In these lab exercises, you will be introduced to a variety of techniques that are very useful in photosynthesis and other research.
The exercises are designed to allow you to improve your ' Experimental Hands ' as you progress from simpler to more complex protocols. Origins and Fate of Light : Students are introduced to the nature of light and its interactions with photosynthetic structures. Lab 01 Spectral Properties of Intact Leaves : Students will measure the reflectance and transmittance spectra of intact photosynthetic leaves.
Lab 02 Visualizing the Photosynthetic Apparatus : Students will examine the chloroplast in situ and in vitro , and be introduced to the technique of chloroplast isolation from photosynthetic tissues. Lab 03 Absorbing Light : Students will collect absorption spectra from chloroplasts in vitro , and from pigments isolated from the chloroplasts.
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Fluorescence and Reaction Centers : Students are introduced to fluorescence spectroscopy techniques. Lab 05 Physiology of Fluorescence : Students measure how modifying the physiology of photosynthesis affects fluorescence emission, a metric of the photochemical mechanisms of reaction centers. Oxygen Electrode: Pathways of Photosynthetic Electron Transport : Students are introduced to the use of the oxygen electrode to assess electron transport chain mechanisms in photosynthesis. Chloroplast Molecular Biology : Students are introduced to chloroplast genome s.
Lab 09 Chloroplast Genome Mapping : Students map the chloroplast genome using restriction endonucleases. Lab 10 Presentations: Pathways of Carbon Dioxide Fixation : Students give a presentation on an enzyme from one of the carbon dioxide fixation pathways. Black Body and Solar Radiation In a general way, there are two types of light sources: 1 Incandescence --The emission of light from hot matter; and 2 Luminescence --The emission of light when 'excited' electrons fall to a lower energy, emitting a photon.
For incandescence, the wavelengths of light that are emitted depend on the temperature. The hotter the material, the shorter the wavelengths of emitted light.
For luminescence, temperature may or may not play a role. The light-emitting diode bulbs used in some of these lab exercises are an example of a luminescent light source, and are cool to touch. The overhead fluorescent lights are another example that can be hot to touch! With the spectrometer, you can actually view the wavelengths of emitted light. For an incandescent bulb, the spectrum is continuous.
For the LED and fluorescent lights, only some wavelengths of light are emitted. In the context of 'natural' photosynthesis, photosynthetic organisms evolved using the sun as the light source. The sun is an example of an incandescent light source, a very hot one Figure 1.
Lectures provide you with the physics-based concepts pertinent to light sources. For the purpose of the lab exercises, you will be introduced to measurement techniques that characterize the capture and utilization of light by the photosynthetic organ. When we consider the capture of light and its conversion to usable energy, the initial steps that we need to explore go beyond the nature of light itself, to how it interacts with the photosynthetic system a leaf for example.
There are three ways that light can interact with a photosynthetic organ such as a leaf Figure 2. The first of these is reflection, simply rebounding off of the leaf surface and therefore never utilized in leaf photosynthesis. Reflectance R comes in various forms.
Photosynthesis Lab Manual (BIOL 4160)
The simplest is regular reflection r r , where the incident light is reflected off of the leaf at the same angle that is, the incident angle is equal to the angle of reflection.
This will occur with very smooth surfaces, but is less likely to happen if the leaf surface is rough as it usually is, at least at a microscopic level. In the latter case, the reflectance is diffuse r d. Figure 3. Pathways of Internal Reflectance Missing from this description is one additional complication: internal reflection. That is, light that enters the leaf, but eventually rebounds back out of the leaf Figure 3.
Absorbance and Transmittance Scales If the light is not reflected, then there are two other outcomes. Transmittance and absorbance are directly related to each other, since both depend upon the ratio of incident light I o to transmitted light I Figure 4. All three outcomes can be described by an overly simple equation:. The equation as written indicates that all the light impinging on the leaf is conserved. Missing is yet another complication! Fluorescence does occur, which is the release of photon energy in the form of longer wavelength light after light absorption.
This is so important in photosynthesis research that we will explore it in a separate lab exercise. In your lab exercises, you will have the opportunity to measure reflectance, transmittance and absorbance, and fluorescence: All four as a function of the wavelength of light. Is this important?
Very much so. For one thing, it reveals the relation between the photosynthetic organ and how it interacts with light, the basic events leading to photosynthesis. For another, it provides the fundamental knowledge necessary for remote sensing of biotic health, by satellites for example. This is how crop productivity is documented annually.
A basic understanding of the fates of light has diverse and economically important relevance. Reflectance Spectra. You may wish to refer to this figure during the lab to relate wavelength and color. Examples of reflectance spectra are shown in Figure 1: Both a leaf upper panel and a flower lower panel.
Your spectra should look similar. Note that the reflectance is shown as a percent for calibrated measurements.
For measurements of reflectance spectra, you will be using a probe that operates as shown in Figure 2. Light is provided from a tungsten-halogen light source an incandescent light source which provides broad spectral 'white light' through a ring of 6 optical fibers.
The reflected light passes through a light reading optical fiber to a diffraction grating that separates the 'colors' wavelengths of the reflected light, thereby creating the wavelength spectrum of reflected light Figure 2. Reflectance Measurement. It is crucial to calibrate the reflectance probe prior to use! This is done using a 'white' reflector called a reflectance standard that reflects all wavelengths of light to adjust 'maximum output', and 'no light' to adjust the 'dark' baseline.
The leaves that you choose to measure are your decision. Make sure you indicate the species, and any special characteristics. For example, is it a grass leaf? A succulent? Is it colored with pigments other than chlorophyll? Some leaves may be 'hairy', and you should take note of this as well, as it may affect the spectral properties of the leaf. Make measurements of reflectance from both the upper surface and the lower surface of the leaves you select.
Are they different? You may notice strong reflection in the infrared region of the spectra. Feel free to compare it with your own reflectance spectrum from your skin! This may provide insight into the very different radiant energy balances of a human and a leaf.
The Origins of Light
You can even test your clothing if you want: to get a better sense of the diversity of reflectance spectra. Once you have obtained reflectance spectra, you will need to measure the absorbance and transmittance spectra. For these, you need to provide a light source on one side of the leaf while you measure the absorbance through the leaf. It is vitally important that you ensure the distance from the light source to the probe is kept constant, while you compare the spectra with the leaf intercepting the light, versus no interception.
Again, you must calibrate the probe before performing your measurements: adjusting the maximum output for the lamp source, and the dark baseline. Also, you need to measure the thickness of the leaf.
The Fates of Light
Absorption depends upon the depth of the material. Doubling the thickness causes a doubling of the absorbance.
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This is very important when you are comparing two different kinds of leaves. A number of different light sources will be provided for you.
Some are specific colors LED lamps , or 'white' incandescent light sources. Choose the white source and two others for your transmittance measurements for the leaves you selected for reflectance measurements.
Please be careful that you do not 'contaminate' your spectra with ambient lighting. Fluorescent lamps emit light at very specific wavelengths. You can minimize contamination using cardboard or light-proof cloth. For leaf absorbance measurements, your light source intensity should not be too high, since it will be difficult to measure absorbance in the very thin leaf, because the ratio of I to I o will be near 1 if the lamp is very bright.
If you can't distinguish the spectra with and without the leaf, put more leaves in the light path. Do measurements of both absorbance and transmittance calibrating the probe each time.
Figure 4 provides a general schematic of the technique. The observations you make should be similar for all lab groups. What will vary is your decision about the photosynthetic leaf you use, and the light sources for absorbance and transmittance.
You have the opportunity to propose your own hypotheses. For example, is infrared reflectance important? Does it differ between a succulent leaf, adapted to desert conditions, and a shade leaf adapted to low light conditions?
What about the position of the leaf in the plant, shaded versus un-shaded? What would you hypothesize?
Does leaf pigmentation besides chlorophyll have an impact?