Characterizing the Two-photon Absorption Properties of Fluorescent Molecules in the 680–1300 nm Spectral Range

Two-photon laser scanning microscopy (2PLSM) is a state-of-the-art technique used for non-invasive imaging deep inside the tissue, with high 3D resolution, minimal out-of-focus photodamage, and minimal autofluorescence background. For optimal application of fluorescent probes in 2PLSM, their two-photon absorption (2PA) spectra, expressed in absolute cross sections must be characterized. Excitation at optimum wavelength will make it possible to reduce the laser power and therefore minimize photodamage. Obtaining 2PA spectra and cross sections requires correcting the two-photon excited fluorescence signals for a combination of laser properties, including the beam spatial profile, pulse duration, and absolute power, at each wavelength of the tuning range. To avoid such tedious day-to-day laser characterization required in the absolute measurement method, a relative method based on independently characterized 2PA reference standards is often used. By carefully analyzing the available literature data, we selected the most reliable standards for both the 2PA spectral shape and cross section measurements. Here we describe a protocol for measuring the 2PA spectral shapes and cross sections of fluorescent proteins and other fluorophores with the relative fluorescence method using these reference standards. Our protocol first describes how to build an optical system and then how to perform the measurements. In our protocol, we use Coumarin 540A in dimethyl sulfoxide and LDS 798 in chloroform for the spectral shape measurements to cover the range from 680 to 1300 nm, and Rhodamine 590 in methanol and Fluorescein in alkaline water (pH 11) for the absolute two-photon cross section measurements.

A 2PA, S (λ) = F 2, S (λ)/f c (λ) = F 2, S (λ)A 2PA, R (λ)/F 2, R (λ) A similar approach is used for evaluating the two-photon cross section values. In this case, the raw fluorescence signal of the sample, measured at a particular excitation wavelength λ ex is corrected for a combination of laser properties by using a 2PA cross section reference standard measured at the same excitation and fluorescence registration conditions. Suppose F 2,S (λ ex , λ reg ) and F 2,R (λ ex , λ reg ) are the two-photon fluorescence signals recorded in a narrow spectral range around registration wavelength, λ reg , with the same excitation conditions for the sample and the reference. To calculate the 2PA cross section, one needs to normalize these signals to the fluorescence quantum efficiencies and concentrations of signal and reference, respectively. Let φ S (λ reg ) and φ R (λ reg ) be the differential quantum efficiencies measured at the same wavelength with the same spectral bandwidth as F 2 signals, but with one-photon excitation. Using a narrow spectral range in fluorescence collection avoids corrections to the spectral sensitivity of the detection system. C S and C R are the corresponding concentrations, used in the two-photon experiment, and calculated using Beer's law: C = OD max /ε max where OD max is the peak optical density and ε max is the peak extinction coefficient. Then the two-photon cross section of the sample reads , eq. (2): σ 2, S λ ex = F 2, S λ ex , λ reg C R φ R λ reg F 2, R λ ex , λ reg C S φ S λ reg σ 2, R λ ex where σ 2,R (λ ex ) is the cross section of the reference.
Although the literature data accumulated over the last few decades for the reference standards start to converge for some of them, significant deviations are still present for the others. The relative method of 2PA characterization was used previously, but the detailed descriptions of the measurement details were often missing and selection of standards was arbitrary, resulting in large variations in the results between different labs. By carefully analyzing the available literature data, we selected the most reliable standards for both the 2PA spectral shape and cross section measurements (see Notes). In our protocol, we use Coumarin 540A in DMSO (de Reguardati et al., 2016) and LDS 798 in chloroform (Makarov et al., 2011) for the spectral shape measurements to cover the range from 680 to 1300 nm. We use Fluorescein in alkaline water (pH 11) ( measurements at selected wavelengths. The standards for the shape were selected because their reported 2PA and 1PA spectra closely overlap, signifying that the reported 2P spectral shapes measurements were reliable. Also, the spectra are very broad and structureless, thus introducing fewer potential errors due to a finite laser spectral shape and small shifts in central wavelength. The standards for the σ 2 were selected based on a close match between two to five independent measurements at a selected wavelength. Using this protocol will make it possible for researchers to characterize the absolute 2PA spectra of new fluorescent probes and sensors in a standardized way and with high reproducibility. Our optical setup ( Figure 1) consists of an automatically tunable (with custom LabView program) femtosecond laser (InSight DeepSee, Spectra Physics) coupled with a photon counting spectrofluorimeter (PC1, ISS). The laser output beam (100-120 fs pulse duration, 680-1,300 nm tuning range, 80 MHz repetition rate, 0.6-1.3 W average power, horizontal polarization) was first attenuated to 100-200 mW and made vertically polarized with a system of a half-wave plate and a Glan-laser polarizer. It then was filtered (with a 645 long pass filter) to remove all residual visible light and was directed with 4 mirrors (M1-M4) to the entrance aperture of the spectrofluorimeter. We use a continuous variable neutral density filter wheel between mirrors M3 and M4 to further attenuate the power to a particular value needed in the experiment. A flip mirror (FM) is used to send an attenuated beam to a power meter (Melles Griot) for monitoring laser power before the sample.
A few re-arrangements were made to the spectrofluorimeter optics to adjust it for the 2PEF measurements. The excitation lamp source (not shown), excitation monochromator (not shown), optional beamsplitter, and two collimating lenses were removed from the excitation path. The neutral density motorized filter wheel with 4 slots (ISS) was added and connected to the step motor of the filter wheel of the left fluorescence registration channel. A NIR achromatic lens, f = 45 mm (Edmund Optics) was inserted into an optical tube (ISS) at the end of excitation path. This lens focuses the laser beam onto the sample, held in a 3 × 3 mm optical cuvette (Starna Cells). To avoid absorption of the laser by the solvent, fluorescence is collected from the first 0.7 mm layer of solution ( Figure 1B). To minimize thermal lensing effects, all dye solutions are stirred during the measurement ( Figure 1B). The left fluorescence detection channel (without monochromator) is used for measurements relative two-photon excitation spectra, because in this case only integrated fluorescence signal is

3.
LabView2018 (National Instruments, https://www.ni.com) Custom Labview program for scanning the laser wavelength could be obtained by sending an e-mail request to mikhail.drobijev@montana.edu

A.
Building optical setup for two-photon absorption measurements

1.
On the optical table set up and fix a femtosecond laser and a spectrofluorimeter, approximately 2 m apart.

2.
Remove the excitation lamp source (Figure 2), the mirror holder with two mirrors from the excitation monochromator ( Figure 3), two lenses (in tubes) (Figures 4 and 5), optional beam splitter ( Figure 6), and optional filter (Figure 7) from the excitation path of the PC1 spectrofluorimeter. The tube carrying the first lens and placed between the monochromator and optional beamsplitter must be removed as well ( Figure 4). The second tube (empty), entering the sample compartment should be kept in place ( Figure 5).

3.
Disconnect the 'Left Emission Wavelength' stepper motor cable from its stepper motor jack inside PC1 (Figure 8).

5.
Set a motorized filter wheel with these ND filters in the excitation optical path of PC1 right after the excitation monochromator and bolt it to the PC1 base (Figures 4 and 8).

6.
Connect the stepper motor of this filter wheel to the stepper motor jack of the 'Left Emission Wavelength' wheel ( Figure 8).

7.
Put the excitation polarizer inside the optical path of the PC1.

8.
Turn on the PC1 and the computer controlling it.

9.
Create a file template in Vinci to measure the excitation power dependence of fluorescence in the left or right emission channels (Power Dependence file), Figure 9.

10.
Create a file template in Vinci to measure the two-photon excitation spectra of the two samples-one set in the Sample and another-in Reference position of the PC1 in one laser scan using left emission channel (Spectral Scan file), Figure 10.

11.
Turn on the laser in the alignment mode (200 mW) at 720 nm. Use safety goggles.

12.
Set the first mirror M1 assembled in the holder right after the laser output (main beam) to turn the beam 90° in horizontal plane, parallel to the table, see Figure 1.

13.
Set first a half-wave plate and second a Glan Laser polarizer assembled in their rotation mounts after the M1 in the laser beam. Make sure the beam passing through both of them freely at its original height (as it came out of the laser).

14.
Turn the half-wave plate fast axis approximately to 45° (turning laser polarization from horizontal to vertical).

15.
Turn the Glan polarizer polarization axis close to vertical position.

16.
Use 3 mirrors (M2-M4) in their holder assemblies to direct the laser beam to the entrance aperture of the spectrofluorimeter (see Figure 1). Use mirrors M2 and M3 to make the beam height close to the height of the entrance aperture of PC1 and parallel to the table. Use M3 and M4 to direct the beam to the center of the entrance aperture of PC1 in the horizontal plane.

17.
Put the continuously variable neutral density filter in the beam after the M3 mirror.

18.
Put the flip mirror FM after M4 and flip it up.

19.
Put the power meter in the laser path after the flip mirror.

20.
Turn the continuously variable neutral density (ND) filter wheel to the maximum transmission position.

21.
Check that the laser power measured with the power meter is ~200 mW.

22.
Turn the continuously variable ND filter to attenuate the power to ~20 mW.

23.
In the VINCI Experiment software (ISS), go to Instrument Control tab and open the excitation shutter.

24.
In the same tab set the Left Emission Wavelength to position 4 (corresponding to empty slot of the excitation ND filter wheel.

25.
With mirrors M3 and M4 align the beam along the axis connecting the center of the entrance aperture of PC1 and the center of the window in the sample holder.

26.
Insert the 45-mm achromatic lens into a 22 mm-to-25 mm diameter adaptor tube (ISS accessory) and slip that adaptor with the lens over a 22-mm ISS tube, with the lens facing toward the sample position ( Figure 11).

27.
Make sure that the laser beam is still pointing at the center of the opening of the sample holder (now with the 45-mm lens in). If necessary slightly re-align the beam with mirrors M3 and M4.

28.
Turn off the laser.

29.
In the right emission channel, insert the Semrock 745/SP filter into a filter slot.

30.
Disconnect the Laser from the computer with InSight GUI software and connect it through an RS 232 Serial port to a PC computer with the custom LabView program for tuning laser wavelength.

31.
Turn on the laser using the LabView program ( Figure 12, Supplemental file 1 Laser Scan).

32.
Tune the laser to 850 nm.

33.
Flip the flip mirror up.

34.
Open the laser shutter with the LabView program.

35.
Adjust the laser power to 200 mW. First make sure that continuously variable ND filter is turned to a fully transmitting position. Then, slowly rotate the half-wave plate (sitting immediately after the laser) until the laser power reads 200 mW. Use this position of the half-wave plate always, unless higher power is needed in experiment.

36.
Move the head of the power meter into the sample compartment and fix it (with the 1" post holder and short post holder base BA1S), such that its sensor is centered inside the round opening of the excitation path and facing the laser beam. Set the excitation polarizer to horizontal (90°) position with the Instrument Control of PC1 software. Adjust the angle of the external Glan Laser polarizer by turning its rotation mount, to a position where the power is minimum. Lock this position with a screw on its rotation mount.

37.
Slide the excitation polarizer out of the excitation optical path.

38.
Calibrate the ND filters at several laser wavelengths in the whole tuning range. Set a first laser wavelength at 680 nm. By using the Instrument Control function in Vinci, change the position of the Left Emission Wavelength filter from 1 to 4 and record the power for all 4 positions. Calculate the ratio of power measured for positions 1-3 to that measured for position 4.

40.
Summarize the data in the Origin Workbook, see Figure 13.

41.
Turn off the laser.

42.
Remove the power meter head from the sample compartment and place it back to its holder after the flip mirror.

43.
Insert the 770/SP filter into a 22 mm-to-25 mm diameter adaptor tube (ISS accessory) and slip that adaptor with the filter over a 22-mm ISS tube (with the PC1 collecting lens in it) used for emission collection through the left emission channel.

45.
Dismount two adaptor holders for 3 × 3 mm cuvettes (unscrew 4 miniature screws holding one side of the holder) and insert a rectangular band of black paper (0.9 × 4 cm) on the side containing 4 screws such that only a narrow slit of ~0.7 mm remains open into each of the holders, see Figure 1B, right, bottom. The slit is supposed to be on the left side of the facet, when looking at the holder.

46.
Mount the holders back, fixing the pieces of paper inside them with 4 screws.

B.
Measuring two-photon excitation spectral shapes using Left Emission Channel

1.
Preparation of samples for two-photon spectral measurements.

a.
Prepare the stock solutions of samples (up to two new samples) and references (LDS798 and Coumarin 540A), see Recipes for preparation of reference solutions, and add 150 μl of them into the 3 × 3 mm optical cuvettes.

b.
Using Lambda 950 Spectrophotometer, measure absorption spectra of all the samples. The optical density in the spectral maximum of all solutions should be in the range of 0.2-1.5 when measured in 3 × 3 mm cuvettes placed in 3-mm adaptor holder.

c.
Put micro stir bars in solutions with reference.

2.
Initial instrument setup preparation. Coumarin 540A solution in Sample holder. Filters 770/SP and 633/SP in the Left Emission Channel.

a.
Turn on the laser in the full power mode.

b.
Start the laser control LabView program (Supplemental file 3 LaserTestVI3.vi) from a PC computer.

c.
Turn on the PC1 spectrofluorimeter.

d.
Insert the adaptor holders for 3 × 3 cuvettes into the sample and reference holders of PC1 such that the masked side of either adaptor holder is directed to the left emission channel when it is set to the measurement (the reference holder can be set to measurement by selecting Reference position of the turret in the Instrument Control of Vinci). Keep the adaptor holders in place during all the measurements.

e.
Set the Sample position to measurement.

f.
In the Instrument Control start stirring both Sample and Reference.

g.
Insert the 3 × 3 mm cuvette with Coumarin 540A solution into the sample holder of PC1.

h.
Turn manually the left emission wavelength filter wheel to position 1 (corresponding to 633/SP filter).

i.
Slide the left emission polarizer in the emission path and set it to Magic Angle position (with Instrument Control function).

j.
Set the laser wavelength to 800 nm.

k.
Adjust the laser power with the continuous ND filter to ~20 mW.

l.
Send the laser beam to PC1 by flipping the flip mirror down.

m.
Make sure that the excitation polarizer in PC1 is moved out of the excitation optical path.

n.
Make sure with the Instrument Control of Vinci that the ND filter wheel in the excitation channel (Left Emission Wavelength) is set to empty position (slot 4).

o.
Open the excitation shutter with Instrument Control.

p.
In a dark room with the sample compartment lid removed, adjust the distance from the 45-mm lens to the sample by sliding the adaptor with the lens in it over the 22-mm tube (fixed) and maximizing the intensity of fluorescence at the left emission channel PMT.

q.
Fix the position of the adaptor (with respect to a 22-mm tube) with a small screw.

3.
Adjusting power range for the samples #1 and #2. First, sample #1 and then sample #2 in Sample holder. Filters 770/SP and 633/SP (or 680/SP, 694/SP or empty) in the Left Emission Channel. Select the second short pass filter such that its cutoff wavelength is larger than fluorescence peak wavelength of samples #1 and #2.

a.
Put the cuvette with the sample #1 solution into the Sample position instead of Coumarin 540A solution.

b.
Cover the sample compartment with the lid.

d.
Repeat the same for the sample #2.

e.
Adjust the laser power with the continuous ND filter such that the maximum signal across the 2PA spectrum of both samples amounts ~1 × 10 6 counts. Typically, this power will be in the range 10-100 mW, depending on sample concentration and two-photon brightness.

b.
Repeat the Step B4a for sample #2.

b.
In the scanning part of the LabView file controlling the laser, set the start wavelength of the scan equal to the largest number found in Steps B4a and B4b.

c.
Calculate the stop wavelength (λ stop ) by taking the longest possible wavelength observed as a long-wavelength edge of one-photon absorption spectrum multiplied by 2 and taking the closest number to it in a sequence of numbers starting at 680 with. Set this number as a stop wavelength.

d.
Set the step equal to 4 nm and the time per step equal to 42 s.

e.
Open the Spectral Scan file, make the notes about the experiment (samples names and positions, filters used, etc.) and start the scan.

f.
Start the laser scan.

g.
Once the scan is finished, remove the cuvettes with the samples from adaptor holders.

b.
Turn manually the left emission wavelength filter wheel to position 4 (empty).

c.
Repeat Steps B5b-B5f with the start wavelength equal to 940 nm in Step B5b and all other settings kept the same in Steps B5b-B5f and making the notes about the reference in Step B5e.

8.
Calculating corrected two-photon excitation spectra of samples #1 and #2 with the programs written in OriginLab.

a.
For a file that contains the two-photon raw spectral data (.ifx) in which two samples were measured simultaneously in the 'Sample' and 'Reference' holders: i.
In OriginLab, create and save an Import Wizard (File > Import > Import Wizard) that will open the file into a workbook so that it looks similar to the example workbook displayed in Figure 14. The data columns that the Vinci program saves are 'Time', 'Sample', 'Intensity', 'IntensityStdError', and 'Real time'.

b.
Add the auto-analysis script to that Import Wizard filter. To do this, first import the file used for the above step, select the filter you just created, and click "Next" through to the last window. Check "Save the filter" as well as "Specify advanced filter options". Save this as a new filter, "2PA_sample_and_reference_autocalculation". Click "Next", which brings you to a window to copy and paste Script 1 (see Supplemental file 2 Origin Script) in the window "Script after each file imported:".

c.
After filling in the appropriate values in the window that pops up (first wavelength, last wavelength, wavelength step, seconds per step), the resulting workbook should look like the following ( Figure 15). Additionally, a graph ( Figure 16) will pop up that shows what the raw data looks like after the script auto-deletes the points when the laser is stabilizing. It should look like data points clustered into two distinct spectra. If there are points that are randomly much lower in intensity than the others, that usually means the script did not work for some reason and the data should be manually sorted. This graph ( Figure 16) is an important visual check that the script worked as it is meant to.

d.
If the samples measured are reference dyes to measure the correction function of the setup, then copy and paste the "correct" reference dye spectra into their corresponding columns (column D for the 'Sample holder', column E for the 'Reference holder). Make sure that the wavelength range you copied matches the wavelength range you measured. Columns F and G are now the correction functions for the 'Sample holder' and the 'Reference holder', respectively. They are set to divide the raw spectra by the correct spectra.
(Correction function data from the 'Sample holder' and the 'Reference holder' should be treated separately.)

i.
First, normalize the short range correction functions by dividing each of them by the mean of the column values in the 940-980 nm range.
ii. Now, find the mean of the row values of the two short range correction functions in the 940-980 nm range and accept it as a part of correction function for this range. iii.
Create the merged correction function.

f.
If the samples measured have unknown 2PA spectra, then copy and paste the correction functions that you measured previously for the sample holder and the reference holder (column D for the 'Sample holder', column E for the 'Reference holder'). Make sure that the wavelength range you copied matches the wavelength range you measured. Columns F and G are now the corrected 2PA spectral shapes for the samples measured in the 'Sample holder' and the 'Reference holder', respectively. They are set to divide the raw spectra by the correction functions.

C.
Measurement of two-photon absorption cross sections of a red fluorescent protein (for example, RGECO1, described in Zhao et al., 2011) relatively to Rhodamine 590 in methanol at 1,060 nm using Right Emission Channel with registration of fluorescence at 600 nm.

1.
Prepare the stock solution of Rhodamine 6G in methanol. Adjust the optical density at 528 nm in 3 × 3 mm cuvette to a number between 0.2 and 0.3. Record the exact number for OD.

2.
Adjust the optical density of the sample in 3 × 3 mm cuvettes to 0.2-0.3. Record the exact number for OD.

3.
Set the laser wavelength at 1,060 nm.

4.
In the right emission channel, slide the polarizer in and set it to the magic angle using Instrument Control function.

5.
Set the right emission monochromator to 600 nm and manually insert the two 2-mm slits into the monochromator.

6.
Put the cuvette with reference Rhodamine 590 solution in the Sample holder of the turret.

7.
Adjust the laser power with the ND continuous filter such that the right emission intensity will be on the order of 50,000-100,000 when the excitation filter wheel is set to empty position (#4).

9.
Repeat Steps C7 and C8 for the sample solution.

10.
Run the Power Dependence file with registration in the Right Emission Channel for Rhodamine 590.

11.
Repeat Step C10 for the sample without changing condition.

12.
Using the OriginLab, plot the power dependencies of fluorescence signals as function of power squared and obtain the ratio F 2, S (λ ex , λ reg )/F 2, R (λ ex , λ reg ) values as ratio of the slopes of linear fits of the sample and reference standard, respectively.

13.
Using known extinction coefficients, calculate the ratio of concentrations:

14.
To measure the φ numbers, dilute the sample and reference solutions to have optical densities, similar to each other at a selected wavelength (530 nm) and falling in the range of 0.05-0.07 in 1-cm optical cuvettes. Record their respective OD values at 530 nm: OD S (530) and OD R (530).

15.
Using LS55 spectrofluorimeter, record fluorescence spectra of the sample and reference upon excitation at 530 nm with excitation slit equal to 5 nm and emission slit-16 nm.

16.
Record the one-photon signals at 600 nm F 1, S (600) and F 1 , R (600) and calculate the ratio:

1.
The variations of spectral profiles, i.e., deviations of relative values at one wavelength when the spectra are normalized at another wavelength (e.g., peak) contain the random and systematic contributions. The random contribution comes from reproducibility of the laser parameters in consecutive scans of the same sample. We have observed that the deviations across the spectra were not larger than 4% in 2-3 consecutive scans. Since two scans (sample and reference standard) are used to calculate the corrected 2PA spectrum, this results in a random error of 6%. The systematic spectral deviations come from the accuracy of the measurement of 2PA spectra of references standards. Those can be estimated to be about 5% for Coumarin 540A (de Reguardati et al., 2016) and ~10% for LDS 798 (Makarov et al., 2011). This results in the total spectral shape errors of 8% in the short wavelength range (680-980 nm) and 12% in the long wavelength range (980-1,300 nm).

2.
The error of the 2PA cross section measurement is a combination of the errors of individual parameters entering eq. (2). The errors of F 2 numbers are estimated from linear fits of the fluorescence intensity vs. power squared plots (usually 1% for both F 2,S and F 2,R ). The errors in differential quantum yields, φ for both sample and reference were determined from 12 independent measurements of one protein and found to be 7%. The errors in concentrations come mostly from the errors in measurements of extinction coefficients of the sample, equal to 5% (Molina et al., 2019). The systematic error, coming from the uncertainty in the 2PA cross section of reference standard is 10% for Rhodamine 590 (see Notes section). Therefore, the total error calculated in quadrature for the σ 2 of the sample measured at one wavelength (1,060 nm) amounts to 15%. This translates to the error of absolute σ 2 value, determined at different wavelengths of ~18% (Molina et al., 2019).

1.
We use the mixture of chloroform/deuterated chloroform (1:2) as solvent for LDS 798 to get rid of chloroform absorption at 1,150 nm. We observed virtually no changes in 1PA spectra of the dyes when going from pure chloroform to the mixture.

2.
The shapes of the one-photon and two-photon absorption spectra of Coumarin When the 2PA spectral shape of Coumarin 540A in DMSO is taken as a reference, the 2PA spectrum of Rhodamine 6G in methanol shows strongly overestimated absorption in the region of 680-750 region (cf. open diamonds and light blue line in Figure 18). This can be due to overestimated values of 2PA of Coumarin 540A, as shown in Figure 17. However, when the 2PA spectrum of Coumarin 540A corrected in the 680-740 nm range with Prodan (see Table 1 for the values) is taken for the A 2PA,R (λ) function, the Rhodamine 6G spectrum (dark green line with triangles in Figure 18 Figure 19).
Since the z-scan data are obtained with absolute method (LDS 798 is a primary standard), we use the corresponding 2PA shape (black squares in Figure 19) for the A 2PA,R (λ) function. To interpolate the A 2PA,R (λ) function to intermediate wavelengths, we use a Gaussian fit to the experimental data between 900 and 1,300 nm, shown by a dashed black line in Figure 19. This spectrum is presented in Table 2. To check the performance of this function, the 2PA spectrum of Rhodamine 590 in methanol was measured relatively to LDS 798 in CHCl 3 /CDCl 3 (1:2) in the 940-1,140 mn region, see Figure 18.

4.
In the measurements of two-photon cross sections, we use optical densities OD < 0.05 at the fluorescence registration wavelength and OD < 0.3 in the absorption peak (in 3-mm cuvette). That is needed to exclude possible re-absorption and reemission effects.

5.
We suggest using Rhodamine 590 in methanol as a reference standard (see Tables 3 and 4 for literature data) for measuring 2PA cross sections at selected wavelengths. Most consistent literature data were collected near 1,060 nm (Table  3). For this wavelength we suggest using σ 2 = 10 ± 1 GM.

6.
We suggest using Fluorescein in water at pH 11 as a reference standard for molecules fluorescing in green part of the spectrum (500-550 nm). Part C of the protocol should be adjusted then to measuring fluorescence in the green region.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.    Removing tube holder with collimating lens (placed right after excitation monochromator) and setting an ISS filter wheel with four slots in place of it    Left: An f = 45 mm achromatic lens is inserted and fixed in a 22-to-25 mm diameter adaptor tube. Right: Achromatic lens in the adaptor tube is slide over the 22-mm empty adaptor tube inside the sample compartment. An example of what the Origin workbook will look like after creating the "2PA_sample_and_reference" Import Wizard filter An example of what the final workbook should look like after opening a file with the "2PA_sample_and_reference_autocalculation" Import Wizard filter This graph pops up after opening a file with the "2PA_sample_and_reference_autocalculation" Import Wizard filter. It is a visual check that the script worked as intended.