontinuum Generation Through Experimental Observation and Mathematica Simulation.” Senior Thesis, 2020.

The goal of this project is to generate new colors of light for two-photon imaging using supercontinuum generation. Supercontinuum generation generates a wide range of frequencies from an ultrashort laser pulse traveling through glass due to the interactions between dispersion and nonlinearities. Dispersion is when a glass fiber has different refractive indices for different wavelengths, so the laser pulse broadens in time as it travels through the fiber because the different frequencies of the pulse travel at different speeds. The pulse experiences nonlinear effects as a result of high-intensity incident light causing the glass fiber to radiate new frequencies, which broadens the width of the pulse in frequency. Supercontinuum generation is the creation of almost white (broad bandwidth) light from the interaction between dispersion and nonlinearities. We have observed supercontinuum generation by sending an 800 nm laser pulse through a FemtoWHITE 800 photonic crystal fiber and measuring the output wavelength spectrum. We then compared the experimental observations to numerical simulations of supercontinuum generation performed in Mathematica. I have explained the equations and Mathematica code that I used to numerically simulate the results of sending a laser pulse through a thin optical fiber, so that this paper can be used as a manual for undergraduate researchers looking to simulate nonlinearities, dispersion, and supercontinuum generation in a photonic crystal fiber.

Nelson, Emma. “Two-Photon Laser Scanning Microscopy Using Two Channels for Imaging Multiple Fluorophores.” Senior Project, 2019.

Two-photon microscopy is an imaging method in which two photons of a near-infrared wavelength are absorbed at a single point in a thick biological sample stained with fluorescent dye. When the photons are absorbed by the sample, a photon of a different wavelength is emitted where it is detected by a photomultiplier tube. The two-photon method allows individual depths of a thick sample to be imaged separately without physically slicing the sample into thin parts. I built a second channel on the existing microscope apparatus so that two different types of fluorescent dye can be detected simultaneously.

Weber Cravioto, Isabel. “Photothermal Detection of Gold Nanoparticles.” Senior Project, 2019.

We present the implementation of a photothermal microscope to image sub-resolution nanoparticles. We utilize the light from a pump laser beam to heat the sample while using a second probe beam to detect the hot spots. The signal detected by the probe beam is weak, and thus we implement a chopper wheel to modulate the pump laser at a known frequency. This in turn modulates the refractive index of the absorbing structure, allowing for the separation of the high frequency signal and low frequency noise. Using a lock-in amplier, we detect this modulation from the probe beam signal, thus extracting information about the location of sub-resolution nanoparticles.

2018 – 2019

On leave

2017 – 2018

Doherty, Bennett. “Building an Automated Laser Alignment System.” Senior Project, 2017.

Laser experimentation requires proper alignment of the laser beam, which is typically done through an iterative manual process. Over the course of an experiment, it is common that a laser will gradually deviate from its original alignment. This deviation can result from ambient temperature fluctuations in the laboratory and changes within the laser itself, necessitating frequent realignment by the experimenter. Making use of quadrant photodiodes, an Arduino microcontroller, routine optics equipment, and standard electronics, this paper demonstrates an automatic laser alignment system that costs a fraction of those available commercially. The system is able to detect deviations in the laser path of (40 ± 20) μrad or greater.

Moskovitz, Emma. “Increasing Sample Rate to Improve Image Quality with a Two-Photon Laser Scanning Microscope.” Senior Project, 2018.

A two-photon laser scanning microscope allows us to look inside a sample layer by layer without making an incision. However, when imaging low-signal samples, such as biological tissue, it is difficult to produce a clear image with our student-built microscope due to noise fluctuations and bleed-through from adjacent points on the sample. We present a modification that oversamples each point on the sample and forms clusters of the multiple values, and then averages the clusters before constructing the final image. This ensures that even if some of the brightness values are corrupted by noise fluctuations or neighboring pixels, their overall contribution to the final brightness value will be negligible when averaged with multiple brightness values. To measure the effectiveness of this modification, we calculate the standard deviation and signal-to-noise ratio of an image produced with the original sampling procedure and compare it to an image produced with the modified procedure. We find that when oversampling 10 times for each pixel, the average standard deviation for each pixel is 1.3 times smaller. We find that the signal-to-noise ratio is 24:61:3 for images captured with the original sampling procedure, and 29:0 1:5 for images captured with clustering sampling procedure.

2016 – 2017

deJong, Max. “Detection Electronics for Scanning Laser Microscopy.” Senior Project, 2016.

Laser scanning microscopes take three dimensional images by focusing a laser beam to a single point in space, and scanning across the sample over a period of time, recording an image pixel by pixel. In each short time window, or pixel, emitted fluorescent light at a certain location is collected within a photomultiplier tube (PMT) which releases an output voltage pulse. This pulse must then be converted into a numerical value so that the pixels can combine to form an image. For this project, I am building an electronic analog circuit that integrates the signal from the PMT, giving a precise quantification of each pixel in the imaged sample in order to reconstruct an image.

Laurence, Colin.  “Shaping Ultrashort Laser Pulses Using an Electrically Tunable Lens.” Senior Thesis, 2017.

Pulse shaping is a method in which the individual wavelengths that make up a laser pulse are variably delayed, which alters the properties of the laser. This has applications for two-photon microscopy, a form of 3D tissue imaging. We use an electrically tunable lens to shape ultrashort pulses. We measure broadening that matches theory to within 300 fs from 180 ± 4% fs to 2400 ± 4% fs, though due to issues with our measurement apparatus, it is possible that the actual range is greater than this.

Laurence, Colin.  “Shaping Ultrashort Laser Pulses Using a Deformable Mirror.” Senior Project, 2016.

In this project, we built an ultrashort pulse shaper. Ultrashort pulses (less than 100 femtoseconds) are formed when a wide bandwidth of frequencies interfere in phase. The primary goal of this project was to manipulate these phase relationships in order to change the shape and width of our laser pulses, which can be done using a deformable mirror. After programming the mirror, we then measured the pulses of the spectral phase shifted laser using autocorrelation. By matching these measurements with simulations and theory, we found that we could reasonably accurately shape pulses over a moderate range by adding second order dispersion, with this range being limited by our equipment.

McNeill, Kirsten.  “Dispersion and Nonlinearities in Optical Fiber Delivery.” Senior Project, 2017.

Depending on laser pulse width, laser power, fiber shape, and fiber core size, the propagation of a pulse through an optical fiber can broaden and even distort the pulse. Chromatic dispersion, in which the refractive index is wavelength-dependent and therefore different wavelengths travel through the fiber at different speeds, dominates in large-mode-area fibers. Nonlinearities primarily occur in step-index single mode fibers. Because the light is confined to a smaller core, the intensity of the light is much greater in step-index single mode fibers, and nonlinear effects change the pulse shape proportional to the laser power. Two different fiber lengths will be used to analyze the relationship between chromatic dispersion effects and the distance through which the pulses travel through the fiber, in addition to developing a computer simulation to predict this broadening. Two types of single-mode fiber cores will be tested, large-mode-area and step-index single mode, to study nonlinearities and dispersion experimentally and through a numerical model.

2015 – 2016

Epstein, Jacob. “Construction and Characterization of an Upright Two-Photon Microscope.”  Senior Thesis, 2016.

Two-photon fluorescence microscopy allows for imaging deep within biological tissue without making an incision. Two-photon fluorescence occurs when two photons are simultaneously absorbed by a dye molecule and a single higher energy photon is released. The low probability of two-photon absorption ensures that fluorescence will only occur where many photons are focused at the same place at the same time. By moving the focus of the laser, a three-dimensional image is produced with micron level resolution. We have built an upright microscope that can image biological samples and created a user-friendly computer program to control it. By measuring the point spread function of the microscope, we have found the lateral resolution to be (1.6 ± 0.1) μm and the axial resolution to be (4.4 ± 0.3) μm.

Epstein, Jacob. “Construction and Characterization of an Upright Two-Photon Microscope.”  Senior Project, 2015.

Two-photon fluorescence microscopy allows for imaging deep within biological tissue without making an incision. Two-photon fluorescence occurs when two photons are simultaneously absorbed by a dye molecule and a single higher energy photon is released. The low probability of two-photon absorption ensures that fluorescence will only occur where many photons are in the same place at the same time, i.e., at the focus of a laser beam. By moving the focus of the laser, a 3D image is produced with micron level resolution. We have built an upright microscope to allow for water immersion samples, and by experimentally measuring its point spread function have found its lateral resolution to be (2.2 ± 0.2) μm.

Gordon, Teddy. “Detection Electronics for a Laser Scanning Microscope.” Senior Project, 2016.

We have designed and built detection electronics for a laser scanning microscope. The microscope operates by scanning a laser across a sample pixel by pixel and recording the fluorescence with a photomultiplier tube. The photons that make up the fluorescence are recorded by a computer in order to digitally re-construct an image. Currently the detection system samples only once per pixel. By using an analog integration technique we hope to improve the resolution. To accomplish this we have constructed an op amp based analog circuit that integrates the voltage over a set window. To maximize the speed and signal-to-noise ratio of the system it was created to fit on a Printed Circuit Board.

Robey, Kyle. “Characterization of a Temporal Focusing Microscope.” Senior Thesis, 2016.

Using thin films of fluorescent dye, I characterize the imaging quality of a temporal focusing microscope. Because two-photon absorption generates very few fluorescent photons, I employ photon counting using a photomultiplier tube (PMT), a comparator to discriminate between signal and noise, and a pulse-counter DAQ board. Using the PMT, I determine that the axial profile of the illumination region is Lorentzian in shape, with a width of 14 μm. After positioning the sample at the focus, I acquire images of the illumination using an sCMOS camera, yielding a lateral profile that is an asymmetric Gaussian with widths of 131 μm and 30.2 μm in each dimension. I then measure the point spread function using 1 μm-diameter fluorescent beads. Both the lateral and axial profiles of the beads are Gaussians, with dimensions of 1.2 μm, 1.2 μm, and 4.2 μm.

Robey, Kyle. “Characterization of a Temporal Focusing Microscope.” Senior Project, 2015.

Using fluorescent dye and fluorescent beads, I characterize the focus of a custom-built temporal focusing microscope. Because these films generate very few fluorescent photons through two photon excitation, counting photons rather than integrating the signal from the photomultiplier tube (PMT) yields improved sensitivity to low signals. A comparator converts PMT pulses above a certain discrimination level to uniform square pulses, and a digital data acquisition board counts these pulses via a LabVIEW program. With temporal focusing, the number of photon counts will peak at the position of the focus. The axial profile is Lorentzian in shape, with a full-width-at-half-maximum (FWHM) of 17 μm. After positioning the sample at the focus, I acquire 2D-images of the illumination profile. The lateral profile is an asymmetric Gaussian with FWHMs of 131 μm and 30.2 μm. Small fluorescent beads are also visible when viewed with a camera, yielding a lateral detection resolution of at least 1 μm.

2014 – 2015

Ballard, J.D. “Creating a 3D Microscope Using Commerical Depth Sensors.” Senior Project, 2015.

Standard microscopes produce two-dimensional images, but to obtain depth information one must physically move the sample. This method is both cumbersome and cannot be done in real time. The Microsoft Kinect is a commercial gaming device that obtains live three-dimensional (3D) data of a scene. This project investigates the feasibility of applying the different Kinect sensors to 3D microscopy. Through reverse engineering we determined that the Kinect v2 uses a time-of-flight method for depth sensing which we could not apply to microscopy due to speed limitations of the electronics. However, we determined that the Kinect v1 uses a structured light method of depth sensing, and we successfully applied this to a single magnifying lens. This setup not only magnified the image 1.8x but also magnified the depth sensing abilities of the Kinect v1 3.6x. We believe that this type of depth sensor could be economically applied to microscopy in the future.

McIntyre, Colin. “Building a Laser Scanning Microscope.” Senior Thesis, 2015.

A multi-photon microscope uses a focused laser beam to create fluorescence images pixel by pixel. This project creates a laser-scanning microscope capable of acquiring high quality, three-dimensional images. To begin, we program rotating galvanometer mirrors to deflect a beam, and rely on a series of lenses to convert the deflection angle to a lateral position on the sample. To calibrate and align this microscope setup, we test our automation programs with an alignment laser. Later on, we use a pulsed, mode locked Titanium-Sapphire laser to excite two photon fluorescence in a basic sample. Construction of an epi-detection system allows us to raster scan a laser focus around a volume several hundred microns in each direction and generate a three-dimensional image. Ultimately we generated a 3D multiphoton image using lens tissue stained with fluorescein.

McIntyre, Colin. “Building a Laser Scanning Microscope.” Senior Project, 2014.

A multi-photon microscope relies on a sharp focus to produce images of a fluorescent sample. This project will develop the infrastructure necessary to produce a high quality image using a simple continuous wave (cw) alignment laser. We use rotating galvanometer mirrors to deflect a beam, and a series of lenses converts the deflection angle to a lateral position on the sample. In addition, this alignment laser allows us to determine the system’s resolution and to calibrate the beam positions with respect to our LabVIEW program. Once the setup and calibration are complete, the apparatus can be readily adapted into a multi-photon system that will raster scan a pulsed laser through a fluorescent sample to generate depth-resolved images.

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