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| public:optical_instruments 2010/07/19 11:38 | public:optical_instruments 2017/05/03 21:53 current | ||
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| **A simple optical system consists a light source (Sun) and an imager (the Eye).** | **A simple optical system consists a light source (Sun) and an imager (the Eye).** | ||
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| - | + | ====== How Do We Generate Light? Where Does It Come From? ====== | |
| - | ====== How Do We Generate Light? / Where Does Light Come From? ====== | + | |
| The light sources are used in photo biology to generate photons with different frequency, energy, temporal and spatial characteristics. Commonly used light sources include lamps, light emitting diodes (LED), and lasers. Lamps are the most common broadband sources while LEDs and lasers are more complex devices that many of their spectral and temporal features may be well controlled. Based on light sources’ spectral bandwidth, they can be classified as broadband sources and narrow band or even line sources. Lamps and LEDs are low fluence broadband illumination sources that are mostly used with monochromator and band pass filters for selected wavelength illumination or as spectroscopic calibration standards. Depending on their temporal characteristics, the light sources can also be categorized into continuous wave (CW) or pulsed. \\ | The light sources are used in photo biology to generate photons with different frequency, energy, temporal and spatial characteristics. Commonly used light sources include lamps, light emitting diodes (LED), and lasers. Lamps are the most common broadband sources while LEDs and lasers are more complex devices that many of their spectral and temporal features may be well controlled. Based on light sources’ spectral bandwidth, they can be classified as broadband sources and narrow band or even line sources. Lamps and LEDs are low fluence broadband illumination sources that are mostly used with monochromator and band pass filters for selected wavelength illumination or as spectroscopic calibration standards. Depending on their temporal characteristics, the light sources can also be categorized into continuous wave (CW) or pulsed. \\ | ||
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| ===== Lamps ===== | ===== Lamps ===== | ||
| - | Lams Pare non coherent sources that usually emit light propagate to all directions. In photo physics research, lamps are used both for illumination and calibration. | + | Lamps are non coherent sources that usually emit light propagate to all directions. In photo physics research, lamps are used both for illumination and calibration. |
| ===== Incandescent Lamps ===== | ===== Incandescent Lamps ===== | ||
| Incandescent lamps are the most common type of light source. Typical incandescent lamps emit light by heating the filament and the emitted light continues broadband spectrum, which can be described by black body radiation. \\ | Incandescent lamps are the most common type of light source. Typical incandescent lamps emit light by heating the filament and the emitted light continues broadband spectrum, which can be described by black body radiation. \\ | ||
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| One main advantage of laser is that both short and long pulsed operation is relatively easy to achieve. Typical nanosecond land picoseconds lasers include solid state lasers and diode lasers. Mode-locked lasers such as Ti: Sapphire may be designed to work at 70-120MHz. \\ | One main advantage of laser is that both short and long pulsed operation is relatively easy to achieve. Typical nanosecond land picoseconds lasers include solid state lasers and diode lasers. Mode-locked lasers such as Ti: Sapphire may be designed to work at 70-120MHz. \\ | ||
| \\ \\ | \\ \\ | ||
| - | ====== How do we control the propagation of light? ====== | + | ====== How Do We Control the Propagation of Light? ====== |
| Upon output from the light source, photons were usually relayed with mirrors and lens to the target and for collection to the detection system. In the last a few years, more and more optical instruments start to use optics fibers for light delivery. \\ | Upon output from the light source, photons were usually relayed with mirrors and lens to the target and for collection to the detection system. In the last a few years, more and more optical instruments start to use optics fibers for light delivery. \\ | ||
| Each of these components is consisted of various basic optics devices such as lens, mirrors, prisms, gratings. A clear understanding of the characteristics of these components and basic optics allows one to asses the capabilities and limitations of these instruments. \\ | Each of these components is consisted of various basic optics devices such as lens, mirrors, prisms, gratings. A clear understanding of the characteristics of these components and basic optics allows one to asses the capabilities and limitations of these instruments. \\ | ||
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| ===== Optical Building Blocks ===== | ===== Optical Building Blocks ===== | ||
| Most of optical instruments are built from many basic optical devices such as lens, mirror, prism, grating, filter, and etc, which control the propagation of light. They are the basic building blocks of optical instruments. \\ | Most of optical instruments are built from many basic optical devices such as lens, mirror, prism, grating, filter, and etc, which control the propagation of light. They are the basic building blocks of optical instruments. \\ | ||
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| - | ====== How do we detect photons? / How do we measure the characteristics of light? ====== | + | ====== How Do We Detect Photons? How Do We Measure the Characteristics of Light? ====== |
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| The photons are converted into electrons via various photo detectors. The number of photons can be correlated to either the electron current or voltage that can be read by various electronics devices such as multi-meter, oscilloscope, or a digitizer interfaced to a computer. \\ | The photons are converted into electrons via various photo detectors. The number of photons can be correlated to either the electron current or voltage that can be read by various electronics devices such as multi-meter, oscilloscope, or a digitizer interfaced to a computer. \\ | ||
| - | + | ===== Photo Diodes: ===== | |
| - | ===== Photo diodes: ===== | + | |
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| Photo diode is the most common point photo detectors. Their operating principle and manufacturing are relatively simple and therefore, they are relatively inexpensive devices that can also be made very small. The working spectral range of photo diodes is determined by the materials used in the P-N junction. In the visible range, silicon photo diodes are the most common. Small active area photo diodes can also be made into high speed photo detectors, e.g. 40GHz. Biased photo diodes usually used very low power while do not have multiplication. Therefore, they are not suitable for low light detection. A new type of photo diode is avalanche photo diode or APD, which has an on board multiplication process that can amplify the photo electrons by a factor of 100. \\ | Photo diode is the most common point photo detectors. Their operating principle and manufacturing are relatively simple and therefore, they are relatively inexpensive devices that can also be made very small. The working spectral range of photo diodes is determined by the materials used in the P-N junction. In the visible range, silicon photo diodes are the most common. Small active area photo diodes can also be made into high speed photo detectors, e.g. 40GHz. Biased photo diodes usually used very low power while do not have multiplication. Therefore, they are not suitable for low light detection. A new type of photo diode is avalanche photo diode or APD, which has an on board multiplication process that can amplify the photo electrons by a factor of 100. \\ | ||
| - | + | ===== Photo Multiplier Tube (PMT) ===== | |
| - | ===== Photo multiplier tube (PMT) ===== | + | |
| PMT is the most common point photo detector for low light detection. PMT usually has a series of anodes after the photo cathode, which converts the incoming photo into electrons. The PMTs are usually much more expensive and bulky than photo diodes due to the complex structure requiring vacuum and need high voltage power supply. However, PMTs can have up to 108 amplification that it they be used for even single photon detection. The temporal response of PMT can be very fast (e.g. 180ps rise time), especially when used with a micro channel plate (MCP). | PMT is the most common point photo detector for low light detection. PMT usually has a series of anodes after the photo cathode, which converts the incoming photo into electrons. The PMTs are usually much more expensive and bulky than photo diodes due to the complex structure requiring vacuum and need high voltage power supply. However, PMTs can have up to 108 amplification that it they be used for even single photon detection. The temporal response of PMT can be very fast (e.g. 180ps rise time), especially when used with a micro channel plate (MCP). | ||
| - | ===== CCD camera: ===== | + | ===== CCD Camera ===== |
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| Charge couple device is an array of photo diodes that provide spatial distribution of light intensity. Although the photo detection is parallel, the read out process is serial on CCD. As illustrated in the following figure, each row of the pixels will be shifted to the read-out register for being read out (digitized via an A/D converter) one pixel at a time. Therefore, CCD is typically not used to directly image high speed (shorter than a few s) events. A more detailed description of CCD devices may be found at: [[http://www.microscopyu.com/articles/digitalimaging/ccdintro.html|Microscopy Primer]]. \\ | Charge couple device is an array of photo diodes that provide spatial distribution of light intensity. Although the photo detection is parallel, the read out process is serial on CCD. As illustrated in the following figure, each row of the pixels will be shifted to the read-out register for being read out (digitized via an A/D converter) one pixel at a time. Therefore, CCD is typically not used to directly image high speed (shorter than a few s) events. A more detailed description of CCD devices may be found at: [[http://www.microscopyu.com/articles/digitalimaging/ccdintro.html|Microscopy Primer]]. \\ | ||
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| {{ :research:optical_instruments:pixel_read_out_screen.jpg?300 }} | {{ :research:optical_instruments:pixel_read_out_screen.jpg?300 }} | ||
| **CCD pixel read out scheme** \\ | **CCD pixel read out scheme** \\ | ||
| - | ===== CMOS camera: ===== | + | ===== CMOS Camera ===== |
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| In CCD cameras, the photon collection process is parallel and the final output is digital. However, the read out process is mostly serial and the charge transfer process is mostly analog before read out. Since 2000, a new family of imaging sensors called complementary metal oxide semiconductor (CMOS) have been emerged into scientific imaging instruments. In CMOS, each pixel has its own read out circuitry that it is considered as the true parallel imaging sensor. Using CMOS technology, large imaging sensors can be made much faster and cheaper. | In CCD cameras, the photon collection process is parallel and the final output is digital. However, the read out process is mostly serial and the charge transfer process is mostly analog before read out. Since 2000, a new family of imaging sensors called complementary metal oxide semiconductor (CMOS) have been emerged into scientific imaging instruments. In CMOS, each pixel has its own read out circuitry that it is considered as the true parallel imaging sensor. Using CMOS technology, large imaging sensors can be made much faster and cheaper. | ||
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| {{ :research:optical_instruments:cmos_addressing.jpg?250 }} | {{ :research:optical_instruments:cmos_addressing.jpg?250 }} | ||
| - | **CMOS addressing** | + | **CMOS addressing** \\ |
| - | + | However, comparing to CCD imagers, current CMOS sensors still lacks sensitivity due to relatively small fill factor (photon sensitive active area vs. total pixel area). \\ | |
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| - | However, comparing to CCD imagers, current CMOS sensors still lacks sensitivity due to relatively small fill factor (photon sensitive active area vs. total pixel area). | + | |
| {{ :research:optical_instruments:cmos_sensor_layout.jpg?250 }} | {{ :research:optical_instruments:cmos_sensor_layout.jpg?250 }} | ||
| - | **CMOS sensor layout** \\ | + | **CMOS sensor layout** \\ \\ |
| - | ===== Intensified CCD camera ===== | + | ===== Intensified CCD Camera ===== |
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| For low signals and time-gated applications, an image intensifier may be used in an Intensified CCD camera (ICCD). The following figure 22 shows the schematic of a typical ICCD system which consists an image intensifier, a set of relay lenses, and a conventional CCD camera. The controlling electronics and computer interface are similar to those of CCD cameras but additional interfacing and time synchronization electronics are required for control of the intensifier. | For low signals and time-gated applications, an image intensifier may be used in an Intensified CCD camera (ICCD). The following figure 22 shows the schematic of a typical ICCD system which consists an image intensifier, a set of relay lenses, and a conventional CCD camera. The controlling electronics and computer interface are similar to those of CCD cameras but additional interfacing and time synchronization electronics are required for control of the intensifier. | ||
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| {{ :research:optical_instruments:intensified_ccd_camera.jpg?350 }} Intensified CCD camera (courtesy of Stanford Computer Optics) | {{ :research:optical_instruments:intensified_ccd_camera.jpg?350 }} Intensified CCD camera (courtesy of Stanford Computer Optics) | ||
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| **A typical gated intensified CCD camera system** \\ | **A typical gated intensified CCD camera system** \\ | ||
| //Reprint with permission from [[http://www.iccd-camera.com/technology_main.htm|Stanford Computer Optics]]// \\ | //Reprint with permission from [[http://www.iccd-camera.com/technology_main.htm|Stanford Computer Optics]]// \\ | ||
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| The core of an ICCD is its image intensifier which consists of a photocathode, a micro channel plate (MCP), and a phosphor screen. In the image intensifier, the photons will go through a photon-electron-amplification-photon process. The basic principle of a typical gated image intensifier is showing in the following figure. Initially, the photons arriving at the photocathode are converted into photo electrons. These electrons will be accelerated by an external electrical field and then amplified when going through the MCP, which maintains the spatial distribution of the electrons. The amplified electrons will be further accelerated to a phosphor screen, which will convert these electrons to photons. Through the coupling lenses or fiber optics coupling bundles, these photons will be collected by the read out CCD camera. \\ | The core of an ICCD is its image intensifier which consists of a photocathode, a micro channel plate (MCP), and a phosphor screen. In the image intensifier, the photons will go through a photon-electron-amplification-photon process. The basic principle of a typical gated image intensifier is showing in the following figure. Initially, the photons arriving at the photocathode are converted into photo electrons. These electrons will be accelerated by an external electrical field and then amplified when going through the MCP, which maintains the spatial distribution of the electrons. The amplified electrons will be further accelerated to a phosphor screen, which will convert these electrons to photons. Through the coupling lenses or fiber optics coupling bundles, these photons will be collected by the read out CCD camera. \\ | ||
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| {{ :research:optical_instruments:typical_mcp_structure.jpg?350 }} | {{ :research:optical_instruments:typical_mcp_structure.jpg?350 }} | ||
| **Schematic of a typical MCP structure** //Courtesy of Stanford Computer Optics// \\ | **Schematic of a typical MCP structure** //Courtesy of Stanford Computer Optics// \\ | ||
| //Reprint with permission from [[http://www.iccd-camera.com/technology_main.htm|Stanford Computer Optics]].// \\ | //Reprint with permission from [[http://www.iccd-camera.com/technology_main.htm|Stanford Computer Optics]].// \\ | ||
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| Because of the build-in amplification, ICCD is capable of detect weak signals with much improve the signal-to-noise ratio. The accelerating electrical fields may also act as a fast shutter, gated ICCDs can be used to image fast events with a shuttle speed as fast as 50-200 ps. The spatial resolution of the ICCD is usually lower than regular CCD limited by the spatial resolution of the MCP. A more detailed description of ICCD technology may be found at [[http://www.iccd-camera.com/technology_main.htm|Stanford Computer Optics]]. \\ | Because of the build-in amplification, ICCD is capable of detect weak signals with much improve the signal-to-noise ratio. The accelerating electrical fields may also act as a fast shutter, gated ICCDs can be used to image fast events with a shuttle speed as fast as 50-200 ps. The spatial resolution of the ICCD is usually lower than regular CCD limited by the spatial resolution of the MCP. A more detailed description of ICCD technology may be found at [[http://www.iccd-camera.com/technology_main.htm|Stanford Computer Optics]]. \\ | ||
| ===== Streak Camera ===== | ===== Streak Camera ===== | ||
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| Currently, the fastest single point photo detector is photo diode with up to 40GHz bandwidth or ~9ps rise time. For MCP-PMT, the fastest commercially available device has bandwidth of about 2GHz or 180ps rise time. In the case of ICCD, the shortest gating window is 50ps. \\ | Currently, the fastest single point photo detector is photo diode with up to 40GHz bandwidth or ~9ps rise time. For MCP-PMT, the fastest commercially available device has bandwidth of about 2GHz or 180ps rise time. In the case of ICCD, the shortest gating window is 50ps. \\ | ||
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| Comparing to these photo detectors, streak camera is the fastest single-shot photo detector with picosecond or sub-picosecond resolution. Streak camera also uses the photon-electron-photon conversion scheme similar to that used in ICCD. The main components of the streak camera also include a photocathode at the input slit, an accelerating electrical field, a phosphor screen, a read out CCD camera with coupling optics. The basic operating principle of a streak camera is shown in figure xxx. When photons arrive at the photocathode, they are converted into photoelectrons and them accelerated by a constant horizontal electrical field along the direction of their travel across the vacuum streak tube. While the electrons travel across the tube, a time varying electrical field is applied vertically perpendicular to their direction of travel. This vertical field is called sweeping field, that changes rapidly in time. Therefore, photo electrons generated at different time at the photocathode will subject to different sweeping electrical field on the vertical direction. Consequently, they will be deflected at different angles while traveling across the steak tube and arrive at different vertical positions on the phosphor screen. Therefore, the steak image spread on the vertical direction can be viewed as a histogram of photons in the time domain. Typical streak camera can have temporal resolution of 2-5 ps. \\ | Comparing to these photo detectors, streak camera is the fastest single-shot photo detector with picosecond or sub-picosecond resolution. Streak camera also uses the photon-electron-photon conversion scheme similar to that used in ICCD. The main components of the streak camera also include a photocathode at the input slit, an accelerating electrical field, a phosphor screen, a read out CCD camera with coupling optics. The basic operating principle of a streak camera is shown in figure xxx. When photons arrive at the photocathode, they are converted into photoelectrons and them accelerated by a constant horizontal electrical field along the direction of their travel across the vacuum streak tube. While the electrons travel across the tube, a time varying electrical field is applied vertically perpendicular to their direction of travel. This vertical field is called sweeping field, that changes rapidly in time. Therefore, photo electrons generated at different time at the photocathode will subject to different sweeping electrical field on the vertical direction. Consequently, they will be deflected at different angles while traveling across the steak tube and arrive at different vertical positions on the phosphor screen. Therefore, the steak image spread on the vertical direction can be viewed as a histogram of photons in the time domain. Typical streak camera can have temporal resolution of 2-5 ps. \\ | ||
| - | + | One of the main advantages of streak camera is its multiplexing capability to acquire multiple channels entering the input slit. A typical application of streak camera is to couple it to a spectrograph such that the horizontal channel measures spectral and vertical channel measures time. In Figure xxx below, a spectrally stretched ultrafast laser pulse is shown on the streak camera. \\ | |
| - | One of the main advantages of streak camera is its multiplexing capability to acquire multiple channels entering the input slit. A typical application of streak camera is to couple it to a spectrograph such that the horizontal channel measures spectral and vertical channel measures time. In Figure xxx below, a spectrally stretched ultrafast laser pulse is shown on the streak camera. \\ | + | |
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| {{ :research:optical_instruments:streak_camera_basic_operation.jpg?350 }} \\ | {{ :research:optical_instruments:streak_camera_basic_operation.jpg?350 }} \\ | ||
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| **A spectrally stretched ultrafast laser pulse measured by a streak camera coupled to a spectrograph.** \\ \\ | **A spectrally stretched ultrafast laser pulse measured by a streak camera coupled to a spectrograph.** \\ \\ | ||
| Streak cameras are usually very expensive compared to other photo detection systems. \\ | Streak cameras are usually very expensive compared to other photo detection systems. \\ | ||
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| - | \\ | + | ====== How Do We Control / Measure the Spectrum of Light? ====== |
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| - | ====== How do we control/measure the spectrum of light? ====== | + | |
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| Generally, we can separate photons spectrally use, 1) band pass filters, 2) a prism, or 3) grating based spectrometers. \\ | Generally, we can separate photons spectrally use, 1) band pass filters, 2) a prism, or 3) grating based spectrometers. \\ | ||
| - | + | ===== Band Pass Filters ===== | |
| - | ===== Band pass filters: ===== | + | |
| - | + | {{ :research:optical_instruments:band_pass_filter.jpg?250 }} \\ | |
| - | Fig. 15 band pass filter \\ | + | **Band Pass Filter** \\ |
| Advantages: simple and cost effective if only a few wavelengths are needed. \\ | Advantages: simple and cost effective if only a few wavelengths are needed. \\ | ||
| Disadvantages: low resolution, little control over bandwidth, difficult to tune the wavelength. \\ | Disadvantages: low resolution, little control over bandwidth, difficult to tune the wavelength. \\ | ||
| - | + | ===== Prisms ===== | |
| - | ===== Prisms: ===== | + | |
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| A prism bends light beam with different wavelengths at different angles based on their refraction index. \\ | A prism bends light beam with different wavelengths at different angles based on their refraction index. \\ | ||
| - | + | {{ :research:optical_instruments:light_dispersion_by_prism.jpg?300 }}\\ | |
| - | Fig. 10: light dispersion by a Prism \\ | + | **Light Dispersion by a Prism** \\ |
| - | + | ===== Spectrometers and Spectrograph ===== | |
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| - | ===== Spectrometers and spectrograph ===== | + | |
| Spectrometers are the mostly used optical instruments in spectroscopy studies that use diffraction gratings to convert the spectral difference of the photons into spatial differences. A common form of spectrometer is the Czerny-Turner design as shown in the following figure. \\ | Spectrometers are the mostly used optical instruments in spectroscopy studies that use diffraction gratings to convert the spectral difference of the photons into spatial differences. A common form of spectrometer is the Czerny-Turner design as shown in the following figure. \\ | ||
| - | Fig. 25: Czerny-Turner Monochromator \\ | + | {{ :research:optical_instruments:czerny-turner_monochromator.png?400 }} \\ |
| + | **Czerny-Turner Monochromator** \\ | ||
| The input light is focused into the entrance slit, which is at the focus plane of the collimating mirror. The collimated light is then diffracted based on its wavelength on the surface of the rotating grating. The light is finally focused on the exit plane by the focusing mirror. At the exit plane, the input light is spreading out linearly to a spectrum. \\ | The input light is focused into the entrance slit, which is at the focus plane of the collimating mirror. The collimated light is then diffracted based on its wavelength on the surface of the rotating grating. The light is finally focused on the exit plane by the focusing mirror. At the exit plane, the input light is spreading out linearly to a spectrum. \\ | ||
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| Based on the output detection system, a spectrometer can be divided into two types, a monochromator or a spectrograph. \\ | Based on the output detection system, a spectrometer can be divided into two types, a monochromator or a spectrograph. \\ | ||
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| In a monochromator, a slit is placed at the focal plane that only allows a narrow portion of the spectrum passing through. The spectral information can be obtained by rotating the grating so the selected portion of the spectrum will pass through the slit. There are two principle applications of a monochromator. One is used as a filter for selected spectrum output as a spectral tunable illumination source. The other one is for analyzing the spectral information of the input light by placing a photosensitive detector behind the exit slit. The spectrum is acquired by sequentially rotating the grating step by step. \\ | In a monochromator, a slit is placed at the focal plane that only allows a narrow portion of the spectrum passing through. The spectral information can be obtained by rotating the grating so the selected portion of the spectrum will pass through the slit. There are two principle applications of a monochromator. One is used as a filter for selected spectrum output as a spectral tunable illumination source. The other one is for analyzing the spectral information of the input light by placing a photosensitive detector behind the exit slit. The spectrum is acquired by sequentially rotating the grating step by step. \\ | ||
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| In a spectrograph, a photosensitive detector array, such as photodiode array or CCD camera, is placed at the exit plane. Therefore, the spatial distribution of the light collected by the detector array corresponds to the intensity variation in wavelength. \\ | In a spectrograph, a photosensitive detector array, such as photodiode array or CCD camera, is placed at the exit plane. Therefore, the spatial distribution of the light collected by the detector array corresponds to the intensity variation in wavelength. \\ | ||
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| ===== Fluorescence Spectroscopy ===== | ===== Fluorescence Spectroscopy ===== | ||
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| Fluorescence spectroscopy instruments are used to excite specimens and detect the fluorescence emission through spectrometer. Fluorescence emission is characterized by its intensity, spectral distribution, and radiative lifetime. In general, fluorescence spectroscopy instrument can be divided into two categories: steady state and time-resolved, which will be described in the following respectively. \\ | Fluorescence spectroscopy instruments are used to excite specimens and detect the fluorescence emission through spectrometer. Fluorescence emission is characterized by its intensity, spectral distribution, and radiative lifetime. In general, fluorescence spectroscopy instrument can be divided into two categories: steady state and time-resolved, which will be described in the following respectively. \\ | ||
| Steady state spectroscopy is one of the most widely used fluorescence techniques. It provides spectroscopic information of the fluorescence emission in terms of its intensity variation across the spectrum. These characteristics include intensity, peak wavelength, and spectral shape. | Steady state spectroscopy is one of the most widely used fluorescence techniques. It provides spectroscopic information of the fluorescence emission in terms of its intensity variation across the spectrum. These characteristics include intensity, peak wavelength, and spectral shape. | ||
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| - | Fig. 26: Dual monochromator excitation-emission fluorescence spectrometer \\ | + | {{ :research:optical_instruments:dual_monochromator_excitation-emission_fluor_spec.png?400 }} \\ |
| + | **Dual monochromator excitation-emission fluorescence spectrometer** \\ | ||
| Typical steady fluorescence emission spectra of Rhodamin B, rose Bengal, 9-cyanoanthracene is shown here. \\ | Typical steady fluorescence emission spectra of Rhodamin B, rose Bengal, 9-cyanoanthracene is shown here. \\ | ||
| - | + | {{ :research:optical_instruments:fluor_emission_spectra_of_3.png?400 }} \\ | |
| - | + | **Fluorescence emission spectra of Rhodamin B, rose Bengal, 9-cyanoanthracene** \\ | |
| - | + | ===== Time-Resolved Fluorescence Spectroscopy ===== | |
| - | Fig. 27 fluorescence emission spectra of Rhodamin B, rose Bengal, 9-cyanoanthracene \\ | + | |
| - | ===== Time-resolved Fluorescence Spectroscopy ===== | + | |
| In addition to fluorescence spectra, time-resolved fluorescence spectroscopy also provides the lifetime of the fluorescence emission intensity decay at specific wavelength. Comparing with the steady state technique, time-resolved fluorescence spectroscopy has the following advantages: | In addition to fluorescence spectra, time-resolved fluorescence spectroscopy also provides the lifetime of the fluorescence emission intensity decay at specific wavelength. Comparing with the steady state technique, time-resolved fluorescence spectroscopy has the following advantages: | ||
| (1) Fluorescence lifetime provides additional information that introduces fundamentally new contrasts for specimen characterization, i.e. it may resolve fluorophores have overlapping fluorescence spectra but different lifetimes. \\ | (1) Fluorescence lifetime provides additional information that introduces fundamentally new contrasts for specimen characterization, i.e. it may resolve fluorophores have overlapping fluorescence spectra but different lifetimes. \\ | ||
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| - | Fig. 28: optical schematics of a pulse sampling time-resolved fluorescence spectroscopy system \\ | + | {{ :research:optical_instruments:optical_schematics_of_a_pulse_sampling_system.png?450 }} |
| - | + | **Optical schematics of a pulse sampling time-resolved fluorescence spectroscopy system** \\ | |
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| Time-resolved fluorescence apparatus usually require far more complex synchronization design. Here is the electronics system design of the above system: \\ | Time-resolved fluorescence apparatus usually require far more complex synchronization design. Here is the electronics system design of the above system: \\ | ||
| + | {{ :research:optical_instruments:electronics_schematics_of_pulse_sampling_system.png?500 }} \\ | ||
| + | **Electronics schematic of a pulse sampling time-resolved fluorescence spectrometer** \\ | ||
| + | Time-resolved fluorescence spectrum of Rhodamin B and 9-cyanoanthracene mixture solution is shown here. Rhodamin B has emission peak at 580nm and lifetime of 3.03ns while 9-cyanoanthracene has emission peak at 445nm and lifetime of 12ns. \\ | ||
| - | Fig. 29: electronics schematic of a pulse sampling time-resolved fluorescence spectrometer \\ | + | {{ :research:optical_instruments:rhodamin_b_and_9-cyanoathracene.png?850 }} \\ |
| + | **Time-resolved fluorescence spectral of the mixture solution of Rhodamin B and 9-cyanoanthracene** \\ | ||
| + | \\ \\ | ||
| - | Time-resolved fluorescence spectrum of Rhodamin B and 9-cyanoanthracene mixture solution is shown here. Rhodamin B has emission peak at 580nm and lifetime of 3.03ns while 9-cyanoanthracene has emission peak at 445nm and lifetime of 12ns. \\ | + | ====== Why Are Lasers Dangerous? ====== |
| + | The light sources and detectors used in optical instruments are potential sources of hazards to human if not properly operated. These hazards primarily come from the optical radiation, high voltage electrical devices, and chemicals. | ||
| + | \\ | ||
| - | + | ===== Laser and Light Radiation Safety ===== | |
| - | + | Optical radiation hazards from the Sun light and high intensity lamps to human have long been discovered since their use in scientific research. They had not been systematically studied, however, before the invention of laser in the 1960s. Comparing with other light sources, the collimated beam of laser is capable of concentrating light energy in a tiny volume and short period of time. In another word, low energy lasers light can have very high irradiance, which is defined as the amount of energy per unit volume and time. \\ \\ | |
| - | + | Lasers are classified into four categories according to their potential optical radiation hazards | |
| - | Fig. 30: time-resolved fluorescence spectral of the mixture solution of Rhodamin B and 9-cyanoanthracene \\ | + | * Class I: not considered as hazards, such as laser scanners used at super market |
| - | \\ | + | * Class II: low power and low risk. Will cause damage only if the person overcomes his/her nature aversion response to continuously stares at the source. The risk is considered very low and is the same as vision damage caused by staring at a high intensity lamp or the Sun. |
| + | * Class III: medium power and moderate risk. Most of laser systems used in photobiology | ||
| + | * Class IIIa: may only lead to visual damage by direct exposure of the laser beam | ||
| + | * Class IIIb: potential damage by both direct exposure and diffused light | ||
| + | * Class IV: high power and high risk. Lasers used for machining and surgery | ||
| + | \\ \\ | ||
| + | Most of the laser systems used in photobiology research belong to Class III that are not capable of causing serious skin injury or vision injury by diffuse reflections under normal use. General precautions while operating these lasers include | ||
| + | * Never direct looking at the laser beam at all wavelengths. | ||
| + | * Track the laser beam propagation. Confine the light within the setup by blocking multiple reflections and exit beam using a beam dumper. Pay extra attention to those working in the invisible spectral range such as ultra violet and inferred. A piece of fluorescence paper is usually used to track UV laser beam propagations and special IR sensitive cards are used to track laser beam in the inferred region. | ||
| + | * Wear proper laser protection goggles while working with them. There are no universal gaggles that will protect you from all lasers. They only working at a specified spectral range. | ||
| + | * Avoid direct exposure of UV light to skin. Studies have shown that excess UV light may cause skin cancers. | ||
| \\ | \\ | ||
| + | ===== Electrical Safety ===== | ||
| + | Except for LED and semiconductor diode lasers, most of the laser systems have high voltage power supplies. Some gas discharge lamps and detector systems such as PMT and intensifiers for CCD or streak cameras also have electrical system with line voltage exceeding tens of thousands of volts. In addition, high energy pulsed laser systems have capacitors charged to several kilovolts with associated energy of hundreds of joules. These high voltages and energies constitute potentially lethal shock hazards. Although most of the complete commercial systems come in enclosed cases that provide some form of protection, most of bench top systems used in research laboratories require periodically maintenance that access to open circuit is needed. Working with the large capacitor banks and very high voltage devices in light sources and detectors requires special attention. Carelessly working around these high voltage electrical devices has the potential of causing severe electrical shock or even possibly result in electrocution. Strict adherence to electrical guidelines and instrument manuals is the key to prevent accidents. \\ | ||
| + | Some simple guidelines are | ||
| + | * Carefully read the operating and maintenance manual supplied by the manufacture. Be familiar with which components have the potential to cause electrical hazards. \\ | ||
| + | * Use only one hand for any manipulation of circuits whenever possible. \\ | ||
| + | * Work in a dry environment. \\ | ||
| + | * Before any work, ground the whole circuit and use a portable multi-meter check the voltage. | ||
| + | \\ | ||
| - | + | ===== Chemical Safety ===== | |
| - | ====== Why are lasers dangerous? ====== | + | Many gas lasers and dye laser systems also contain highly toxic chemicals, which are also fire hazards in many cases. Proper face masks and skin protection should be worn during maintenance procedures. In some particular types of laser systems, respiratory devices should also have been used. \\ \\ |
| - | + | ||
| - | + | ||
| - | The light sources and detectors used in optical instruments are potential sources of hazards to human if not properly operated. These hazards primarily come from the optical radiation, high voltage electrical devices, and chemicals. \\ | + | |
| - | + | ||
| - | ===== Laser and light radiation safety ===== | + | |
| - | + | ||
| - | Optical radiation hazards from the Sun light and high intensity lamps to human have long been discovered since their use in scientific research. They had not been systematically studied, however, before the invention of laser in the 1960s. Comparing with other light sources, the collimated beam of laser is capable of concentrating light energy in a tiny volume and short period of time. In another word, low energy lasers light can have very high irradiance, which is defined as the amount of energy per unit volume and time. \\ | + | |
| - | + | ||
| - | Lasers are classified into four categories according to their potential optical radiation hazards: \\ | + | |
| - | Class I: not considered as hazards such as laser scanners used at super market \\ | + | |
| - | Class II: low power and low risk. Will cause damage only if the person overcomes his/her nature aversion response to continuously stares at the source. The risk is considered very low and is the same as vision damage caused by staring at a high intensity lamp or the Sun. \\ | + | |
| - | Class III: medium power and moderate risk. Most of laser systems used in photobiology \\ | + | |
| - | Class IIIa: may only lead to visual damage by direct exposure of the laser beam; \\ | + | |
| - | Class IIIb: potential damage by both direct exposure and diffused light; \\ | + | |
| - | Class IV: high power and high risk. Lasers used for machining and surgery. \\ | + | |
| - | + | ||
| - | Most of the laser systems used in photobiology research belong to Class III that are not capable of causing serious skin injury or vision injury by diffuse reflections under normal use. General precautions while operating these lasers include: \\ | + | |
| - | 1. Never direct looking at the laser beam at all wavelengths. \\ | + | |
| - | 2. Track the laser beam propagation. Confine the light within the setup by blocking multiple reflections and exit beam using a beam dumper. Pay extra attention to those working in the invisible spectral range such as ultra violet and inferred. A piece of fluorescence paper is usually used to track UV laser beam propagations and special IR sensitive cards are used to track laser beam in the inferred region. \\ | + | |
| - | 3. Wear proper laser protection gaggles while working with them. There are no universal gaggles that will protect you from all lasers. They only working at a specified spectral range. \\ | + | |
| - | 4. Avoid direct exposure of UV light to skin. Studies have shown that excess UV light may cause skin cancers. \\ | + | |
| - | + | ||
| - | ===== Electrical safety ===== | + | |
| - | + | ||
| - | Except for LED and semiconductor diode lasers, most of the laser systems have high voltage power supplies. Some gas discharge lamps and detector systems such as PMT and intensifiers for CCD or streak cameras also have electrical system with line voltage exceeding tens of thousands of volts. In addition, high energy pulsed laser systems have capacitors charged to several kilovolts with associated energy of hundreds of joules. These high voltages and energies constitute potentially lethal shock hazards. Although most of the complete commercial systems come in enclosed cases that provide some form of protection, most of bench top systems used in research laboratories require periodically maintenance that access to open circuit is needed. Working with the large capacitor banks and very high voltage devices in light sources and detectors requires special attention. Carelessly working around these high voltage electrical devices has the potential of causing severe electrical shock or even possibly result in electrocution. Strict adherence to electrical guidelines and instrument manuals is the key to prevent accidents. Some simple guidelines are listed here: \\ | + | |
| - | 1. Carefully read the operating and maintenance manual supplied by the manufacture. Be familiar with which components have the potential to cause electrical hazards. \\ | + | |
| - | 2. Use only one hand for any manipulation of circuits whenever possible. \\ | + | |
| - | 3. Work in a dry environment. \\ | + | |
| - | 4. Before any work, ground the whole circuit and use a portable multi-meter check the voltage. \\ | + | |
| - | + | ||
| - | + | ||
| - | ===== Chemical safety ===== | + | |
| - | + | ||
| - | Many gas lasers and dye laser systems also contain highly toxic chemicals, which are also fire hazards in many cases. Proper face masks and skin protection should be worn during maintenance procedures. In some particular types of laser systems, respiratory devices should also been used. | + | |
| - | + | ||
| ===== Additional Readings ===== | ===== Additional Readings ===== | ||
| - | |||
| - | |||
| * David Sliney and Myron Wolbarsht, “Safty with Lasers and Other Optical Sources, A Comprehensive Handbook”, Plenum Press, New York/London, 1980. \\ | * David Sliney and Myron Wolbarsht, “Safty with Lasers and Other Optical Sources, A Comprehensive Handbook”, Plenum Press, New York/London, 1980. \\ | ||
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