Lalwani Balasingam Olazarra Saffari - Revision history

imported>Student221: /* Clinical Background */

2019-12-13T18:57:46Z

Clinical Background

← Older revision		Revision as of 18:57, 13 December 2019
Line 11:		Line 11:
	Cancers of the head and neck can be life-altering and even deadly, as well as difficult to detect early. While the 5-year survival rate is high, these cancers can often go unnoticed until a very late stage [12]. As a result, there is a strong and urgent need for better tools to detect head and neck cancers.		Cancers of the head and neck can be life-altering and even deadly, as well as difficult to detect early. While the 5-year survival rate is high, these cancers can often go unnoticed until a very late stage [12]. As a result, there is a strong and urgent need for better tools to detect head and neck cancers.

	We were driven to find a label-free method for autofluorescence imaging of cholesteatomas. Cholesteatomas are non-malignant, non-cancerous growths that typically form behind the tympanic membrane [1, 2]. They are similar to cysts, and often occur in people who are prone to otitis media (or middle ear infections), and many are in fact congenital [2, 3]. Our objective is to create a label-free method to characterize biomolecular changes in the keratinized epithelial tissue by exploiting the molecular specificity of autofluorescence spectroscopy. Specifically, we will measure the spectral signatures of biological molecules like NADH and elastin, both of which are naturally ~~found~~ in the body and can fluoresce upon absorption of ultraviolet (UV) or visible light. Ultimately, being able to quickly and reliably visualize these cholesteatomas is desirable for early diagnosis and complete resection of the lesions, in order to better preserve the patient’s hearing ability.		We were driven to find a label-free method for autofluorescence imaging of cholesteatomas. Cholesteatomas are non-malignant, non-cancerous growths that typically form behind the tympanic membrane [1, 2]. They are similar to cysts, and often occur in people who are prone to otitis media (or middle ear infections), and many are in fact congenital [2, 3]. Our objective is to create a label-free method to characterize biomolecular changes in the keratinized epithelial tissue by exploiting the molecular specificity of autofluorescence spectroscopy. Specifically, we will measure the spectral signatures of biological molecules like NADH and elastin, both of which are naturally occurring in the body and can fluoresce upon absorption of ultraviolet (UV) or visible light. Ultimately, being able to quickly and reliably visualize these cholesteatomas is desirable for early diagnosis and complete resection of the lesions, in order to better preserve the patient’s hearing ability.

	===Technical Background===		===Technical Background===

imported>Student221: /* Introduction */

2019-12-13T18:52:13Z

Introduction

← Older revision		Revision as of 18:52, 13 December 2019
Line 1:		Line 1:
	==Introduction==		==Introduction==

	This quarter, our team worked on characterizing fluorophores purchased by the Valdez Lab in the Department of Otolaryngology, Head and Neck Surgery at the Stanford School of Medicine to determine optimal materials for cholesteatoma visualization. The fluorophores we measured include elastin, keratin, NADH, and collagen. Our inspiration to pursue a project focused on using fluorescence to better image cholesteatomas was inspired by work being done in the Valdez lab as well as oral imaging work being done by Dr. Joyce E. Farrell and Zheng Lyu in Stanford's Department of Electrical Engineering. We hope our work can shed some light on which fluorophores may be best for producing a label-free method in congenital cholesteatoma detection via autofluorescence microscopy. To aid these efforts, our team worked on characterizing and analyzing such fluorophores to determine the optimal selections for clinical use given their respective absorption and emission spectra. Following these measurements, we designed a fluorophore matrix that accommodated ~~the~~ fluorophores ~~to enable~~ further measurements and characterization to be performed using the OralEye imaging system developed by Dr. Farrell and Zheng. While our group focused on using this matrix for measurements of our chosen fluorophores, the matrix was designed to be customizable for users ~~and allow~~ them to insert and remove their own fluorophores of interest. While our project allowed us to gain a great deal of information about ~~the~~ fluorophores and expand upon our own understanding of how light interacts with materials, we plan on refining our methods and taking further measurements in the year to come.		This quarter, our team worked on characterizing fluorophores purchased by the Valdez Lab in the Department of Otolaryngology, Head and Neck Surgery at the Stanford School of Medicine to determine optimal materials for cholesteatoma visualization. The fluorophores we measured include elastin, keratin, NADH, and collagen. Our inspiration to pursue a project focused on using fluorescence to better image cholesteatomas was inspired by work being done in the Valdez lab as well as oral imaging work being done by Dr. Joyce E. Farrell and Zheng Lyu in Stanford's Department of Electrical Engineering. We hope our work can shed some light on which fluorophores may be best for producing a label-free method in congenital cholesteatoma detection via autofluorescence microscopy. To aid these efforts, our team worked on characterizing and analyzing such fluorophores to determine the optimal selections for clinical use given their respective absorption and emission spectra. Following these measurements, we designed a fluorophore matrix that accommodated our selected fluorophores, which enabled further measurements and characterization to be performed using the OralEye imaging system developed by Dr. Farrell and Zheng. While our group focused on using this matrix for measurements of our chosen fluorophores, the matrix was designed to be customizable for users by allowing them to insert and remove their own fluorophores of interest. While our project allowed us to gain a great deal of information about our selected fluorophores and expand upon our own understanding of how light interacts with materials, we plan on refining our methods and taking further measurements in the year to come.

	==Background==		==Background==

imported>Student221: /* Acknowledgements */

2019-12-13T18:11:08Z

Acknowledgements

← Older revision		Revision as of 18:11, 13 December 2019
Line 214:		Line 214:

	==Acknowledgements==		==Acknowledgements==
	We would like to express our appreciation for the countless hours that Professor Brian A. Wandell, Dr. Joyce E. Farrell, and Zheng Lyu spent on making this course truly exceptional. Across our team, the class has inspired several new interests, including presenting magneto-sensor research at the next imaging ~~conference~~, exploring a career in ophthalmology, and continuing to refine our fluorophore results in the year to come. We are tremendously grateful for your time, energy, and investment in our team. It brought us great joy and honor to be your students.		We would like to express our appreciation for the countless hours that Professor Brian A. Wandell, Dr. Joyce E. Farrell, and Zheng Lyu spent on making this course truly exceptional. Across our team, the class has inspired several new interests, including presenting magneto-sensor research at the next imaging symposium, exploring a career in ophthalmology, and continuing to refine our fluorophore results in the year to come. We are tremendously grateful for your time, energy, and investment in our team. It brought us great joy and honor to be your students.

imported>Student221: /* Appendix II */

2019-12-13T18:10:32Z

Appendix II

← Older revision		Revision as of 18:10, 13 December 2019
Line 207:		Line 207:
	==Appendix II==		==Appendix II==

	Anand and Persiana worked on characterizing the fluorophores by measuring the absorption and emission spectra of each fluorophore. They used a fluorescence spectrometer (~~fluorimeter~~) in ~~the chemistry department~~ that can measure wavelengths from the UV range to 1,000 nanometers ~~as well as~~ has a sub-nanometer image resolution. Following fluorimeter training, preparation of samples, and recording of measurements, Anand and Persiana analyzed the data and interpreted it for the writing of this report and the presentation.		Anand and Persiana worked on characterizing the fluorophores by measuring the absorption and emission spectra of each fluorophore. They used a fluorescence spectrometer (fluorometer) in Stanford's Department of Chemistry that can measure wavelengths from the UV range to 1,000 nanometers. Additionally, Haribo's Fluorolog fluorometer has a sub-nanometer image resolution. Following fluorimeter training, preparation of samples, and recording of measurements, Anand and Persiana analyzed the data and interpreted it for the writing of this report and the presentation.

	Based on the results of the fluorophore absorption and emission spectra, Sofie and Ramya constructed a matrix containing ~~the optimal~~ water-soluble fluorophores. The design was modeled after that of Connect Four, and was 3D-printed ~~on campus,~~ in the Huang basement. Following construction of the matrix, Sofie and Ramya proceeded to take measurements with the OralEye Camera in Dr. Farrell’s lab. They then subsequently plotted their results using MATLAB scripts found in Appendix I.		Based on the results of the fluorophore absorption and emission spectra, Sofie and Ramya constructed a matrix containing these water-soluble fluorophores. The design was modeled after that of a Connect Four, and was 3D-printed at Stanford in the Huang building's basement. Following construction of the matrix, Sofie and Ramya proceeded to take measurements with the OralEye Camera in Dr. Farrell’s lab. They then subsequently plotted their results using MATLAB scripts found in Appendix I.

	We all contributed to the final in-class presentation and our Wiki page submission.		We all contributed to the final in-class presentation and our Wiki page submission.

imported>Student221: /* Future Work */

2019-12-13T18:07:55Z

Future Work

← Older revision		Revision as of 18:07, 13 December 2019
Line 173:		Line 173:

	==Future Work==		==Future Work==
	In the future, we plan to evaluate the effects of different concentrations on our ability to distinguish a fluorescence signal from the background reflected light signal. Our experiments with the fluorometer indicate that concentration can have a noticeable effect on our ability to measure fluorescence. Assuming a similar dependency on concentration in our OralEye camera set-up, we would like to perform many experiments to find the “sweet-spot” concentration value that gives us measurable fluorescence. By the same token, we would like to perform more measurements on the fluorometer by exciting our fluorophores at the 385 nm wavelength, to better compare the measurements between the OralEye Camera and the emission spectra. This will also help us find emission spectra that are consistent with those recorded in previous literature.		In the future, we plan to evaluate the effects of different concentrations on our ability to distinguish a fluorescence signal from the background reflected light signal. Our experiments with the Fluorolog fluorometer indicate that concentration can have a noticeable effect on our ability to measure fluorescence, in addition to the excitation wavelength itself. Assuming a similar dependency on concentration in our OralEye camera set-up, we would like to perform many experiments to find the “sweet-spot” concentration value that gives us measurable fluorescence. By the same token, we would like to perform more measurements on the fluorometer by exciting our fluorophores at the 385 nm wavelength, to better compare the measurements between the OralEye Camera and the emission spectra. This will also help us find emission spectra that are consistent with those recorded in previous literature.

	In order to acquire more meaningful data using the OralEye camera set-up, we must address the issue of measuring weak fluorescence in the presence of a relatively strong reflected light signal. One obvious way to do this is to implement a more effective filter in order to prevent excessive reflected light from “leaking” into our fluorescence measurements. One possibility would be to apply a filter with an even higher threshold, such as a Y48 or Y50 filter (with cut-off wavelengths of ~480 nm and ~500 nm, respectively), to further reduce the impact of the light reflected from the 325 nm light source. As mentioned earlier, another possibility would be to increase or decrease the concentration of the fluorophore samples we measure; doing so might increase the signal produced by the fluorophores, and thus reduce the issues associated with the weak signal we saw in our earlier measurements. By exploring these sorts of solutions, we feel confident that a robust set of measurements can be acquired for our selected fluorophores.		In order to acquire more meaningful data using the OralEye camera set-up, we must address the issue of measuring weak fluorescence in the presence of a relatively strong reflected light signal. One obvious way to do this is to implement a more effective filter in order to prevent excessive reflected light from “leaking” into our fluorescence measurements. One possibility would be to apply a filter with an even higher threshold, such as a Y48 or Y50 filter (with cut-off wavelengths of ~480 nm and ~500 nm, respectively), to further reduce the impact of the light reflected from the 325 nm light source. As mentioned earlier, another possibility would be to increase or decrease the concentration of the fluorophore samples we measure; doing so might increase the signal produced by the fluorophores, and thus reduce the issues associated with the weak signal we saw in our earlier measurements. By exploring these sorts of solutions, we feel confident that a robust set of measurements can be acquired for our selected fluorophores.

imported>Student221: /* Results of OralEye Camera Measurements */

2019-12-13T18:05:24Z

Results of OralEye Camera Measurements

← Older revision		Revision as of 18:05, 13 December 2019
Line 121:		Line 121:

	===Results of OralEye Camera Measurements===		===Results of OralEye Camera Measurements===
	In order to understand the data we collected for our fluorophore measurements, we plotted the emission spectra corresponding to each of our fluorophores as a function of wavelength. This plot can be seen in Figure 8. In inspecting this plot, we made two primary observations. First, we noted that all four of our fluorophores demonstrated a sharp increase in measured fluorescence at a wavelength of approximately 425 nm, peaking shortly thereafter. The fact that all four of our fluorophores peaked in this region was not necessarily worrisome ~~of and~~ by itself, as we expected very little signal below 425 nm due to the presence of the Y44 filter, which is ideally ~~designed to~~ only pass light corresponding to wavelengths greater than 425 nm. As such, it seemed reasonable that we would see a spike in all four of the signals at wavelengths beyond this threshold. The second observation we made was that the peak magnitude of the measured fluorescence for three of our four fluorophores was nearly identical (exact values for maxima are specified in the legend of Figure 8). This feature of the plot was slightly more concerning, as it suggested the possibility that we were measuring the same signal for all of our fluorophores despite the fact that we expected different emission spectra. This raised concerns regarding the integrity of our data, and pointed to the possibility that we measured reflected light rather than a true fluorescence signal.		In order to understand the data we collected for our fluorophore measurements, we plotted the emission spectra corresponding to each of our fluorophores as a function of wavelength. This plot can be seen in Figure 8. In inspecting this plot, we made two primary observations. First, we noted that all four of our fluorophores demonstrated a sharp increase in measured fluorescence at a wavelength of approximately 425 nm, peaking shortly thereafter. The fact that all four of our fluorophores peaked in this region was not necessarily worrisome by itself, as we expected very little signal below 425 nm due to the presence of the Y44 filter, which should ideally only pass light corresponding to wavelengths greater than 425 nm. As such, it seemed reasonable that we would see a spike in all four of the signals at wavelengths beyond this threshold. The second observation we made was that the peak magnitude of the measured fluorescence for three of our four fluorophores was nearly identical (exact values for maxima are specified in the legend of Figure 8). This feature of the plot was slightly more concerning, as it suggested the possibility that we were measuring the same signal for all of our fluorophores despite the fact that we expected different emission spectra. This raised concerns regarding the integrity of our data, and pointed to the possibility that we measured reflected light rather than a true fluorescence signal.

	In order to understand the data we collected for our fluorophore measurements, we plotted the emission spectra corresponding to each of our fluorophores as a function of wavelength. This plot can be seen in Figure 8. In inspecting this plot, we made two primary observations. First, we noted that all four of our fluorophores demonstrated a sharp increase in measured fluorescence at a wavelength of approximately 425 nm, peaking shortly thereafter. The fact that all four of our fluorophores peaked in this region was not necessarily worrisome of and by itself, as we expected very little signal below 425 nm due to the presence of the Y44 filter, which is ideally designed to only pass light corresponding to wavelengths greater than 425 nm. As such, it seemed reasonable that we would see a spike in all four of the signals at wavelengths beyond this threshold. The second observation we made was that the peak magnitude of the measured fluorescence for three of our four fluorophores was nearly identical (exact values for maxima are specified in the legend of Figure 8). This feature of the plot was slightly more concerning, as it suggested the possibility that we were measuring the same signal for all of our fluorophores despite the fact that we expected different emission spectra. This raised concerns regarding the integrity of our data, and pointed to the possibility that we measured reflected light rather than a true fluorescence signal.

	[[File:fig8.png\|center]]		[[File:fig8.png\|center]]
Line 151:		Line 150:


	After establishing that we did not measure a robust fluorescence signal for our fluorophores, we wanted to determine whether or not we were able to effectively measure the stronger, more robust fluorescence signal associated with our fluorescent calibration slides. This would help us to clarify whether our undesirable fluorophore measurements were rooted in the weak fluorescence of those fluorophores, or a general inability to measure fluorescence using our previously-described experimental configuration and measurement schema. Just as we had done in our fluorophore measurement analysis, we began ~~this process~~ by plotting the emission spectra of the calibration slides alongside the filtered reflected light spectrum. From this initial plot, pictured in Figure 12, it immediately became clear that the fluorescence signal associated with the calibration slides was far more robust when compared with the signal associated with the reflected light than the fluorophores were. That is, the magnitude of the fluorescence signal for the calibration slides was much larger than the magnitude of the signal associated with the reflected light at the wavelengths we were interested in. This indicated that the signals we were measuring at or above 425 nm were likely due to actual fluorescence rather than reflected light. As such, we concluded that our experimental configuration could, in fact, measure fluorescence provided that the fluorescence produced by the sample was greater in magnitude than that of the reflected light.		After establishing that we did not measure a robust fluorescence signal for our fluorophores, we wanted to determine whether or not we were able to effectively measure the stronger, more robust fluorescence signal associated with our fluorescent calibration slides. This would help us to clarify whether our undesirable fluorophore measurements were rooted in the weak fluorescence of those fluorophores, or a general inability to measure fluorescence using our previously-described experimental configuration and measurement schema. Just as we had done in our fluorophore measurement analysis, we began by plotting the emission spectra of the calibration slides alongside the filtered reflected light spectrum. From this initial plot, pictured in Figure 12, it immediately became clear that the fluorescence signal associated with the calibration slides was far more robust when compared with the signal associated with the reflected light than the fluorophores were. That is, the magnitude of the fluorescence signal for the calibration slides was much larger than the magnitude of the signal associated with the reflected light at the wavelengths we were interested in. This indicated that the signals we were measuring at or above 425 nm were likely due to actual fluorescence rather than reflected light. As such, we concluded that our experimental configuration could, in fact, measure fluorescence provided that the fluorescence produced by the sample was greater in magnitude than that of the reflected light.

	[[File:fig12.png\|center]]		[[File:fig12.png\|center]]

imported>Student221: /* Results from Fluorometer Measurements */

2019-12-13T18:01:38Z

Results from Fluorometer Measurements

← Older revision		Revision as of 18:01, 13 December 2019
Line 62:		Line 62:

	===Results from Fluorometer Measurements===		===Results from Fluorometer Measurements===
	We found the following spectra for the NADH, collagen, elastin, and keratin fluorophores using Haribo’s ~~fluorolog~~. Additionally, we chose to excite these fluorophores at specific wavelengths based on work being conducted in the Valdez lab and methods used in previous literature [4, 7, 8, 9].		We found the following spectra for the NADH, collagen, elastin, and keratin fluorophores using Haribo’s Fluorolog. Additionally, we chose to excite these fluorophores at specific wavelengths based on work being conducted in the Valdez lab and methods used in previous literature [4, 7, 8, 9].

	'''NADH'''		'''NADH'''
Line 88:		Line 88:
	'''Elastin'''		'''Elastin'''

	Figure 6 summarizes the absorption and emission spectra we recorded for elastin various specified wavelengths.		Figure 6 summarizes the absorption and emission spectra we recorded for elastin at various specified wavelengths.

	[[File:elastin.png\|800px\|center]]		[[File:elastin.png\|800px\|center]]
Line 94:		Line 94:
	<div align="center">'''Figure 6''': The leftmost plot illustrates the absorption spectrum of elastin at an emission of 415 nm. The following two plots (in the middle and the right) are emission spectra where elastin is excited at 325 nm. Note that, on the middle emission spectrum, the concentration of elastin was halved before being excited at 325 nm.</div>		<div align="center">'''Figure 6''': The leftmost plot illustrates the absorption spectrum of elastin at an emission of 415 nm. The following two plots (in the middle and the right) are emission spectra where elastin is excited at 325 nm. Note that, on the middle emission spectrum, the concentration of elastin was halved before being excited at 325 nm.</div>

	These results are nearly, though not completely, consistent with results ~~from~~ reported in ~~previously-published~~ literature [8]. While the absorption spectrum is relatively close to what has been seen in previous findings, the emission spectrum for elastin typically is more prominent at around 400 nm. The rightmost emission spectrum is closer to this value, but is just a hair shy. Additionally, these peaks are broader than typical spectra for elastin.		These results are nearly, though not completely, consistent with results reported in previous literature [8]. While the absorption spectrum is relatively close to what has been seen in previous findings, the emission spectrum for elastin typically is more prominent at around 400 nm. The rightmost emission spectrum is closer to this value, but is just a hair shy. Additionally, these peaks are broader than typical spectra for elastin.


Line 110:		Line 110:
	'''Troubleshooting the Measurements and Adjusting our Protocol'''		'''Troubleshooting the Measurements and Adjusting our Protocol'''

	One of the main reasons why our spectra may differ from those reported for the same fluorophores in previous literature may be due to cross-contamination of the fluorophores. Before beginning our experiment, we were prepared to measure our samples using cuvettes with beveled edges. ~~This~~ produced a great deal of scattering, ~~and~~ so we switched ~~over~~ to using clear cuvettes without any beveled edges. Raman peaks can be detected by changing the excitation spectrum by a few nanometers and observing if there is a shift in the emission peak by the same amount. If so, then we knew that this was indeed a Raman peak. Raman peaks generally occur close to the excitation wavelength; and by implementing a low pass filter cut-off above the Raman peak, we can make sure that we are measuring the true signal of the fluorophore. While this technique allowed us to eliminate the presence of Raman peaks for samples measured in the beveled cuvettes, switching over to the clear cuvettes proved to be the best solution for eliminating scattering. Unfortunately, we did not have enough of these clear cuvettes for each sample, so we had to re-use ~~the~~ clear cuvette for numerous samples. While we tried our best to clean and remove all fluorophore remnants from previous trials, we would be remiss to not acknowledge the potential for any lingering fluorophores to interfere with the measurements of subsequent fluorophores.		One of the main reasons why our spectra may differ from those reported for the same fluorophores in previous literature may be due to cross-contamination of the fluorophores. Before beginning our experiment, we were prepared to measure our samples using cuvettes with beveled edges. These cuvettes produced a great deal of scattering, so we switched to using clear cuvettes without any beveled edges. This technique allowed us to eliminate the presence of Raman peaks for samples measured in the beveled cuvettes; therefore, switching over to the clear cuvettes proved to be the best solution for eliminating scattering. Unfortunately, we did not have enough of these clear cuvettes for each sample, so we had to re-use one clear cuvette for numerous samples. While we tried our best to clean and remove all fluorophore remnants from previous trials, we would be remiss to not acknowledge the potential for any lingering fluorophores to interfere with the measurements of subsequent fluorophores.

			Raman peaks can be detected by changing the excitation spectrum by a few nanometers and observing if there is a shift in the emission peak by the same amount. If there is such a shift, then we knew that we were witnessing a Raman peak. This was how we identified the cuvette type as being the main culprit for our scattering problems. Raman peaks generally occur close to the excitation wavelength; and by implementing a low pass filter cut-off above the Raman peak, we can make sure that we are measuring the true signal of the fluorophore.

	In addition to this Raman scattering (as seen in the case of the beveled cuvette), we also needed to address the second-harmonic light arising from the excitation source. The second harmonic excitation is when light from the excitation source (a white light with a grating filter on top) measures not only the wavelength of interest (for example, 300 nm) but also the second harmonic of that wavelength (600 nm). The second harmonic is then recorded as a very strong and narrow peak. To avoid this issue, we made sure that the high pass filter in the Fluorolog's fluorescence settings is set to 10 nm below the second harmonic.		In addition to this Raman scattering (as seen in the case of the beveled cuvette), we also needed to address the second-harmonic light arising from the excitation source. The second harmonic excitation is when light from the excitation source (a white light with a grating filter on top) measures not only the wavelength of interest (for example, 300 nm) but also the second harmonic of that wavelength (600 nm). The second harmonic is then recorded as a very strong and narrow peak. To avoid this issue, we made sure that the high pass filter in the Fluorolog's fluorescence settings is set to 10 nm below the second harmonic.

imported>Student221: /* Measurements with the OralEye Camera */

2019-12-13T17:53:58Z

Measurements with the OralEye Camera

← Older revision		Revision as of 17:53, 13 December 2019
Line 40:		Line 40:
	===Measurements with the OralEye Camera===		===Measurements with the OralEye Camera===

	~~In order to~~ determine ~~whether~~ we ~~were able to~~ measure fluorescence using the OralEye camera, we built a matrix that not only accommodated our water-soluble fluorophores, but also accommodated standard fluorescent microscope slides (and similarly-prepared, alternative fluorophores identified by the user). A three-dimensional rendering of the matrix, which was modeled after standard color calibration targets, is pictured in Figure 2. Performing measurements using the fluorescent calibration targets allowed us to determine whether we were able to measure any fluorescence at all, while performing similar measurements using our chosen fluorophores allowed us to attempt to measure and compare their respective fluorescence signals with the results obtained using the fluorometer.		To determine if we could measure fluorescence using the OralEye camera, we built a matrix that not only accommodated our water-soluble fluorophores, but also accommodated standard fluorescent microscope slides (and similarly-prepared, alternative fluorophores identified by the user). A three-dimensional rendering of the matrix, which was modeled after standard color calibration targets, is pictured in Figure 2. Performing measurements using the fluorescent calibration targets allowed us to determine whether we were able to measure any fluorescence at all, while performing similar measurements using our chosen fluorophores allowed us to attempt to measure and compare their respective fluorescence signals with the results obtained using the fluorometer.


Line 48:		Line 48:


	Prior to performing the fluorophore fluorescence measurements, we prepared four standard glass microscope slides: one for each of the respective fluorophore solutions. Sample preparation varied depending on the state in which the fluorophores were provided, but was largely consistent with the sample preparation described in the fluorometer measurements section. For keratin and elastin, which were provided in liquid form, we dissolved approximately ~~20uL~~ of the fluorophore in ~~1mL~~ of DI water. For NADH and collagen, the concentrations were slightly less straightforward. The NADH provided took the form of a collection of non-uniform beads; to prepare this sample, we dissolved approximately five of the beads in 20uL of distilled water. Finally, the collagen was presented as a cotton-ball-like substance; to prepare this sample, we tore off a small portion of the collagen substance, then dissolved it in 20uL of distilled water. After the fluorophore solutions were prepared, we sealed them by placing glass cover slides on top.		Prior to performing the fluorophore fluorescence measurements, we prepared four standard glass microscope slides: one for each of the respective fluorophore solutions. Sample preparation varied depending on the state in which the fluorophores were provided, but was largely consistent with the sample preparation described in the fluorometer measurements section. For keratin and elastin, which were provided in liquid form, we dissolved approximately 20 uL of the fluorophore in 1 mL of DI water. For NADH and collagen, the concentrations were slightly less straightforward. The NADH provided took the form of a collection of non-uniform beads; to prepare this sample, we dissolved approximately five of the beads in 20uL of distilled water. Finally, the collagen was presented as a cotton-ball-like substance; to prepare this sample, we tore off a small portion of the collagen substance, then dissolved it in 20uL of distilled water. After the fluorophore solutions were prepared, we sealed them by placing glass cover slides on top.

	[[File:Exp_schematic.png\|center\|500px\|]]		[[File:Exp_schematic.png\|center\|500px\|]]
Line 56:		Line 56:


	Our four fluorophore samples were then installed in the aforementioned matrix in order to ~~begin performing~~ fluorescence measurements using the OralEye camera. We fixed the matrix to an optical stand, which was then fixed to an optical table. The optical stand allowed us to move the matrix vertically in order to capture data corresponding to both the top and bottom row samples. The OralEye camera was installed on a track that allowed us to move the camera/light source horizontally in order to illuminate and capture data corresponding to both the left and right column samples. In order to actually capture the emission spectra, we situated a spectroradiometer approximately one meter from the matrix. Because the spectroradiometer, alone, does not have a dynamic range high enough to distinguish a weak emitted fluorescence signal from a strong reflected light signal, we equipped the spectroradiometer with a Y44 longpass filter. By installing this filter, we were able to filter out a large fraction of the signal produced due to light reflected from the 385 nm light source. (Further discussion regarding the efficacy of this filter with respect to our particular results can be found in the “Results” and “Conclusions” sections.)		Our four fluorophore samples were then installed in the aforementioned matrix in order to perform fluorescence measurements using the OralEye camera. We fixed the matrix to an optical stand, which was then fixed to an optical table. The optical stand allowed us to move the matrix vertically in order to capture data corresponding to both the top and bottom row samples. The OralEye camera was installed on a track that allowed us to move the camera/light source horizontally in order to illuminate and capture data corresponding to both the left and right column samples. In order to actually capture the emission spectra, we situated a spectroradiometer approximately one meter from the matrix. Because the spectroradiometer, alone, does not have a dynamic range high enough to distinguish a weak emitted fluorescence signal from a strong reflected light signal, we equipped the spectroradiometer with a Y44 longpass filter. By installing this filter, we were able to filter out a large fraction of the signal produced due to light reflected from the 385 nm light source. (Further discussion regarding the efficacy of this filter with respect to our particular results can be found in the “Results” and “Conclusions” sections.)

	==Results==		==Results==

imported>Student221: /* Fluorometer Measurements with Haribo's Fluorolog Device */

2019-12-13T17:50:03Z

Fluorometer Measurements with Haribo's Fluorolog Device

← Older revision		Revision as of 17:50, 13 December 2019
Line 25:		Line 25:
	===Fluorometer Measurements with Haribo's Fluorolog Device===		===Fluorometer Measurements with Haribo's Fluorolog Device===

	The fluorometer we used for the measurement of the fluorophores was made available to us ~~courtesy of~~ the Stanford Department of Chemistry in Stauffer 1. Figure 1 is an image of the machine ~~that~~ we used to measure the emission and absorption spectra of the fluorophores.		The fluorometer we used for the measurement of the fluorophores was made available to us by the Stanford Department of Chemistry (machine located in Stauffer 1). Figure 1 is an image of the Fluorolog machine we used to measure the emission and absorption spectra of the fluorophores.


Line 34:		Line 34:


	The fluorometer’s excitation wavelength ~~range~~ from 250 nm to 400 nm, while the emission spectrum has a range ~~between~~ 250 nm to 1000 nm. Therefore, ~~we felt that~~ this tool would be particularly useful in our measurements as its dynamic range met the specifications required by our fluorophores.		The fluorometer’s excitation wavelength ranges from 250 nm to 400 nm, while the emission spectrum has a range of 250 nm to 1000 nm. Therefore, this tool would be particularly useful in our measurements, as its dynamic range met the specifications required by our fluorophores.

	As for the fluorophores, we ~~tried to keep~~ the concentrations consistent within each cuvette ~~so that we could~~ effectively compare spectra between NADH, elastin, collagen, and keratin. For our liquid samples (keratin and elastin, which was mixed with deionized (DI) water), we put ~30 ul into 1.5 mL of DI water in a clear cuvette. For NADH, which came in the form of small "dip-n-dot"-like samples, we placed between 3-5 dots (depending on the size of the dots) in 1.5 mL of DI water in the cuvette. Finally, for collagen, which arrived as a cotton-ball-like material, we evenly divided the sample into 5 pieces and placed 1 piece in the cuvette (with 1.5 mL of DI water) for spectral measurements. Based on this concentration level, we set the ~~fluorolog’s~~ slit size to 3.0 mm in order to produce emission spectra that did not have disproportionately high intensities. As a reference, concentrated solutions are typically those that look cloudy in the cuvette; and those solutions that are more heavily concentrated must have a smaller slit width in order to appropriately measure the fluorescence at a reasonable intensity.		As for preparing the fluorophores themselves, we kept the concentrations consistent within each cuvette in order to effectively compare spectra between NADH, elastin, collagen, and keratin. For our liquid samples (keratin and elastin, which was mixed with deionized (DI) water), we put ~30 ul into 1.5 mL of DI water in a clear cuvette. For NADH, which came in the form of small "dip-n-dot"-like samples, we placed between 3-5 dots (depending on the size of the dots) in 1.5 mL of DI water in the cuvette. Finally, for collagen, which arrived as a cotton-ball-like material, we evenly divided the sample into 5 pieces and placed 1 piece in the cuvette (with 1.5 mL of DI water) for spectral measurements. Based on this concentration level, we set the Fluorolog’s slit size to 3.0 mm, in order to produce emission spectra that did not have disproportionately high intensities. As a reference, concentrated solutions are typically those that look cloudy in the cuvette; and those solutions that are more heavily concentrated must have a smaller slit width in order to appropriately measure the fluorescence at a reasonable intensity.

	===Measurements with the OralEye Camera===		===Measurements with the OralEye Camera===

imported>Student221: /* Methods */

2019-12-13T17:45:20Z

Methods

← Older revision		Revision as of 17:45, 13 December 2019
Line 21:		Line 21:
	==Methods==		==Methods==

	We acquired four fluorophores (NADH, collagen, elastin, and keratin), then measured the fluorescence signal ~~produced by~~ each using ~~both~~ a fluorometer and the OralEye camera provided by Dr. Farrell. The purpose of our initial fluorescence measurements, which were performed using a fluorometer, was to determine whether we ~~were able to~~ measure emission spectra that were consistent with those recorded in the literature for each of our fluorophores. Secondary measurements, which were performed using the OralEye camera, sought to determine whether we were able to effectively measure similar emission spectra using the 385 nm excitation light source associated with the OralEye camera.		We acquired four fluorophores (NADH, collagen, elastin, and keratin), then measured the fluorescence signal of each using a fluorometer and the OralEye camera, which was provided by Dr. Farrell. The purpose of our initial fluorescence measurements, which were performed using a fluorometer, was to determine whether we could measure emission spectra that were consistent with those recorded in the literature for each of our fluorophores. Secondary measurements, which were performed using the OralEye camera, sought to determine whether we were able to effectively measure similar emission spectra using the 385 nm excitation light source associated with the OralEye camera.

	===Fluorometer Measurements with Haribo's Fluorolog Device===		===Fluorometer Measurements with Haribo's Fluorolog Device===