Preliminary Written Report: Light Detection with Ultra-High Dynamic Range
Group 35
Dohyun Kim, Leran Firer, and Eric Kleinberg
Client: Dr. Silva
9/26/12
Background:
Specialized chemical or voltage sensing domains of proteins in cells often times respond to specific environmental stimuli. Until recently it has been difficult to study and observe such proteins in action: the output of their response was typically known, but the processes were ambiguous. To better observe protein movements and conformational changes (which occur on a timescale of microsecond to nanosecond), fluorescence microscopy techniques were developed. Specifically, a floure molecule is attached to a protein molecule to be observed. Light is constantly input into the system as a stimulus for fluorescence. When a change in protein structure or function occurs, the attached floure fluoresces at a known wavelength (often 576nm in calcium channel studies) which is focused onto a photodiode system. The photodiode produces a current often in the range of picoamps, while the background signals are often on the order of microamps.
Due to the size of the background noise, this current signal must then be filtered and amplified via an integrating amplifier circuit: typically a Bessel Filter. To reduce noise from the PIN diode itself, the light must be tightly focused to the limit at which diffraction effects begin to distort the signal, the diffraction limit spot. To reduce thermal noise from the PIN diode, it must be cooled, typically via a Peltier system (also known as a thermoelectric cooler).
Changes in the emissions of labeled residues are rapid: absorption occurs in the femtosecond range, while emission occurs in the nanosecond range, orders of magnitude faster than known gating processes. As such, researchers can now observe such processes in real time. Experiments have previously used ~576 nm fluorescence light to characterize calcium channel protein kinetics.
Our client Jon Silva currently studies channel gating via oocytes. He specifically applies flourescence microscopy to research the ion channel movements and conformational changes while oocytes are patch clamped. This research has many applications within BME, particularly in understanding cardiac diseases/disorders such as Long QT3 syndrome.
Existing Solutions:
A cursory search for existing photodiode systems brought up the PhotoMax 200 by Dagan Corporation. This was used by Dr. Chris S. Gandhi and Dr. Riccardo Olcese as an example system used in fluorescence microscopy. The system cost ranges from $10,850-13,900 depending on configuration. The PhotoMax 200 utilizes a two stage Peltier cooled PIN diode headstage along with a four pole Bessel filter, which can serve as an inspiration for our design. Of interest is the notably low cost of the components when considering the total cost of the system, since typical PIN diodes retail for roughly $300, and Peltier Systems retail between $50-100. Beyond potential cost savings, a custom design has the advantage of more detailed specifications: manufacturers often omit certain statistics from their specification sheets such as focused light spot size or the specific temperature of the included Peltier Systems.
The optical focusing system must be able to focus a laser beam with a 600 nm wavelength to a diffraction limited spot onto a PIN Diode as to minimize noise. This can be achieved using many different types of lenses. Singlet lenses are simple and easy to use, but their performance is limited by spherical and chromatic aberrations, astigmatism, and other distortions. Thus, multi element lenses, designed using combinations of singlet elements, are necessary to minimize the number of aberrations during measurement. One excellent solution is an achromatic doublet lens system, which involves two singlet lens elements cemented together to nearly eliminate the spherical and longitudinal aberration components. Achromatic lenses exhibit an interesting behavior in that the shorter the back focal length (distance from the second lens to focal point), the tighter the focal spot. Deeper research into this phenomenon will help determine the ideal specifications of the lens we will ultimately implement.
Project Scope:
1. Filtering System (Electronics)
a. Design electrical system using a Pin Diode of our choice that is capable of accurately amplifying a signal 0.1% in magnitude relative to the background noise, a feat that manufacturers claim to be unreasonably difficult, but which Professor Silva has apparently demonstrated. Must condition signal to have high signal to noise ratio. Some possible design directions/inspirations include: reducing size/capacitance, biasing by -15 V, and implementation of variable resistance to control current into OP Amp. Target bandwidth for the filter should be 5-10kHz. Client recommended specifically using an Analog Bessel Filter.
b. Biasing will likely be done with some sort of power source (ex: battery). The power source is not constrained by the headstage as wires can be lead through the headstage.
Figure1. Head Stage of the Electrical System
2. Cooling System
a. The Pin Diode system produces a cleaner signal at lower temperatures. As such, client has requested a cooling system. He has suggested implementation of a Peltier type system (0 ~ -20oc) or similar such systems, though we are free to design a custom system if necessary. The chosen system should fit the head stage.
3. Light Focusing System
a. Client has requested design of an improved optical focusing system. Calculation is required for ideal area of focused light spot: specifically, there is a tradeoff between how much light reaches the PIN Diode versus noise. Client has requested either a custom focusing system, or off the shelf equivalent if cost is reasonable.
Specific Design Requirements:
Table 1: Specifications
System
Requirements
Electrical Filtration System
· Accurately filter and amplify a signal of .1% magnitude relative to background.
· Bias signal to -15V.
· Minimize capacitance to ensure high signal to noise ratio.
Peltier Cooling System
· Cool the Electrical Filtration System to optimal temperature dependent on PIN diode (~-20 C).
· Fit the SM1 mount used for PIN diode properly.
Light Focusing System
· Focus ~600nm laser light to diffraction limited spot.
Cost
· <$1000
Preliminary Analysis:
Table 2: Pugh Chart for PIN Diode
Weight
PIN_10AP
PIN_10DP(I)/SB
PIN_APD032
PIN_FD07
PIN_FD15
PIN_HR(s)008(L)
PIN-RD100(A)
Ultra Low
Noise
10
10
10
10
10
10
10
10
Low Capacitance
8
0
0
10
10
10
10
10
Temperature Range
5
6
6
10
9
9
9
7
Responsivity at 600nm
9
7
8
6
6
6
7
9
Diode Activation Area
8
-
-
-
-
-
-
-
High Speed Circuit
4
0
0
2
2
2
2
2
Total
193
200
282
287
287
296
296
Scores for the Size of Diode Activation Area were not assigned yet since it requires the decision made on focusing system. It will be updated once Leran Firer finishes his analysis.
Table 3: PIN Diode Specifications
PIN Diode Type
Ultra Low Noise
Low Capacitance
Temperature Range (oC)
Responsitivity at 600nm (A/W)
Activation Area (mm2)
High Speed Circuit
PIN_10AP
✔
X
0 – 70
0.27 - 0.4
100
X
PIN_10DP(I)/SB
✔
X
-10 – 60
0.33 – 0.4
100
X
PIN_APD032
✔
✔
-60 – 100
7.5 @ 850nm
0.5
✔
PIN_FD07
✔
✔
-40 – 100
0.3
7.1
✔
PIN_FD15
✔
✔
-40 – 100
0.3
14.9
✔
PIN_HR(s)008(L)
✔
✔
-40 – 100
0.32
0.04
✔
PIN-RD100(A)
✔
✔
-20 – 60
0.4
100
X
Figure 2: 4th order Bessel Bandpass Filter Schematics
Actual Calculations
- Center Frequency FM= 7.5KHz
- Bandwidth B= 5kHz
- Q= FM/B = 1.5
- Center Gain Km = 1 (absolute value); it’s is an unity gain filter
- From the Coefficient of the 4th order Filter Table a1 = 1.3617
b1 = 0.6180
α = 1.2711 (at Q = 1.5)
- Fm1 = FM/ α = 5900.4
Fm2 = FM* α = 9533.3
-
- C = 10 nF
-
-
Figure 3: 4th Order Bessel Bandpass Filter Designed with Commercially Available Resistors and Capacitors
Design Schedule:
Figure 4: Preliminary Design Schedule
Team Organization:
Table 4: Organization
Team Member
Specific Role
Eric Kleinberg
Research and Design of Peltier (TEC) Cooling System
Leran Firer
Research and Design of Optical Focusing System
Dohyun Kim
Research and Design of Electrical Circuitry
The team met and decided on September 11, 2012 to split up the research and design. Dr. Silva requested the design of three things: an electrical filter/amplifier, a cooling system for the filter, and a focusing system. As such, the team decided that each design could be tackled independently and that we would later reconvene to verify each other’s designs.
Literature Searches/Sources:
A.J. Horne and D. Fedida, Use of Voltage Clamp Fluorimetry in Understanding Potassium Channel Gating: A Review of Shaker Fluorescence Data, Can J Physiol Pharmacol. 2009 Jun;87(6):411-8
The study of the potassium channel gating and protein conformation change with emission of 576 nm wavelength gave us the deep understanding of the application of fluorescence microscopy.
Born, Max, and Emil Wolf. 7th ed. N.p.: Cambridge UP, 1999. Google Books. Web. 10 Sept. 2012. <http://books.google.com/books?hl=en>.
A book about the principles of optics to understand the basics of optical phenomenon as well as the different means to manipulate light via devices.
Chris S. Gandhi, The Voltage-Clamp Fluorometry Technique, Methods Mol Biol. 2009 ;491 :213-31 18998096
This article introduces the history of the fluorescence microscopy and its usage in the field of biology. It explains central design elements of fluorescence microscopy such as photodiode, voltage-clamped/patch-clamped cell bath in given wavelength. It also describes the 1st experiment conducted with commercial fluorescence microscopy – PhotoMax200. It was the primary existing solution of the project.
Custom Electric, Thermal Interfaces and Thermal Interface Materials, http://www.customthermoelectric.com/TIMs.html
This website provides a solid resource both on general peltier cooler module design, as well as some of the less discussed details such as mounting and thermal interfacing.
Davidson, Michael W., and Mortimer Abramowitz. "Optical Microscopy." (n.d.): n. pag. Olympus. Olympus America, Inc. Web. Sept. 2012. <http://micro.magnet.fsu.edu/primer/pdfs/microscopy.pdf>.
A paper about optical microscopy that discusses microscope objectives, eyepieces, condensers, and optical aberrations that are relevant to our fluorescence microscopy design.
Kugelstadt, Thomas. "Chapter 16: Active Filter Design Techniques." Op Amps for Everyone. Amsterdam: Newnes/Elsevier, 2009. 16-1-6-66.
This chapter illustrates the general concepts of active filter design in frequency domain. Each active filter introduced in this chapter –Butterworth, Shneck, and Bessel – are broadly explained its properties with its coefficient. This was the main source of the Bessel Filter resistance and capacitance calculation
Sigworth, F.J. “Chapter 4: Electron Design of the Patch Clamp.” Single-Channel Recording, B. Sakmann, E. Neher (eds.), DOI 10.1007/978-1-4419-1229-9_4, © Springer Science+Business Media, LLC 2009
This chapter illustrates experimental usage of patch clamp and its design. While it delivers the general concepts of the current-voltage properties of cell, it also explains background noise generated in the system. The equations of noise described in this chapter were the primary sources of the noise analysis.
Sunex. "Achromatic Doublet." Optics-Online. N.p., n.d. Web. http://optics-online/ach.asp.
An achromatic doublet lens manufacturing competitor, with details pertaining to the lens’s physical nature as well as a listing of the commercially available lenses and their specifications.
Sunex. "Lens, Lens System and Optical Aberrations." Cartage. N.p., n.d. Web. <http://www.cartage.org.lb/en/themes/sciences/physics/optics/Optical/Lens/Lens.htm>.
An article that discusses the different singlet lens elements, and the aberrations encountered with their use. This introduces the advantage of multi-element lenses that can focus light to a tighter spot without the spherical and chromatic aberrations in singlet lenses.
Thor Labs. "Visible Achromatic Doublets." THORLABS. N.p., n.d. Web. <http://www.thorlabs.de/newgrouppage9.cfm?objectgroup_id=120>.
An achromatic doublet lens manufacturer, with details pertaining to the lens’s features, applications, and damage thresholds. There is also a listing of the commercially available lenses and their specifications.
US5054896: Existing patent of a continuously focusable microscope incorporating an afocal variator optical system that uses an achromatic doublet lens.
Group 35
Dohyun Kim, Leran Firer, and Eric Kleinberg
Client: Dr. Silva
9/26/12
Background:
Specialized chemical or voltage sensing domains of proteins in cells often times respond to specific environmental stimuli. Until recently it has been difficult to study and observe such proteins in action: the output of their response was typically known, but the processes were ambiguous. To better observe protein movements and conformational changes (which occur on a timescale of microsecond to nanosecond), fluorescence microscopy techniques were developed. Specifically, a floure molecule is attached to a protein molecule to be observed. Light is constantly input into the system as a stimulus for fluorescence. When a change in protein structure or function occurs, the attached floure fluoresces at a known wavelength (often 576nm in calcium channel studies) which is focused onto a photodiode system. The photodiode produces a current often in the range of picoamps, while the background signals are often on the order of microamps.
Due to the size of the background noise, this current signal must then be filtered and amplified via an integrating amplifier circuit: typically a Bessel Filter. To reduce noise from the PIN diode itself, the light must be tightly focused to the limit at which diffraction effects begin to distort the signal, the diffraction limit spot. To reduce thermal noise from the PIN diode, it must be cooled, typically via a Peltier system (also known as a thermoelectric cooler).
Changes in the emissions of labeled residues are rapid: absorption occurs in the femtosecond range, while emission occurs in the nanosecond range, orders of magnitude faster than known gating processes. As such, researchers can now observe such processes in real time. Experiments have previously used ~576 nm fluorescence light to characterize calcium channel protein kinetics.
Our client Jon Silva currently studies channel gating via oocytes. He specifically applies flourescence microscopy to research the ion channel movements and conformational changes while oocytes are patch clamped. This research has many applications within BME, particularly in understanding cardiac diseases/disorders such as Long QT3 syndrome.
Existing Solutions:
A cursory search for existing photodiode systems brought up the PhotoMax 200 by Dagan Corporation. This was used by Dr. Chris S. Gandhi and Dr. Riccardo Olcese as an example system used in fluorescence microscopy. The system cost ranges from $10,850-13,900 depending on configuration. The PhotoMax 200 utilizes a two stage Peltier cooled PIN diode headstage along with a four pole Bessel filter, which can serve as an inspiration for our design. Of interest is the notably low cost of the components when considering the total cost of the system, since typical PIN diodes retail for roughly $300, and Peltier Systems retail between $50-100. Beyond potential cost savings, a custom design has the advantage of more detailed specifications: manufacturers often omit certain statistics from their specification sheets such as focused light spot size or the specific temperature of the included Peltier Systems.
The optical focusing system must be able to focus a laser beam with a 600 nm wavelength to a diffraction limited spot onto a PIN Diode as to minimize noise. This can be achieved using many different types of lenses. Singlet lenses are simple and easy to use, but their performance is limited by spherical and chromatic aberrations, astigmatism, and other distortions. Thus, multi element lenses, designed using combinations of singlet elements, are necessary to minimize the number of aberrations during measurement. One excellent solution is an achromatic doublet lens system, which involves two singlet lens elements cemented together to nearly eliminate the spherical and longitudinal aberration components. Achromatic lenses exhibit an interesting behavior in that the shorter the back focal length (distance from the second lens to focal point), the tighter the focal spot. Deeper research into this phenomenon will help determine the ideal specifications of the lens we will ultimately implement.
Project Scope:
1. Filtering System (Electronics)
a. Design electrical system using a Pin Diode of our choice that is capable of accurately amplifying a signal 0.1% in magnitude relative to the background noise, a feat that manufacturers claim to be unreasonably difficult, but which Professor Silva has apparently demonstrated. Must condition signal to have high signal to noise ratio. Some possible design directions/inspirations include: reducing size/capacitance, biasing by -15 V, and implementation of variable resistance to control current into OP Amp. Target bandwidth for the filter should be 5-10kHz. Client recommended specifically using an Analog Bessel Filter.
b. Biasing will likely be done with some sort of power source (ex: battery). The power source is not constrained by the headstage as wires can be lead through the headstage.
Figure1. Head Stage of the Electrical System
2. Cooling System
a. The Pin Diode system produces a cleaner signal at lower temperatures. As such, client has requested a cooling system. He has suggested implementation of a Peltier type system (0 ~ -20oc) or similar such systems, though we are free to design a custom system if necessary. The chosen system should fit the head stage.
3. Light Focusing System
a. Client has requested design of an improved optical focusing system. Calculation is required for ideal area of focused light spot: specifically, there is a tradeoff between how much light reaches the PIN Diode versus noise. Client has requested either a custom focusing system, or off the shelf equivalent if cost is reasonable.
Specific Design Requirements:
Table 1: Specifications
System
Requirements
Electrical Filtration System
· Accurately filter and amplify a signal of .1% magnitude relative to background.
· Bias signal to -15V.
· Minimize capacitance to ensure high signal to noise ratio.
Peltier Cooling System
· Cool the Electrical Filtration System to optimal temperature dependent on PIN diode (~-20 C).
· Fit the SM1 mount used for PIN diode properly.
Light Focusing System
· Focus ~600nm laser light to diffraction limited spot.
Cost
· <$1000
Preliminary Analysis:
Table 2: Pugh Chart for PIN Diode
Weight
PIN_10AP
PIN_10DP(I)/SB
PIN_APD032
PIN_FD07
PIN_FD15
PIN_HR(s)008(L)
PIN-RD100(A)
Ultra Low
Noise
10
10
10
10
10
10
10
10
Low Capacitance
8
0
0
10
10
10
10
10
Temperature Range
5
6
6
10
9
9
9
7
Responsivity at 600nm
9
7
8
6
6
6
7
9
Diode Activation Area
8
-
-
-
-
-
-
-
High Speed Circuit
4
0
0
2
2
2
2
2
Total
193
200
282
287
287
296
296
Scores for the Size of Diode Activation Area were not assigned yet since it requires the decision made on focusing system. It will be updated once Leran Firer finishes his analysis.
Table 3: PIN Diode Specifications
PIN Diode Type
Ultra Low Noise
Low Capacitance
Temperature Range (oC)
Responsitivity at 600nm (A/W)
Activation Area (mm2)
High Speed Circuit
PIN_10AP
✔
X
0 – 70
0.27 - 0.4
100
X
PIN_10DP(I)/SB
✔
X
-10 – 60
0.33 – 0.4
100
X
PIN_APD032
✔
✔
-60 – 100
7.5 @ 850nm
0.5
✔
PIN_FD07
✔
✔
-40 – 100
0.3
7.1
✔
PIN_FD15
✔
✔
-40 – 100
0.3
14.9
✔
PIN_HR(s)008(L)
✔
✔
-40 – 100
0.32
0.04
✔
PIN-RD100(A)
✔
✔
-20 – 60
0.4
100
X
Figure 2: 4th order Bessel Bandpass Filter Schematics
Actual Calculations
- Center Frequency FM= 7.5KHz
- Bandwidth B= 5kHz
- Q= FM/B = 1.5
- Center Gain Km = 1 (absolute value); it’s is an unity gain filter
- From the Coefficient of the 4th order Filter Table a1 = 1.3617
b1 = 0.6180
α = 1.2711 (at Q = 1.5)
- Fm1 = FM/ α = 5900.4
Fm2 = FM* α = 9533.3
-
- C = 10 nF
-
-
Figure 3: 4th Order Bessel Bandpass Filter Designed with Commercially Available Resistors and Capacitors
Design Schedule:
Figure 4: Preliminary Design Schedule
Team Organization:
Table 4: Organization
Team Member
Specific Role
Eric Kleinberg
Research and Design of Peltier (TEC) Cooling System
Leran Firer
Research and Design of Optical Focusing System
Dohyun Kim
Research and Design of Electrical Circuitry
The team met and decided on September 11, 2012 to split up the research and design. Dr. Silva requested the design of three things: an electrical filter/amplifier, a cooling system for the filter, and a focusing system. As such, the team decided that each design could be tackled independently and that we would later reconvene to verify each other’s designs.
Literature Searches/Sources:
A.J. Horne and D. Fedida, Use of Voltage Clamp Fluorimetry in Understanding Potassium Channel Gating: A Review of Shaker Fluorescence Data, Can J Physiol Pharmacol. 2009 Jun;87(6):411-8
The study of the potassium channel gating and protein conformation change with emission of 576 nm wavelength gave us the deep understanding of the application of fluorescence microscopy.
Born, Max, and Emil Wolf. 7th ed. N.p.: Cambridge UP, 1999. Google Books. Web. 10 Sept. 2012. <http://books.google.com/books?hl=en>.
A book about the principles of optics to understand the basics of optical phenomenon as well as the different means to manipulate light via devices.
Chris S. Gandhi, The Voltage-Clamp Fluorometry Technique, Methods Mol Biol. 2009 ;491 :213-31 18998096
This article introduces the history of the fluorescence microscopy and its usage in the field of biology. It explains central design elements of fluorescence microscopy such as photodiode, voltage-clamped/patch-clamped cell bath in given wavelength. It also describes the 1st experiment conducted with commercial fluorescence microscopy – PhotoMax200. It was the primary existing solution of the project.
Custom Electric, Thermal Interfaces and Thermal Interface Materials, http://www.customthermoelectric.com/TIMs.html
This website provides a solid resource both on general peltier cooler module design, as well as some of the less discussed details such as mounting and thermal interfacing.
Davidson, Michael W., and Mortimer Abramowitz. "Optical Microscopy." (n.d.): n. pag. Olympus. Olympus America, Inc. Web. Sept. 2012. <http://micro.magnet.fsu.edu/primer/pdfs/microscopy.pdf>.
A paper about optical microscopy that discusses microscope objectives, eyepieces, condensers, and optical aberrations that are relevant to our fluorescence microscopy design.
Kugelstadt, Thomas. "Chapter 16: Active Filter Design Techniques." Op Amps for Everyone. Amsterdam: Newnes/Elsevier, 2009. 16-1-6-66.
This chapter illustrates the general concepts of active filter design in frequency domain. Each active filter introduced in this chapter –Butterworth, Shneck, and Bessel – are broadly explained its properties with its coefficient. This was the main source of the Bessel Filter resistance and capacitance calculation
Sigworth, F.J. “Chapter 4: Electron Design of the Patch Clamp.” Single-Channel Recording, B. Sakmann, E. Neher (eds.), DOI 10.1007/978-1-4419-1229-9_4, © Springer Science+Business Media, LLC 2009
This chapter illustrates experimental usage of patch clamp and its design. While it delivers the general concepts of the current-voltage properties of cell, it also explains background noise generated in the system. The equations of noise described in this chapter were the primary sources of the noise analysis.
Sunex. "Achromatic Doublet." Optics-Online. N.p., n.d. Web. http://optics-online/ach.asp.
An achromatic doublet lens manufacturing competitor, with details pertaining to the lens’s physical nature as well as a listing of the commercially available lenses and their specifications.
Sunex. "Lens, Lens System and Optical Aberrations." Cartage. N.p., n.d. Web. <http://www.cartage.org.lb/en/themes/sciences/physics/optics/Optical/Lens/Lens.htm>.
An article that discusses the different singlet lens elements, and the aberrations encountered with their use. This introduces the advantage of multi-element lenses that can focus light to a tighter spot without the spherical and chromatic aberrations in singlet lenses.
Thor Labs. "Visible Achromatic Doublets." THORLABS. N.p., n.d. Web. <http://www.thorlabs.de/newgrouppage9.cfm?objectgroup_id=120>.
An achromatic doublet lens manufacturer, with details pertaining to the lens’s features, applications, and damage thresholds. There is also a listing of the commercially available lenses and their specifications.
US5054896: Existing patent of a continuously focusable microscope incorporating an afocal variator optical system that uses an achromatic doublet lens.