Typical optical instruments used in photo physics include: spectrometers, microscopes, power/energy meters, and imaging systems. These instruments are made up from modules such as light sources, mirrors, lens, filters, fibers, photo detectors, cameras.

In Photo physics, optical instruments are used to:
1. Generate light with specific characteristics (light sources)
2. Deliver light to the targets (mirrors, lens, filters, fibers)
3. Collect light back from the target (emitted, reflected, scattered, or transmitted)
4. Analyzed the collected light signal via a detection system (spectroscopy, imaging, etc).

Light is electromagnetic radiation that can be described as particles and waves. Experimentally, the characteristics of light include energy, spectrum, spatial distribution, and temporal distribution.

Intensity is used to describe the brightness of light. Quantitatively, intensity may be measured in terms of power (energy per unit time or J∙s-1) or fluence (J∙s-1∙m-2). Intensity may be measured by an optical energy or power meter (link to laser power/energy meter).

When light propagates in space, we sometimes not only need to know its overall power (as measured by a power meter), but also it’s the the spatial distribution of its energy, i.e. the light distributed in an area and/or some times in a volume. In practice, two-dimensional distribution is usually measured using a beam profiler.
The following figure is an example of the profile of a typical laser beam intensity spatial distribution. The intensity at each pixel is color coded (shown in color bar on the top right corner) from blue to red, while purple is designated as zero and white is for saturation of the photo detector.

Typical 2-D spatial distribution of a non-Gaussian laser beam measured by a beam profiling system. The intensity is shown in color with the mapping on the top right corner. The white curves are horizontal and vertical intensity profile at the two black dashed lines.

One of the most important characters of light is its spectral distribution, or spectrum. As you might already know, light has dual characters: wave and particle. From the wave point of view, the spectrum is the energy distribution of light as a function of its wavelength. From the particle point of view, spectrum is the energy distribution histogram of photons. The spectrum may be measured by a monochromator or spectrograph. In may photo physics experiments, spectral selection may be achieved using glass filters, prisms, and other devices.

Emission spectra of several light emitting diodes

Another important character of light is its distribution over time. The temporal profile of light is usually called pulse. Temporal characters of light are pulse duration, pulse shape, and repetition rate. Instrument used to measure temporal profile of light pulses include oscilloscope (when used with a photo detector such as photo diode or photomultiplier tube), streak cameras, and intensified cameras.

A pulse train of light detected by a photo diode and a digital oscilloscope

When light is described using electromagnetic waves, the phase relation between different wavelets are called coherence. When two wavelets have the same frequency and a fixed phase relation, they are called coherent. Coherent light can interfere with each other.

Light are transverse electromagnetic waves, the orientation of its oscillation plane is described by polarization. The simplest polarization state is called linear polarization where the EM wave oscillating plane does not change. Many light sources including sun light are random polarized, which means there is no fixed correlation between the polarization states of the wavelet originated from the light sources. Polarization may be detected by ellipsometers and manipulated by linear polarizers and wave plates.

How light propagate in space can be described by its divergence. Lasers are usually collimated with very little divergence. LEDs usually are not collimated with a relatively small divergence and light emitted by lamps are usually omni-directional.

Typical optical instruments used photo physics include light sources, light delivery and collections devices (lens, mirror, fiber, apertures, windows, etc), photo detectors (photo diodes, photo multiplier tubes, cameras etc.), and analysis devices (spectrometers, filters, imagers, etc.)
Light are usually generated by light sources. The typical light sources used in optical instruments include various types of lasers, lamps, and light emitting diodes (LED). 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.
One of the most robust, simple optical systems is the Sun light illumination with observation of human eyes.

A simple optical system consists a light source (Sun) and an imager (the Eye).

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.

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 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.
Tungsten- Halogen lamp is a typical type of incandescent lamp that the filament is made of Tungsten and Halogen vapor is used to regenerate the filament. Tungsten-Halogen lamps emit from ultra violet to near infrared. Quartz (fused silicon) windows are usually used for sources used for ultra violet illumination. The continuous broadband spectrum of Tungsten-Halogen emission spectrum may be used as calibration source for absolute spectral response.
Emission spectrum of halogen lamp

Another important family of lamps is gas discharge lamp in which gas atoms are ionized and lead to electronics transitions. Compare to incandescent lamps, gas discharge lamps have more significant narrow band atomic emission lines that may be used as wavelength calibration sources.

Emission spectrum of room light showing mercury lines
Most of incandescence lamps are continuous wave (CW) sources. Gas discharge lamps can be made into flash lamps such as xenon flash lamps that generate pulsed operation usually in the microseconds to milliseconds regime.
For broad band sources such as lamps, selection of illumination spectrum is achieved using monochromator or band pass filters.

Light Emission Diode (LED) is a semiconductor PN junction that can emit non-coherent light when biased in forwarded direction with narrower bandwidth than lamps. Comparing to lamps, LED has the following advantages:

• More compact and with low electrical power consumption
  
• Simple electronics that have much flexible control over the light emission
  
• Economical and reliable
  
• Generate CW to nanosecond pulse outputs
  
• Narrower bandwidth comparing with lamps (10-200 nm)
  
• Small angle of divergence for more directional illumination
  
• Can be made into LED arrays
  

However, LEDs are still incoherent sources with relatively low optical power output.

LED emission spectra

Laser is an acronym for Light Amplification of Stimulated Emission of Radiation. It is a device that produces highly collimated, monochromatic, high intensity light, which is usually also coherent. Although lasers are highly inefficient light sources in terms of converting electrical power into optical power, they are the sources with the most control, which includes directional, spectral, phase relation, polarization, and temporal. He-Ne Laser emission lines
Typical lasers are monochromatic sources with very narrow emission lines. Typically, the laser lines are characteristics of the laser medium.
Laser spectra: Animation Bar (see web page)

Dye laser are continuously tunable from UV to the visible and near infrared (300nm-900nm) within the tuning range (~20nm) of a particular dye. Their operation requires a pumping laser usually in the UV, such as nitrogen lasers and harmonic generation of solid state lasers. Dye lasers usually operate with relatively low power and require cumbersome maintenance.
Another type of tunable laser is ultrafast lasers such as Ti: Sapphire lasers.

Recently, semiconductor lasers and fiber lasers have been developed as the future generation of lasers. They are compact, low cost and can be modulated with high repetition rate. In most cases, they are simple to operate and so far only have limited wavelength range, not collimated.
Emission spectrum of a 405nm diode laser

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.

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.

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.

• Based on refraction, they are usually used to focus or collimate light beams
  

	Double Convex Lens
	Double Concave Lens

• based on reflection, they are usually used to steering light propagation.

• based on light with different wavelength have different behaviors while passing through glass, mostly being used to separate wavelengths
  

Illustration of light dispersion by a prism

• mostly being used to separate wavelengths based on diffraction.
  

Diffraction Grating

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 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.

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).

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: Microscopy Primer.

CCD pixel read out scheme

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.

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).
CMOS sensor layout

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. Intensified CCD camera (courtesy of Stanford Computer Optics) A typical gated intensified CCD camera system
Reprint with permission from Stanford Computer Optics
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.

Schematic of a typical MCP structure Courtesy of Stanford Computer Optics
Reprint with permission from 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 Stanford Computer Optics.

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.
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.

Basic operating principle of a streak camera. Reprint with permission from Optronis.


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.

Generally, we can separate photons spectrally use, 1) band pass filters, 2) a prism, or 3) grating based spectrometers.

Band Pass Filter

Advantages: simple and cost effective if only a few wavelengths are needed.
Disadvantages: low resolution, little control over bandwidth, difficult to tune the wavelength.

A prism bends light beam with different wavelengths at different angles based on their refraction index.

Light Dispersion by a Prism

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.

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.
Based on the output detection system, a spectrometer can be divided into two types, a monochromator or a spectrograph.
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 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.

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. A simple steady state fluorescence spectroscopy apparatus is shown in the following figure. A Xeon lamp is used as the light source. An excitation monochromator is used to select the exciting wavelength. The excitation light is focused at the sample and the fluorescence light emitting from the sample is collected by a cylindrical lens and focused at the input slit of monochromator. A PMT is placed at the exit slit of the monochromator for detection. The analog output of the PMT is measured by an A/D convert. The steady state spectrum is acquired via rotating the grating in the emission monochromator across the desired spectral band.

Dual monochromator excitation-emission fluorescence spectrometer
Typical steady fluorescence emission spectra of Rhodamin B, rose Bengal, 9-cyanoanthracene is shown here.

Fluorescence emission spectra of Rhodamin B, rose Bengal, 9-cyanoanthracene

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.
(2) Fluorescence decay is independent of fluorescence emission intensity given sufficient signal-to-noise ratio (SNR). Therefore, the lifetime of the decay is independent of the presence of endogenous chromophores in tissue (such as hemoglobin) or excitation-collection geometry. (3) Fluorescence lifetime is sensitive to microenvironmental parameters in tissue such as pH, enzymatic activity, and temperature. Therefore, time-resolved measurements may reflect the variation of these parameters.
A typical fluorescence lifetime spectroscopy system is shown here. Optical fibers are used to deliver excitation light and collect fluorescence emission. A dual output spectrograph can simultaneously acquire steady state fluorescence spectrum through the CCD camera and time-resolved fluorescence emission through the PMT.

Optical schematics of a pulse sampling time-resolved fluorescence spectroscopy system
Time-resolved fluorescence apparatus usually require far more complex synchronization design. Here is the electronics system design of the above system:

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.

Time-resolved fluorescence spectral of the mixture solution of Rhodamin B and 9-cyanoanthracene

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.

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

• 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.

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.

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.

• David Sliney and Myron Wolbarsht, “Safty with Lasers and Other Optical Sources, A Comprehensive Handbook”, Plenum Press, New York/London, 1980.
  

• J. R. Lakowicz, “Principles of Fluorescence Spectroscopy,” Plenum Press, New York/London, 1999.
  

• M. Bass, E. W. Stryland, D. R. Williams, W. L. Wolfe, “Handbook of Optics,” McGraw Hill, New York, 1995.
  

• W. Demtroder, “Laser Spectroscopy: Basic Concepts and Instrumentation,” Springer, Berlin, 2003.