Assessment of UV Excilamps for Wound Sterilization - Columbia

Assessment of UV Excilamps for
Wound Sterilization
Hamin Jeon
Emory University
Mentors: Alan Bigelow and Gerhard Randers-Pehrson
Radiological Research Accelerator Facility (RARAF)
Summer of 2011
The Outline
Background information (why excilamps?)
 The spectra of KrBr and KrCl excilamps
 The filter selection process
 Light attenuation through different media
 Cell inactivation with unfiltered KrCl excilamp
 The Outline
Background information (why excilamps?)
 The spectra of KrBr and KrCl excilamps
 The filter selection process
 Light attenuation through different media
 Cell inactivation with unfiltered KrCl excilamp
 Surgical Site Infections
A major cause of illness
 Among about 27 million surgical procedures, the
infections number around 500,000 per year (˜2%)
 Sources are: airborne bacteria, contaminated
operating room surfaces, contaminated instruments,
organisms from a surgical team in an operating room
 Ultraviolet (UV) irradiation is proven to be effective
for preventing infections
 Low-Pressure Mercury Lamps vs. Excilamps
Mercury lamps
 Typically used for UV irradiation procedures
 Emit UV light in a wide range of wavelengths: UVA (320 to 400 nm),
UVB (290 to 320 nm), UVC (220 to 290 nm), etc
 UVA: can cause sunburn on human skin and cataracts in eyes
 UVB: can cause damage to DNA
 Need UV protective gear: expensive, cumbersome
Excilamps
 UV produced from an excited molecule complex
 Emit UV light of single primary peak wavelength
  KrBr (206nm)
KrCl (222nm)
High Wavelength vs. Low Wavelength
   UV light of higher wavelengths are more penetrating than
that of lower wavelengths
A bacterial cell diameter: normally less than 1µm
A human cell diameter: between 10 to 30 µm
So, Why KrBr and KrCl Excilamps?
   Emit UV light of low wavelength
High enough wavelength to kill bacteria, but low
enough wavelength to not damage human cells
Possibly much safer to use excilamps than to use
mercury lamps, but also as effective
The Outline
Background information (why excilamps?)
 The spectra of KrBr and KrCl excilamps
 The filter selection process
 Light attenuation through different media
 Cell inactivation with unfiltered KrCl excilamp
 KrBr and KrCl Spectra from the
Manufacturer’s Manual
The spectrum of KrBr excilamp
The spectrum of KrCl excilamp
Ozone Production
 Can smell ozone when the excilamps are turned on
 Ozone is produced the most around 184 nm
 Emission of light of below 200nm wavelength?
A Spectrometer & a Nitrogen Purge
   Use the spectrometer specifically ordered to:
detect any signal below 200nm and verify the
spectra of KrBr and KrCl excilamps
Light of wavelengths below 200nm may get
absorbed by moisture or oxygen in air
A nitrogen purge may be applied to get a better
signal below 200nm
KrBr and KrCl Excilamps Spectra Without a
Nitrogen Purge and with Optical Fiber
The spectrum of KrBr excilamp
Oh, no….
The spectrum of KrCl excilamp
KrBr Excilamp Spectrum Without Fiber
Optics but with a Nitrogen Purge
“Zoomed-in” KrBr and KrCl Excilamps Spectra
The spectrum of KrBr excilamp
The spectrum of KrCl excilamp
Summary of Problems & Possible Solutions
 Problems:
 KrBr
excilamp spectrum obtained with the spectrometer
different from the one from the manufacturer
 Insignificant signal below 200nm
 Possible solutions
 Communications
with the excilamp and spectrometer
manufacturers
 Using a calibration mercury lamp
(THE TIEBREAKER!)
KrBr and KrCl Excilamps’ Power Density
Measurement for Different Distances
The Outline
Background information (why excilamps?)
 The spectra of KrBr and KrCl excilamps
 The filter selection process
 Light attenuation through different media
 Cell inactivation with unfiltered KrCl excilamp
 Short- and Long-Pass Filters vs. a Bandpass Filter
Filters from Omega Optical, Inc.
 For KrBr
 A combination
of short-pass (210 SP) and long-pass
(218DCLP) filters vs. bandpass (206NB6) filter
 For KrCl
 A combination
of short-pass (220AF10) and long-pass
(233DCLP) filters vs. bandpass (222NB7) filter
KrBr, A Combination of 210 SP and 218DCLP
Filters vs. 206NB6 Filter
210 SP and 218 DCLP, transmittance vs. wavelength
206 NB6, transmittance vs. wavelength
16
25
14
20
Transmittance (%)
Transmitance (%)
12
10
8
6
15
10
4
5
2
0
0
150
170
190
210
230
250
Wavelength (nm)
270
290
310
170
180
190
200
210
220
Wavelength (nm)
230
240
250
KrCl, A Combination of 220AF10 and
233DCLP Filters vs. 222NB7 Filter
222NB7, transmittance vs. wavelength
30
30
25
25
20
20
Transmittance (%)
Transmittance (%)
220AF10 and 233DCLP, transmittance vs. wavelength
15
10
5
15
10
5
0
0
200
220
240
260
Wavelength (nm)
280
300
200
210
220
230
Wavelength (nm)
240
250
208NB6 & 224NB7 Bandpass Filters
Chose the dish diameter of 3cm and the distance between
the lamp and the dish as 4cm
 Maximum angle of incidence (AOI)
of about 20 degrees
 The performance varies at different
AOI
 Peak wavelengths with a half cone 20
degrees AOI are around 203.6nm and
219.6nm
 Requested filters that transmit light of 2nm
higher peak wavelengths at normal incidence
 Intensity [AU]
Intensity [AU]
Expected Performance
YAY!
YAY!
Quartz Dish
 Quartz coverslip
 No
 fluorescence, low absorption (˜6% attenuation)
Quartz dish
 inner
diameter: 12.51mm
 inner area: 1.229 cm2
 height of the dish: 18.76 mm
 inner volume of the dish: 2.306 cm3
 0.2 ml of volume increment used
The Outline
Background information (why excilamps?)
 The spectra of KrBr and KrCl excilamps
 The filter selection process
 Light attenuation through different media
 Cell inactivation with unfiltered KrCl excilamp
 The Experimental Setup
Light Attenuation Through Distilled Water
Light Attenuation Through Phosphate
Buffered Saline (PBS)
Light Attenuation Through Small Airway Basal
Medium (SABM)
The Outline
Background information (why excilamps?)
 The spectra of KrBr and KrCl excilamps
 The filter selection process
 Light attenuation through different media
 Cell inactivation with unfiltered KrCl excilamp
 Irradiation and Incubation
 Total of 7 cell dishes irradiated for 0 s (two dishes),
2 s, 5 s, 15 s, 30 s, 60 s, but placed under the
excilamp for total of 60s
 Cells were not placed in SABM during irradiation
 After the irradiation, put in an incubator for two days
The Experimental Setup
Hemocytometry (Initial Assay Attempt)
After incubation, trypsin and trypan blue dye applied
to one 0 s dish and 60 s dish
 Dead cells: fully stained
 Cell counting with hemocytometer: # of fully stained
cells vs. # of unstained cells
 0s cells: no fully stained cells, cells were alive
 60s cells: did not detach from the dish, which was
not good
 Microscopy (Final Assay)
Cell fixing with methanol
 Observation of cell morphology after irradiation
 Hoffman modulation contrast imaging: enhances
the contrast in unstained biological specimen by
varying light intensity
 DAPI (4',6-diamidino-2-phenylindole):
Fluorescent stain that binds to DNAs, allows for
nuclei detection
 Images of Cells Obtained by Using Hoffman
Modulation Contrast Imaging
Cells irradiated for 0 s
Cells irradiated for 2 s
Cells irradiated for 15 s
Cells irradiated for 5 s
Cells irradiated for 30 s
Images of Cells Obtained by Using
Fluorescence Microscopy with DAPI Stain
Cells irradiated for 0 s
Cells irradiated for 2 s
Cells irradiated for 15 s
Cells irradiated for 5 s
Cells irradiated for 30 s
Analysis of the Images
Round and small structures indicate compact cell
nuclei which might be related to cell death
 Ruptured cell nuclei also might be related to cell
death
 Without using a filter, the excilamp could kill the
cells
 Need the filter, with which we could kill bacteria
but not damage human cell deaths
 Acknowledgements
Alan W. Bigelow: for overseeing and supporting the
progresses I have made
 Gerhard Randers-Pehrson: for providing extensive advice in
any physical analysis
 Charles R. Geard: for overseeing and supporting any
biological setups and procedures
 Brian Ponnaiya: for helping any biological assessments
 Bo Zhang: for preparing cells for us
 Stephen Marino: the RARAF manager
 John Parsons: for providing me with the opportunity to
work at Nevis Lab this Summer!
 References
1. "DAPI Nuclear Counterstain." http://www.piercenet.com/browse.cfm?fldID=01041204
 2. Nichols, Ronald L. "Preventing Surgical Site Infections: A Surgeon's Perspective." March 05, 2009. http://www.cdc.gov/ncidod/
eid/vol7no2/nichols.htm
 3. Fitzwater, Janet. "Bacteriological Effect of Ultraviolet Light on a Surgical Instrument Table.” Public Health Rep. 76. 2 (1961),
97, http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC1929616/
 4. Zeman, Gary. "Ultraviolet Radiation." July 06, 2011.http://www.hps.org/hpspublications/articles/uv.html
 5. Allen, Jeannie. "Ultraviolet Radiation: How It Affects Life on Earth." September 6, 2001. http://earthobservatory.nasa.gov/
Features/UVB/
 6. "bacteria." March 15, 2008.http://www.sizes.com/natural/bacteria.htm
 7. Machalek, Alisa Z. "Chapter 1: An Owner's Guide to the Cell." April 22, 2011. http://publications. nigms.nih.gov/insidethecell/
chapter1.html
 8. "Fuller Ozone Generators." http://www.fulleruv.com/ozone_generators.html
 9. Padley, Paul . "The Fresnel Equations." Oct 14, 2005. http://cnx.org/content /m12904/ latest/
 10. Garbett, Ian. "Light attenuation and exponential laws." January 1, 2001.http://plus.maths.org/ content/light-attenuation-andexponential-laws
 11. Hardy, Kate. "Cell death in the mammalian blastocyst.” Molecular Human Reproduction 3. 10 (1997), 919, http://
www.ncbi.nlm.nih.gov/pubmed/9395266.
 12. Sosnin, Edward A., Eva Stoffels, Michael V. Erofeev, Ingrid E. Kieft, and Sergey E. Kunts. "The Effects of UV Irradiation and
Gas Plasma Treatment on Living Mammalian Cells and Bacteria: A Comparative Approach.” IEEE TRANSACTIONS ON
PLASMA SCIENCE 32. 4 (2004), 1547, http://ieeexplore.ieee.org/xpls/abs_all.jsp?arnumber=1341520.
 13. Krebsfaenger, Niels. "Micronucleus Assay." www.genpharmtox.de/downloads/ AssaySheetMikronucleusAssay.pdf
 14. Giannini, Gina T., John T Boothby, and Eric E Sabelman. "Infected wound model development of an in vitro biomaterialprotected wound infection model to study microbial activity and antimicrobial treatment through microdialysis.” Adv Skin
Wound Care 23. 8 (2010), 358-64, http://www.ncbi.nlm.nih.gov/pubmed/20664329.
 Backup slides
206NB6 Spectral Calculation
222NB7 Spectral Calculation
Future Objectives
To obtain the expected filtered spectra from both
KrBr and KrCl excilamps
 To design a safer UV box with enclosed front
 Tests with in-vitro 3-D human skin system, in-vitro
wound infection model and in-vivo nude mouse
model
 Clinical trials
