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Lab 04 Determining Excitation and Emission Spectra

Photosynthesis 13 Fluorescence.png

General Objective

To measure and compare the absorbance and emission spectra of isolated chloroplasts with the absorbance and emission spectra of the chlorophyll pigments.

Chloroplast Isolation

Solutions

STK (pre-chilled on ice):

  • Sucrose (FW 342.3) 0.6 M
  • Tricine (FW 179.2) 0.05 M (Tricine buffer has a pK of 8.15. The pH of the media is adjusted to 7.5 with KOH/HCl, as required.)
  • KCl (FW 74.55) 0.02 M (mimics the ionic environment of the cytoplasm)

TK (prechilled on ice):

  • As above, without sucrose (used to dilute your stock chloroplast suspensions)

Ac/W:

  • 80% acetone, 20% H2O (to isolate chlorophyll pigments)

Milk (dilute):

  • a small aliquot of whole milk in H2O (1/100) (the fat globules are effective at scattering light)

Other materials: Spinach, Mortar and pestle (pre-chilled on ice), Algal cultures, Glass homogenizer (pre-chilled on ice), Cheesecloth (to filter the homogenates), Centrifuge and centrifuge tubes (to pellet the chloroplasts), and Miscellany (beakers, cuvettes, No. 1 filter paper etc.)

Isolation Procedure

Rinse spinach in tap water, remove midribs with scissors (or razor, but scissors are easier and faster) and cut the remaining tissue into pieces of about 2 cm width and length.

About 10 grams of spinach should be placed in a pre-chilled mortar and pestle with 50-100 ml of pre-chilled STK. Grind in a mortar and pestle, ensuring that all leaves are compacted down and effectively homogenized. The homogenate is poured through three layers of cheesecloth into a pre-chilled beaker in ice. The cheesecloth can be squeezed gently to release more chloroplasts. Centrifuge for 1–2 min at 500 X g to spin down debris. The supernatant should be centrifuged for 10 min at 10,000 X g to spin down the chloroplast into a pellet. Decant the supernatant and gently disperse the pellet into about 5 ml of STK. Keep the chloroplast suspension on ice.

If algal cells are provided... when you harvest the algal cultures, the technique for isolating chloroplasts will vary depending on the type of algae. If you are provided with Chara australis, it is as simple as cutting one end of the internode cell (with small scissors) and squeezing the cell sap (with chloroplasts) onto a microscope slide. If you are provided with Eremosphaera viridis, the large cells need to be broken apart. This is done with a glass mortar and pestle. The cells are decanted from the culture flask into a 50 ml disposable centrifuge tube, allowed to settle (this will take less than 5 minutes at "1 X g"). The supernatent (culture media) is carefully poured off (into the sink) and the cells re-suspended in STK. Then, they are poured into the glass homogenizer. The plunger is pushed to the bottom while twisting it. This is repeated 4 times, at which point the cells will have been disrupted, releasing the chloroplasts. The homogenate is filtered through multiple layers of cheesecloth, and the filtrate (chloroplast rich) is used as is for microscope visualization.

When performing fluorescence measurements, the amount of fluorescence will depend upon the concentration of the chloroplasts. So, it is important to ensure the same amount of chloroplasts is used for all measurements. Normally, the chlorophyll concentration is used to standardize chloroplast concentrations. Note that the spectrometer configuration for absorbance measurements is different from the configuration for fluorescence.

Initial Absorbance Setup.png
So, do your absorbance measurements first, then re-configure the setup for fluorescence. In SpectraSuite, you can select particular wavelengths to obtain the absorbance (shown at the bottom of the graph). For fluorescence, there is no calibration required (you are measuring relative intensities). The integration time can be adjusted to get a good signal.


Standardizing Chloroplast Concentration

Absorbance (sometimes called optical density, abbreviated OD) is a logarithmic measure of light absorption: If light of an intensity Io enters the sample and a lesser intensity I exits: A = log10(Io/I). This logarithmic measure has the virtue that it is proportional to the concentration of light-absorbing material in the sample. In a laboratory, a path length through the sample of 1 cm is the common convention. Thus, your cuvettes are 1 cm in width. In some research fields, absorbance is defined as the natural logarithm of the ratio Io/I (A = ln((Io/I), but OD (OD = log10(Io/I) is the more common measure, and what is measured by your Ocean Optics spectrometer. An additional source of confusion is that the equation is sometimes shown in the equivalent form OD = -log10(I/Io). Because it's easy to lose the negative sign, I prefer log10(Io/I).

Add 0.05 ml of the chloroplast suspension to 5.0 ml of the Ac/W, vortex, and filter through No. 1 Whatman filter paper directly into a test-tube (or 10 ml graduate cylinder). Ac/W alone should be used as your control for absorbance measurements. The extracted pigment is labile. It will breakdown spontaneously as well as photobleach in the light so absorbance measurements should be done without unnecessary delay and assuring the filtrate is not exposed to direct light. Determine the absorbance (OD --Optical Density) at 662 nm and 645 nm. To measure the measure the absorbance at these specific values, click on the absorbance spectrum graph. A box will appear below the graph; change the value to 645 nm and read the OD to the right of the box. Repeat for 662 nm. If OD662 is greater than 1.0 or less than 0.2, adjust the amount of chloroplast suspension accordingly in a new measurement. Concentrations of chlorophylls a and b in the Ac/W extract can be calculated from the following equations:

Chl a = 12.7 (OD662) – 2.7 (OD645) (micrograms / ml)

Chl b = 23.0 (OD645) – 4.7 (OD662) (micrograms / ml)


Calculate the concentrations of chlorophylls in the stock suspension based upon the dilution factor you used to make the Ac/W extract. For example, if you used 0.05 ml of suspension (in 5.0 ml of Ac/W), the dilution factor is 5.05 / 0.05, or 100–fold. Adjust the volume of the chloroplast suspension to a concentration of about 0.1 to 0.5 mg/ml Chl using STK. A sample calculation can be found at the end of the lab exercise [1].

Be sure to save your chlorophyll pigments (on ice) for measurements of excitation/emission spectra of pigments (to compare with chloroplasts).

For most experiments, the stock suspension of chloroplasts will be diluted into TK solution to a final density of 10 micrograms/ml Chl. However, chloroplasts should always be stored concentrated since this improves their viability outside of their native environment.

Fluorescence Spectra Measurements --Setup

Figure 1. Fluorescence Setup
Fluorescence setup.png

The instrumentation setup includes the computer running the SpectraSuite software (Figure 1). The computer is connected to the spectrometer via a USB cable. One of the fiber optic leads is connected to the spectrometer. The other is connected to the light source (a high intensity tungsten halogen lamp). The light source can be hot, so be careful touching the housing. The fiber optic cables can be damaged by overbending, so be gentle. The two fiber optic leads are connected at the cuvette holder at right angles. The wavelength of the excitation light is adjusted by sliding the filter carefully --the scattered light peak will be observable as part of the SpectraSuite spectrum.

There are some crucial points to bear in mind when you setup the apparatus.

  • Make sure you shield the cuvette from extraneous illumination. Otherwise, scattered light from the fluorescence lighting will 'contaminate your spectra. You should be able to determine whether this is a problem by monitoring the spectra.
  • Be careful when sliding the excitation filter to adjust the excitation wavelength. This is a very expensive interference filter, so two hands are better than one. Let your lab mate collect the spectra on the computer, as required).
There are lots of possible variants that you may wish to do. For example, dilute milk is a good way to confirm the scattered light peak. Cells will scatter the light the most, chloroplasts to some degree, isolated pigments very little (why to you think this is so?). You can even try other light sources --such as the LED lamps-- by shining them down on the cuvette containing your sample.


Determining Excitation and Emission Spectra

Figure 2. Fluorescence Scans. Here are examples of fluorescence scans chloroplasts and acetone/water extracted chlorophyll to give you an idea about the kind of scans you will see with your preparations. Note that chloroplasts scatter light much more than chlorophyll does. Why?
Fluorescence scans.png

After placing the 10 microgram/ml chloroplast suspension in the cuvette provided , measure the excitation and emission spectra for your chloroplast suspension. The full spectra will be observed on the spectrometer. If the signal is low, you can increase the integration time to collect more photons per spectrum. Slide the linear bandpass filter to change the excitation wavelength. You should be able to observe it on the spectrum (due to light scattering) as a moving peak. Do the same for a chlorophyll suspension in Ac/W. Figure 2 shows an example of the spectra you should be able to obtain.


Calculate the excitation state energy for Chl* based upon the peak excitation and emission wavelengths. Note that the energy at a specific wavelength is E(λ) = hc/λ, where h is Planck’s constant (1.584 • 10-37 kcal-sec), c is the speed of light (3.00 • 108 m/sec in vacuum, but about 2.25 • 108 m/sec in water), and λ is the wavelength. Assure your units are correct.

Calculate the energy available from chlorophyll in a chloroplast for both excitation and emission. How does this compare to the energy of ATP formation from ADP and Pi (about 8 kcal/mole)? You may need to use Avogadro’s number (6.023 • 1023 molecules/mole) and the molecular weight of chlorophyll (about 800). Other data for chloroplasts is available in your textbook (Lawlor, Table 4.1) (for example, an average chloroplast of volume 3.3 • 10–17 m3 contains 9 • 10–13 g of chlorophyll, and [ATP] is about 1.5 mM).

You can do the calculations on a ‘per mole’ or ‘per molecule’ basis.



Calculation of Chlorophyll Concentration

The following example is fictitious, but shows you how to calculate chlorophyll concentration and dilution factors. Take 0.05 ml of your chloroplast suspension (ensuring it is well-mixed before drawing the aliquot) and add to 5 ml of Ac/W. In the spectrometer, you measure blank (Ac/W alone) values of 'zero', and sample absorbances of:
OD662: 0.5
OD645: 0.2
Filling in the blanks of the equations

Chl a = 12.7 (OD662) – 2.7 (OD645) (micrograms / ml) Chl a = (12.7)(0.5) - (2.7)(0.2) = 5.81 micrograms/ml
Chl b = 23.0 (OD645) – 4.7 (OD662) (micrograms / ml) Chl b = (23.0)(0.2) - (4.7)(0.5) = 2.25 micrograms/ml
Total chlorophyll is8.06 micrograms/ml, or 0.00806 mg/ml


Since the dilution was 100-fold, your suspension chlorophyll is 0.806 mg/ml

Now, suppose you want a final chlorophyll suspension of 0.05 mg/ml in a total volume of 5 ml (for fluorescence measurements):

(X/5)*(0.806) = 0.05 (in units of mg/ml)

The algebra..... X = (5)(0.05)/0.806 = 0.31 ml

So..... add 0.31 ml of suspension and 4.7 ml of your suspension medium to reach 0.05 mg/ml

If you want a final chlorophyll suspension of 0.50 mg/ml in a total volume of 5 ml (for oxygen electrode measurements):
X = (5)(0.5)/0.806 = 3.1 ml. So add 3.1 ml of your suspension with 1.9 ml of suspension medium.

Keep your concentrated chloroplast suspension (on ice)! As noted, high concentrations improve stability.

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