Team:UANL Mty-Mexico/Contributions/Light Machine

This page explains extensively how to build a "Light Machine".

Overview of the "Light Machine"

We built a special machine that allows us to properly excite both photoreceptors (CcaS/CcaR and Cph8) independently/simultaneously using specific wavelengths of 535 and 650 nm, respectively. This "Light Machine" was based mainly on previous publications that work with light [1][2][3].

Light Machine overview.

Materials

• Two 100 W halogen lamp (Philips)
• Green and Red bandpass interference filters
*We got ours from Edmunds Optics.
Description: Interference Bandpass Filter 535nm ±2, 25mm Catalog Number: NT65-700.
And Interference Bandpass Filter 650nm ±2, 25mm Catalog Number: NT65-715
• 37°C incubator with thermometer hole on top
• Adjustable shelves
• Two Support Stands/Clamps
• Two Emitter-Receptor Pieces
• T-45° Block
• Aluminum tubes (33 mm X 180 mm & 33 mm X 90 mm) mirror like finish inside
• Two mirrors 50 mm X 50 mm
• Two porcelain sockets T4 base
• 2 Small Fans

Picture from the inside of LightMachine

Emitter. 100% aluminium

Emitter with porcelain socket and halogen bulb.

T 45 block. In wood and aluminum

Receptor. 100% Aluminium

Distance overview of "Light Machine"

Light Machine Diagram.

Instructions

• First of all, collect or make all the materials shown above
• Wallpaper completely the inside of the incubator with black paper.
• Clear the thermometer hole in the top of incubator such that light can pass through.
• Place the T-45° Block at the top of the incubator on the thermometer hole and fix it with a plastic ring.
• Place two mirrors on T-45°Block.
• Immobilize the bandpass filter inside a plastic 1' coupling with small plastic ring.
• Join the desired coupling to the small aluminum pipe of T-45° Block.
• Immobilize the emitter-receptor pieces at both sides of incubator, we use adjustable shelves.
• Join the aluminum tube to the Emitter-Receptor.
• Leave proper distance (two finger rule) between coupling and aluminum pipe.
• Immobilize aluminum tubes with support stands.
• Place the aluminum tube in Emitter-Receptor piece and place the pipes parallel to the T-45° Block.
• Turn on the lamps; adjust the positioning of mirrors such that a clear and homogeneous light pattern appears in the center of your incubator.
Notes:
• Have fans near Aluminum Pieces, they become very hot over time, prevents halogen lamp and filter fails.
• Our incubator has a 1 3/4' hole.
• When placing T-45° Block ensure to cover all light penetrable areas.
• Buy more than 2 halogen lamps; they break easily.
• Be careful when handling the halogen lamps, they heat in a very short time.
• We bought 4 BP filters 532, 575, 650, 710 nm respectively.
• Make experiments in dark room.
• Clean mirrors when possible.
• Measure light intensity before starting test.

Before starting making measurements it is necessary to understand the following concepts:

Radiometry: is the measurement of optical radiation from a physical point of view, includes the regions commonly called the ultraviolet, the visible and the infrared. Two out of many typical units encountered are Watt and Joule.

Photometry: is the measurement of visible light, which is detectable by the human eye. These measurements tend to be subjective. Typical photometric units include lumens, lux and candelas.

Photometry is almost the same as radiometry, except that radiometry includes the entire optical radiation spectrum, while photometry is limited to the visible spectrum as defined by the response of the eye[4][6].

Irradiance (a.k.a. flux density) is a SI derived unit and is measured in W/m2. Irradiance is power per unit area incident from all directions in a hemisphere onto a surface that coincides with the base of that hemisphere.

Illuminance (a.k.a. luminous flux density) is another SI unit and is measured in lux. Illuminance is the total luminous flux incident on a surface per unit area. (Is the photometric equivalent of irradiance)[5].

**In a few words you need to measure in photometric units (lux) and convert them to radiometric units (W/m²) or measure directly in W/m²

There are two typical kinds of devices to measure light intensities once having assembled the "Light Machine":

Photometer - is an instrument for measuring Illuminance (Photometric units), then this value will be converted into Irradiance (W/m2).

**Cheaper measurement device and works properly.

Photometer.

Spectrometer – is an instrument for measuring Irradiance.

**Is the device that has everything but it costs much more

Spectrometer.

Determining light Intensities

The intensity of light was measured in lux units, lumens per square meter using an Easy View Light Meter (Model EA31) calibrated photometer, which later were converted to power units of Watts per square meter.

The light beam was divided into zones, which underwent an average of intensities in the X, Y-axis to determine the intensity of that area. Intensity averages were calculated before each test. Samples were placed in the zone that better fits the desired intensity.

The Bandpass interference filters have a 10 nm transmission window with a peak at the emission wavelength. Precise light wavelengths and intensities are very important to achieve the desired response of photoreceptors.

How to convert to from lux to W/m2

Radiometric and photometric units can be converted into each other[6]. Several factors, such as wavelength and mono/multicromatic light source must be take into account.

The conversion between photometric and radiometric units, for a monochromatic light source, is given by the following equation:

K(λ) = K_m*V(λ)

Where:

K(λ) - Radiant flux (lm/W)

V(λ) - Photo tropic spectra luminous efficiency function. Corresponds to the sensitivity of the human eye and its function of the wavelength of light (Fig 2 Appendix B)[6]

K_m – Scaling Factor: 683 lm/W

**Remember that this formula is effective only for monochromatic light sources (Multicromatic light sources are a bit more complicated).

**Observe measurement device modality (Photopic or Scotopic)**

 

Example of conversion from photometric to radiometric units for a 532 nm wavelength:

K(λ) = K_m* V(λ)

K(532 nm) = 683 lm/W *V(532nm)

K(532 nm) = 683 lm/W * 0.862

K(532 nm) = 588.746 lm/W

 

1 W = 588.76 lm @ 532 nm

1 W/m2 = 588.79 lm/m² ; 1 lux = 1 lm/m²

1 lux = 1/588.79 W/m² = 1.69 mW/m² @ 532 nm

Now calculate your respective intensities for your experiment.

Correlation W/m2 to Lux @710nm

Intensity/Wavelength

The light intensity on the sample depends mainly on 3 factors:

• Distance - Is inversely proportional to the light output
• Lamp light output - Depending on the type of lamp, it will produce different wavelength intensities.
• Specific Wavelength.

Consider the light loss during the pathway.

It is very important to have the correct intensity/wavelenght (W/m² for @ nm) for each photoreceptor, otherwise it may produce unwanted results.

1) Two-color optical control of gene expression in E. coli. Light intensity transfer functions of strains carrying each sensor alone or both sensors. Strains expressing the green sensor only CcaS/CcaR (green circles), red sensor only Cph8 (red squares), or both (gray circles) were exposed to varying intensities of 532 nm or 650 nm light. 2) Spectral transfer functions. E. coli carrying the green or red sensor was exposed to saturating levels of a given light wavelength, and Miller assays were conducted. Figures taken from Tabor JJ et al. (2010) J. Mol. Biol. 405: 315-324.

• Tabor JJ, Levskaya A, Voigt CA. (2010). Multichromatic Control of Gene Expression in Escherichia coli. J. Mol. Biol. 405: 315-324.
• Levskaya A, et al. (2005). Synthetic 674 biology: engineering Escherichia coli to see light. Nature. 438: 441–442.
• http://openwetware.org/wiki/LightCannon.
• www.optics.arizona.edu/Palmer/rpfaq/rpfaq.htm.
• www.helios32.com/Measuring%20Light.pdf
• Photonfocus AG, Application Note AN008. 12/2004 V1.1 "Photometry versus Radiometry". http://www.photonfocus.com/upload/application_notes/AN008_e_V1_1_PhotometryVersusRadiometry.pdf