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May 2019

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Characterizing the Two-photon Absorption Properties of Fluorescent Molecules in the 680-1300 nm Spectral Range
680-1300 nm波段荧光分子双光子吸收特性的表征   

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Abstract

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.

Keywords: Two-photon absorption (双光子吸收), Two-photon laser scanning microscopy (双光子激光扫描显微镜), Two-photon brightness (双光子亮度), Two-photon spectra (双光子光谱), Cross sections (截面), Fluorescent proteins (荧光蛋白), Fluorophores (荧光基团), Two-photon reference standards (双光子参考标准)

Background

Two-photon laser scanning microscopy (2PLSM) is an advanced imaging technique used in neuroscience, cancer biology, immunology, and other areas of biosciences (Denk et al., 1990; Xu et al., 1996; Zipfel et al., 2003; Helmchen and Denk, 2005). Compared to fluorescence confocal microscopy, which is based on one-photon excitation, 2PLSM provides deeper interrogation of tissues with less out-of-focus photodamage and less autofluorescence background. For optimal application of 2PLSM, information about the brightness of fluorescent probes upon two-photon excitation at different laser wavelengths (i.e., two-photon absorption and excitation spectra) is necessary. That information helps researchers choose the right probe(s) for a particular application (e.g., the brightest probe excitable with optimum wavelength within the near-infrared tissue transparency window, or a combination of probes for multicolor imaging). There are several methods to measure the two-photon absorption spectra and absolute cross section values (Rumi and Perry, 2010), including a direct one, based on nonlinear transmission (z-scan), and indirect ones, such as two-photon excited fluorescence (2PEF). Here we choose the 2PEF approach because it is much more sensitive, requiring much lower concentration of fluorophores, and all the probes used in 2PLSM are intrinsically fluorescent. In contrast to one-photon (1P) excitation, two-photon (2P) excitation efficiency strongly depends on the laser beam spatial and (pulse) temporal profiles. Since these characteristics usually vary from wavelength to wavelength across the laser tuning range, careful corrections for these variations are necessary for extracting pure molecular two-photon absorption (2PA) spectral shape and absolute cross section values (σ2). To obtain these properties one can use either an absolute or a relative method. In the absolute measurement method, the laser properties such as power, pulse duration and shape, and spatial beam profile (i.e., intensity dependence on x, y, and z coordinates inside the sample solution) must be characterized at each wavelength. Then, a function representing a combination of these parameters at each laser wavelength should be constructed and applied for correction of raw data. The spectra obtained using this absolute method (Xu and Webb, 1996; Albota et al., 1998; Makarov et al., 2008; Makarov et al., 2011; de Reguardati et al., 2016) can be considered as reference standards for application in relative method.

To avoid tedious characterization of laser properties on a day-to-day basis (necessary in absolute method of measurement), we use in our protocol a relative method based on previously characterized reference standards. In this method, the raw 2PA spectra are collected for the sample, F2,S(λ), and for the reference, F2,R(λ), (indices S and R are used for sample and reference, respectively, here and throughout) in the same conditions of excitation, and then the correction function fc(λ) is calculated using the known 2PA spectrum of the reference A2PA,R(λ) and its raw spectrum F2,R(λ):



This correction function is then applied to the raw spectrum of the sample to get its corrected, “true” spectrum (Makarov et al., 2008), eq. (1):



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 λex 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 F2,Sex, λreg) and F2,Rex, λreg) are the two-photon fluorescence signals recorded in a narrow spectral range around registration wavelength, λreg, 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 φSreg) and φRreg) be the differential quantum efficiencies measured at the same wavelength with the same spectral bandwidth as F2 signals, but with one-photon excitation. Using a narrow spectral range in fluorescence collection avoids corrections to the spectral sensitivity of the detection system. CS and CR are the corresponding concentrations, used in the two-photon experiment, and calculated using Beer’s law: C = ODmaxmax where ODmax is the peak optical density and εmax is the peak extinction coefficient. Then the two-photon cross section of the sample reads (Makarov et al., 2008), eq. (2):



where σ2,Rex ) 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) (Xu et al., 1995; Xu and Webb, 1996; Makarov et al., 2008; de Reguardati et al., 2016) and Rhodamine 6G in methanol (Hermann and Ducuing, 1972; Bradley et al., 1972; Penzkofer and Leupacher, 1987; Albota et al., 1998; Makarov et al., 2008; de Reguardati et al., 2016) for the absolute σ2 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 σ2 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 x 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 required, and the right fluorescence channel (with monochromator) is used for measuring the two-photon cross sections.


Figure 1. Schematic of experimental setup. A. General layout. A femtosecond InSight DeepSee (SpectraPhysics) laser beam is steered with a system of mirrors (M1-M4) to the entrance aperture of a photon counting spectrofluorimeter PC1 (ISS). Automatic wavelength-to-wavelength tuning of the laser is controlled with a custom LabView program and the fluorescence signal acquisition is accomplished with the ISS Vinci software. B. Top: Schematic of the geometrical arrangement of the 3 x 3 mm cuvette with the sample solution (top view). The laser beam (dark red) is focused with an f = 45 mm lens into a frontal part of the solution. The fluorescence is collected through a side, directed to the left emission channel through a mask. The mask is a narrow rectangle of black paper fixed inside the cuvette holder that allows only the fluorescence from the front 0.7 mm of solution to get out. Bottom: A cuvette with a sample solution inside the holder is shown from the side of the holder having a mask (left image); the same cuvette taken out of holder having a micro stir bar on its bottom (right image).

Materials and Reagents

  1. 15 ml screw cap glass vials (Kesell, catalog number: BLP025, package of 8)
  2. 200 µl gel pipette tips (Fisher Scientific, BasixTM, catalog number: 13-611-116)
  3. Thick black construction paper
  4. Coumarin 540A dye (Exciton, catalog number: 05450)
  5. LDS 798 dye (Exciton, catalog number: 07980)
  6. Rhodamine 590 dye (Exciton, catalog number: 05901)
  7. Fluorescein sodium salt, analytical standard (Sigma-Aldrich, catalog number: 30181-100MG)
  8. Chloroform, CHCl3 (Fisher Scientific, HPLC grade, catalog number: C607-1)
  9. Deuterated chloroform, CDCl3 (Sigma-Aldrich, catalog number: 151858-10x0.75ML)
  10. Dimethyl sulfoxide, C2H6OS (Fisher Chemical, Certified ACS grade, catalog number: 151874-10x0.75)
  11. Methanol, CH3OH (Fisher Chemical, HPLC grade, catalog number: A452-4)
  12. LDS 798 dye solution in CHCl3:CDCl3 (1:2) mixture (see Recipes)
  13. Coumarin 540A dye solution in DMSO (see Recipes)
  14. Rhodamine 590 dye solution in methanol (see Recipes)

Equipment

  1. Optical Table: Sealed Hole Table Top with Tuned Damping (Newport, model: RS 2000TM), set on High Performance Laminar Flow Isolators (4 pieces) (Newport, model: S 2000)
  2. Femtosecond pulsed laser (InSight DeepSee Dual, Newport Spectra-Physics)
  3. Photon Counting Spectrofluorimeter (ISS, model: PC1TM)
  4. Silver coated mirrors (Newport, model: New Focus 5103 (5 pcs.))
  5. Stainless steel optical posts (Thorlabs, catalog numbers: TR3 (4pcs.) and TR4 (7 pcs.))
  6. Standard ½” diameter, 3” long postholders (Thorlabs, catalog number: PH3 (11 pcs.))
  7. Standard ½” diameter 1” long postholder (Thorlabs, catalog number: PH1 (1pc.))
  8. Post holder bases (Thorlabs, catalog numbers: BA1 (11 pcs.) and BA1S (1pc.))
  9. Bolts and nuts. ¼”–20 Hardware Kit (Thorlabs, catalog number: HW-KIT2)
  10. New Focus mirror mount (Newport, catalog number: 9773 (5 pcs.))
  11. Flipper Optic Mount (Newport, model: New Focus 9891)
  12. Mounted achromatic half wave plate (690-1,200 nm) (Thorlabs, catalog number: AHWP10M-980)
  13. Rotation Mount for Ø1" Optics (Thorlabs, catalog number: RSP1 (2 pcs.))
  14. Unmounted Glan-Laser Polarizer, Ø10 mm CA, Uncoated (Thorlabs, catalog number: GL10P)
  15. SM1 Lens Tube Mount for 8 mm and 10 mm Mounted Polarizing Prisms (Thorlabs, catalog number: SM1PM10)
  16. Mounted Continuously Variable ND Filter, Ø50 mm, OD: 0.04-4.0, ARC: 650-1,050 nm (Thorlabs, catalog number: MDC-50C-4M-B)
  17. Broadband Energy/Power Meter (Melles-Griot, Model 13PEM001)
  18. 25 mm Dia. x 45 mm FL, NIR II Coated, Achromatic Lens (Edmund Optics, model: ACH-NIR 25 x 45 NIR-II, Stock: 45-802)
  19. 22 mm-to-25 mm inner diameter lens tube adaptors, ISS Accessory, 2 pieces
  20. ND New Focus reflective filters (Newport, catalog numbers: 5240 (OD 1), 5235 (OD 0.5), and 5233 (OD 0.3))
  21. 4-slot filter wheel with a stepper motor, ISS Accessory
  22. Short pass filters, 633/SP (Semrock, catalog number: BSP01-633R-25), 680/SP (Semrock, catalog number: FF01-680/SP-25), 694/SP (Semrock, catalog number: FF02-694/SP-25), 745/SP (Semrock, catalog number: FF01-745/SP-25), 770/SP (Semrock, catalog number: FF01-770/SP-25) 
  23. Long pass filter 645/LP (Thorlabs, catalog number: FGL 645) 
  24. RS 232 Serial cable (15 ft)
  25. Micro, square, stopper top special optical glass cuvettes, 3 x 3 mm (Starna Cells, catalog number: 23-3.45-SOG-3, quantity 6)
  26. 3-mm SOG cuvette adaptor holder (Starna Cells, catalog number: FCA3, quantity 4)
  27. 1 cm standard glass fluorometer rectangular cells (Starna Cells, catalog number: 23-G-10, quantity 4)
  28. Micro spinbar magnetic stir bar, 5 x 1 mm (Thomas Scientific, catalog number: 1207Q08, quantity 6)
  29. UV/Vis spectrophotometer (PerkinElmer, LAMBDA 950)
  30. Luminescence Spectrometer (PerkinElmer, LS55)
  31. 200 µl pipette (Gilson, model: Pipetman Classic P200, catalog number: F123601)

Software

  1. OriginPro 2017 (OriginLab, https://www.originlab.com)
  2. Vinci 3 (ISS, http://www.iss.com/)
  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

Procedure

  1. 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).


      Figure 2. Entrance aperture of the PC1 ISS spectrofluorimeter after the excitation lamp source was removed. A flip mirror is used for steering the laser beam into a powermeter head. Red dashed arrow shows the direction of laser beam propagation here and in Figures 3-7.


      Figure 3. Removing mirror holder with two mirrors from excitation monochromator compartment


      Figure 4. Removing tube holder with collimating lens (placed right after excitation monochromator) and setting an ISS filter wheel with four slots in place of it


      Figure 5. Changes in the sample compartment. A focusing lens is removed from the tube lens holder pointing toward the sample. 3-mm cuvette adaptors are inserted in the Sample and Reference cuvette holders. In the Left Emission Channel, an additional adaptor tube holder (25-mm) with a short-pass filter (770/SP) is slide onto a 22-mm tube lens holder.


      Figure 6. Removing optional beam splitter


      Figure 7. Removing optional excitation filter

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


      Figure 8. Electrically disconnecting Left Emission Wavelength filter wheel from its stepper motor and connecting an additional filter wheel in excitation path with the stepper motor of the Left Emission Wavelength filter wheel

    4. In a spare motorized filter wheel with 4 slots (ISS accessories) insert New Focus ND reflective filters with OD = 1, OD = 0.5, and OD = 0.3 in the slots # 1, 2, and 3, respectively. Leave the slot # 4 empty.
    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. For that, go to Experiment in Vinci software, then click on Single Point, and in the drug menu select Intensity. Right-click on the block called Fixed Parameter, click on Remove tab, and select Left Emission Wavelength box, then click OK. Right click on the block called Variable Parameters, click on Add tab and select Left Emission Wavelength function then click OK. In the Variable Parameters, Left Emission Wavelength box, click on the Edit List and call filter #1–0.1T, #2–0.3T, #3–0.5T, and #4–empty. In the Time Base window, select 10 s, and in Number of Iterations, select 1. Save this file as “Power Dependence”.


      Figure 9. Upper left part of the screenshot showing a Vinci 3 template file for the power dependence measurement in the Sample position in both left and right emission channels

    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. For that, go to Experiment in Vinci software, then click on Slow Kinetics, and in the drug menu select Intensity. Right-click on the block called Variable Parameters and add a Sample function. In the Time Base window, select 1.0 s, and in Time window in Variable parameters select 0 s for Start, 6,500 s for Stop, and 6.00 for Step. Select 200 min for ‘Measure Dark every __ minutes’. In the block Fixed Parameters under Left Emission Wavelength select empty in the Value box. Save the file as “Spectral Scan”.


      Figure 10. Upper left part of the screenshot showing a Vinci 3 template file for the spectral scan of two samples at once (set in the Sample and Reference positions)

    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).


      Figure 11. Placing a 45-mm achromatic lens into an excitation optical pathway inside the sample compartment. 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.

    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).


      Figure 12. Front view of the custom-made LabView program to scan the laser from the initial to the final wavelength with a particular step and dwell time

    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.
    39. Repeat Step A38 for a set of wavelengths (680, 690, 710, 730, 780, 800, 810, 840, 900, 940, 960, 1,000, 1,050, 1,060, 1,100, 1,200, 1,300 nm) in the laser tuning range.
    40. Summarize the data in the Origin Workbook, see Figure 13.


      Figure 13. Origin workbook containing a look up calibration table for the transmission properties of ND filters in the filter wheel set in the excitation path at several laser wavelengths

    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.
    44. In the Left Emission Filter Wheel with 4 slots (disconnected from its electric jack) insert the Semrock 633/SP, 694/SP, and 680/SP filters in slots 1, 2, and 3, respectively. Leave slot 4 empty.
    45. Dismount two adaptor holders for 3 x 3 mm cuvettes (unscrew 4 miniature screws holding one side of the holder) and insert a rectangular band of black paper (0.9 x 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.

  2. Measuring two-photon excitation spectral shapes using Left Emission Channel
    1. Preparation of samples for two-photon spectral measurements.
      1. 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 x 3 mm optical cuvettes.
      2. 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 x 3 mm cuvettes placed in 3-mm adaptor holder.
      3. 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.
      1. Turn on the laser in the full power mode.
      2. Start the laser control LabView program (Supplemental file 3 LaserTestVI3.vi) from a PC computer.
      3. Turn on the PC1 spectrofluorimeter.
      4. Insert the adaptor holders for 3 x 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.
      5. Set the Sample position to measurement.
      6. In the Instrument Control start stirring both Sample and Reference.
      7. Insert the 3 x 3 mm cuvette with Coumarin 540A solution into the sample holder of PC1.
      8. Turn manually the left emission wavelength filter wheel to position 1 (corresponding to 633/SP filter).
      9. Slide the left emission polarizer in the emission path and set it to Magic Angle position (with Instrument Control function).
      10. Set the laser wavelength to 800 nm.
      11. Adjust the laser power with the continuous ND filter to ~20 mW.
      12. Send the laser beam to PC1 by flipping the flip mirror down.
      13. Make sure that the excitation polarizer in PC1 is moved out of the excitation optical path.
      14. 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).
      15. Open the excitation shutter with Instrument Control.
      16. 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.
      17. 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.
      1. Put the cuvette with the sample #1 solution into the Sample position instead of Coumarin 540A solution.
      2. Cover the sample compartment with the lid.
      3. Measure the maximum signal at several wavelengths across the 2PA spectrum, e.g., 700, 800, 900, 1,000, 1,100, 1,200 nm (using left PMT).
      4. Repeat the same for the sample #2.
      5. Adjust the laser power with the continuous ND filter such that the maximum signal across the 2PA spectrum of both samples amounts ~1 x 106 counts. Typically, this power will be in the range 10-100 mW, depending on sample concentration and two-photon brightness.
    4. Checking the power dependence of fluorescence at different wavelengths for 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.
      1. Using the Power Dependence file, check that the power dependence of sample #1 fluorescence is quadratic (power exponent between 1.95 and 2.05) at wavelengths in the short wavelength region of spectrum, where the one-photon excitation can contribute. Find the shortest wavelength by using discrete steps of 20 nm (i.e., 680, 700, 720 nm, etc.), at which the signal is quadratic.
      2. Repeat the Step B4a for sample #2.
    5. Scanning 2PA spectra of samples #1 and #2. Sample #1–in Sample position and sample #2–in Reference position. Filters 770/SP and 633/SP (or 680/SP, 694/SP or empty) in the Left Emission Channel.
      1. Put cuvettes with sample #1 in the Sample position and with sample #2 in the Reference position of the sample compartment turret.
      2. 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.
      3. Calculate the stop wavelength (λstop) 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.
      4. Set the step equal to 4 nm and the time per step equal to 42 s.
      5. Open the Spectral Scan file, make the notes about the experiment (samples names and positions, filters used, etc.) and start the scan.
      6. Start the laser scan.
      7. Once the scan is finished, remove the cuvettes with the samples from adaptor holders.
    6. Scanning 2PA spectra of Coumarin 540A solution. One cuvette with Coumarin 540A solution–in Sample position and another cuvette with Coumarin 540A solution–in Reference position. Filters 770/SP and 633/SP in the Left Emission Channel.
      1. Put two cuvettes with the same solution of Coumarin 540A in DMSO into the Sample and Reference positions of the turret.
      2. Repeat Steps B5b-B5g with the stop wavelength equal to 980 nm in Step B5c and all other settings kept the same in Steps B5b-B5f and making the notes about the reference in Step B5e.
    7. Scanning 2PA spectra of LDS 798 solution. One cuvette with LDS 798 solution–in Sample position and another cuvette with LDS 798 solution–in Reference position. Filter 770/SP in the Left Emission Channel.
      1. Put two cuvettes with the same solution of LDS 798 in chloroform/deuterated chloroform, 1:2, into the Sample and Reference positions of the turret.
      2. Turn manually the left emission wavelength filter wheel to position 4 (empty).
      3. 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.
      1. 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:
        1. 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'.
        2. Name this Import Wizard filter "2PA_sample_and_reference".


          Figure 14. An example of what the Origin workbook will look like after creating the "2PA_sample_and_reference" Import Wizard filter

      2. 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:".
      3. 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.


        Figure 15. An example of what the final workbook should look like after opening a file with the "2PA_sample_and_reference_autocalculation" Import Wizard filter


        Figure 16. An example of the graph showing what the raw data looks like after the script auto-deletes the points when the laser is stabilizing. 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.

      4. 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.
      5. Merge the short range correction functions separately measured with the reference dyes Coumarin 540A in DMSO (λstart-980 nm) and LDS798 in 2:1 CDCl3:CHCl3 (940 nm-λstop) into full range correction function (λstartstop). (Correction function data from the 'Sample holder' and the 'Reference holder' should be treated separately.)
        1. 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.
        2. 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.
        3. Create the merged correction function. For the λstart-936 nm range, paste the normalized correction function measured with Coumarin 540A in DMSO. For the 940 nm-980 nm range, paste the average correction function from the previous step. For the 980 nm-λstop range, paste the normalized correction function measured with LDS798 in 2:1 CDCl3:CHCl3.
      6. 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.

  3. 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 x 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 x 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).
    8. Record this power. 
    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 F2, Sex, λreg)/F2, Rex, λreg) 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: ODS (530) and ODR (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 F1, S (600) and F1, R (600) and calculate the ratio:



    17. Using eq. (2), all the ratio values obtained in Steps C12-C16, and Rhodamine 590 cross section σ2(1060) = 10 GM, calculate the 2PA cross section of the sample.

Data analysis

  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 F2 numbers are estimated from linear fits of the fluorescence intensity vs. power squared plots (usually 1% for both F2,S and F2,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 σ2 of the sample measured at one wavelength (1,060 nm) amounts to 15%. This translates to the error of absolute σ2 value, determined at different wavelengths of ~18% (Molina et al., 2019).

Notes

  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 540A in DMSO closely match each other in the region of 745-1,000 nm, if the 1PA is plotted versus one-photon wavelength doubled (de Reguardati et al., 2016; Figure 17). In the shorter wavelength range, 680-745 nm the relative 2PA values presented in (de Reguardati et al., 2016) systematically exceed the 1PA values. We have measured independently the 2PA spectrum of Coumarin 540A in DMSO, using Prodan in DMSO as a reference standard (de Reguardati et al., 2016) in the region of 680-800 nm, and obtained much better correlation between the 2PA and 1PA spectral shapes of Coumarin 540A, cf. red and black lines in Figure 17.


    Figure 17. Spectral shapes on 1PA and 2PA spectra of Coumarin 540A in DMSO. The 1PA spectrum measured in this work, black line, and 2PA spectrum (de Reguardati et al., 2016), blue line, are normalized at their peak values. The short wavelength part of the 2PA spectrum (680-800 nm), red line, was measured in this work, using Prodan in DMSO (de Reguardati et al., 2016) as a standard.

      To double check the spectral shape of Coumarin 540A (A2PA,R(λ) function) in a broad range from 680 to 1,000 nm, we measured the 2PA spectrum of Rhodamine 590 (= Rhodamine 6G) in methanol versus Coumarin 540A in DMSO and compared it to the known literature data (Figure 18). The data from Hermann and Ducuing (1972), Makarov et al. (2008), de Reguardati et al. (2016) were presented as measured, in absolute cross section values (see Footnote a and b to the Table 5 below for the Hermann and Ducuing data). The data from Albota et al. (1998), Rodriguez et al. (2009), Rodriguez and Chiesa (2011), Rodriguez and Chiesa (2012) were scaled to a value of 74 GM at 820 nm that matches the measurements of Makarov et al. (2008), de Reguardati et al. (2016). The spectrum from Wakebe and Van Keuren (1999), originally presented as relative spectrum for Rhodamine 6G in ethanol, is shifted 17 nm to the red (in 2PA wavelengths) and scaled to a value of 34 GM at 844, matching the measurements of Makarov et al. (2008), de Reguardati et al. (2016). In almost the whole region of Coumarin 540A absorption, from 690 to 960 nm, there is a good correlation between the spectral shapes measured in Albota et al. (1998) and de Reguardati et al. (2016), therefore we conclude that these spectra present the best approximation to the 2PA spectrum of Rhodamine 6G. This conclusion is supported by good matches of other data to these spectra in different spectral regions or at separate wavelengths: Hermann and Ducuing data at 694 and 1,060 nm; Wakebe and Van Keuren data–in the 806-1,106 nm region; Makarov et al. (2008) data–in the 720-900 and 1,060-1,100 nm regions; Rodriguez et al. (2007) data–in the 720-860 nm region.

    Table 1. Correct 2PA spectral shape of Coumarin 540A in DMSO, with 680-760 nm data corrected by Prodan in DMSO (de Reguardati et al., 2016)

    Wavelength

    Relative value

    Wavelength

    Relative value

    Wavelength

    Relative value

    Wavelength

    Relative value

    680

    0.135

    760

    0.376

    840

    0.990

    920

    0.523

    684

    0.117

    764

    0.408

    844

    0.996

    924

    0.478

    688

    0.113

    768

    0.445

    848

    0.999

    928

    0.434

    692

    0.112

    772

    0.483

    852

    1.00

    932

    0.390

    696

    0.112

    776

    0.522

    856

    0.998

    936

    0.348

    700

    0.115

    780

    0.561

    860

    0.994

    940

    0.307

    704

    0.118

    784

    0.600

    864

    0.985

    944

    0.268

    708

    0.124

    788

    0.639

    868

    0.973

    948

    0.232

    712

    0.135

    792

    0.678

    872

    0.955

    952

    0.199

    716

    0.146

    796

    0.715

    876

    0.934

    956

    0.169

    720

    0.160

    800

    0.751

    880

    0.908

    960

    0.143

    724

    0.176

    804

    0.786

    884

    0.881

    964

    0.120

    728

    0.194

    808

    0.819

    888

    0.850

    968

    0.100

    732

    0.215

    812

    0.851

    892

    0.816

    972

    0.0832

    736

    0.234

    816

    0.881

    896

    0.780

    976

    0.0683

    740

    0.256

    820

    0.908

    900

    0.741

    980

    0.0556

    744

    0.279

    824

    0.931

    904

    0.700

    984

    0.0448

    748

    0.303

    828

    0.952

    908

    0.657

    988

    0.0355

    752

    0.325

    832

    0.968

    912

    0.613

    992

    0.0274

    756

    0.350

    836

    0.980

    916

    0.568

    996

    0.0203


      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 A2PA,R(λ) function, the Rhodamine 6G spectrum (dark green line with triangles in Figure 18) matches quite well the literature spectra of Albota et al. (1998) and de Reguardati et al. (2016) in the region of 680-980 nm. Therefore we conclude that the corrected 2PA spectrum of Coumarin 540A in DMSO is a good approximation for the A2PA,R(λ) function when the corrected spectrum of Coumarin 540A in DMSO is used as a reference in the range of 680-980 nm.

    Table 2. Correct 2PA spectral shape of LDS 798 in chloroform from 936-1,300 nm; values are from fitting a Gaussian to the LDS 798 z-scan data from Makarov et al., 2011

    Wavelength

    Relative value

    Wavelength

    Relative value

    Wavelength

    Relative value

    Wavelength

    Relative value

    936

    0.116

    1028

    0.410

    1120

    0.847

    1212

    0.978

    940

    0.124

    1032

    0.428

    1124

    0.864

    1216

    0.970

    944

    0.132

    1036

    0.446

    1128

    0.879

    1220

    0.961

    948

    0.141

    1040

    0.465

    1132

    0.894

    1224

    0.950

    952

    0.150

    1044

    0.484

    1136

    0.907

    1228

    0.937

    956

    0.159

    1048

    0.503

    1140

    0.920

    1232

    0.923

    960

    0.169

    1052

    0.522

    1144

    0.933

    1236

    0.908

    964

    0.180

    1056

    0.542

    1148

    0.944

    1240

    0.892

    968

    0.191

    1060

    0.561

    1152

    0.954

    1244

    0.874

    972

    0.203

    1064

    0.581

    1156

    0.963

    1248

    0.855

    976

    0.215

    1068

    0.601

    1160

    0.972

    1252

    0.835

    980

    0.227

    1072

    0.621

    1164

    0.979

    1256

    0.813

    984

    0.240

    1076

    0.641

    1168

    0.985

    1260

    0.791

    988

    0.253

    1080

    0.661

    1172

    0.991

    1264

    0.767

    992

    0.267

    1084

    0.681

    1176

    0.994

    1268

    0.742

    996

    0.282

    1088

    0.701

    1180

    0.997

    1272

    0.717

    1000

    0.296

    1092

    0.720

    1184

    0.999

    1276

    0.692

    1004

    0.311

    1096

    0.740

    1188

    1.00

    1280

    0.665

    1008

    0.327

    1100

    0.759

    1192

    0.999

    1284

    0.638

    1012

    0.343

    1104

    0.777

    1196

    0.998

    1288

    0.611

    1016

    0.359

    1108

    0.795

    1200

    0.995

    1292

    0.584

    1020

    0.376

    1112

    0.813

    1204

    0.991

    1296

    0.556

    1024

    0.393

    1116

    0.831

    1208

    0.985

    1300

    0.529




    Figure 18. Two-photon absorption spectra (logarithmic scale) of Rhodamine 6G in merthanol or ethanol. The data of Hermann and Ducuing (1972)–black stars, Makarov et al. (2008)–blue circles, de Reguardati et al. (2016)–light blue line, are presented in absolute σ2 values (see footnotes a and b to Tables 3-5 after Table 5). The data of Albota et al. (1998)–red circles, Rodriguez et al. (2009)–ping right triangles; Rodriguez and Chiesa (2011)–pink left triangles, Rodriguez and Chiesa (2012)–pink up triangles are scaled (see text), and Wakebe and Van Keuren (1999)–orange circles are scaled and shifted (see text). The (scaled) spectrum obtained here using Coumarin 540A in DMSO 2PA spectrum (de Reguardati et al., 2016) as a standard, is shown by open diamonds. The spectrum measured in this work (scaled) relatively to Coumarin 540A in DMSO in 740-1,000 nm region and relatively to Prodan in DMSO in 680-740 nm region is shown by dark green down triangles connected with continuous line. The spectrum obtained in this work relatively to LDS 798 in chloroform/deuterated chloroform, is shown by purple diamonds connected with continuous line.

  3. In Makarov et al. (2011), the 2PA spectra of the LDS 798 dye in chloroform were obtained using fluorescence (relatively to Styryl 9M [Makarov et al., 2008]) and z-scan (absolute measurement) techniques. The shapes of the one-photon and two-photon absorption spectra are very broad, structureless, and closely match each other in the region of 900-1,300 nm, if the 1PA is plotted versus one-photon wavelength doubled (Makarov et al., 2011; Figure 19).


    Figure 19. Spectral shapes of 1PA (purple line) (measured here) and 2PA spectra of LDS 798 in chloroform, green open squares–using fluorescence technique relatively to Styryl 9M, and black squares–using z-scan and absolute method (Makarov et al., 2011). All spectra are normalized at their peak values. Black dashed line shows a Gaussian fit to the z-scan data in the region from 900 to 1,300 nm that is used as an A2PA,R(λ) function here. This figure is adapted from Makarov et al. (2011).

      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 A2PA,R(λ) function. To interpolate the A2PA,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 CHCl3/CDCl3 (1:2) in the 940-1,140 mn region, see Figure 18. The data (purple line) match well the spectra of Albota et al. (1998); Wakebe and Van Keuren (1999), and de Reguardati et al. (2016) and overlaps well with the spectrum measured using Coumarin 540A (dark green line) in the region 940-980 nm. This corroborates our choice of LDS 798 as a reference standard for the spectral shape in the 940-1,300 nm region.

  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 re-emission 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 σ2 = 10 ± 1 GM.

    Table 3. Literature data for the two-photon cross section of Rhodamine 6G in methanol or ethanol in the 1,054-1,064 nm region. Most of the values (column no. 7) group around 10 GM. The average of all 11 measurements provides σ2 = 12 ± 2 GM (average ± SE of mean). The underlined values are obtained either with mode-locked lasers or using the methods where the uncertainty in the second-order time correlation function of laser intensity g(2) was removed. Out of these six measurements, the lowest (4.2 GM) (Kaatz and Shelton, 1999), was obtained by using Hyper Rayleigh scattering technique to calibrate the laser properties, including g(2). The authors found that this method gave underestimated values of σ2, compared to those obtained with standard calibration against one-photon fluorescence. Therefore, the suggested value of σ2 is calculated by excluding this measurement and averaging the numbers selected in italic in column 7.

    Wavelength

    (nm)

    Solvent

    Conc.

    (M)

    Pulse

    Original

    σ2 (GM)

    Correction 1

    σ2 (GM)

    Correction 2

    σ2 (GM)

    (final)

    Ref.

    1060

    C2H5OH

    n/a

    ns

    12.9 ± 6

    25.8 ± 12a

    10.1 ± 4.7b

    Hermann and Ducuing, 1972

    1060

    C2H5OH

    10-3

    60 ns

    5.5

    11a

    5.5c

    Bradley et al., 1972

    1060

    C2H5OH

    10-4

    30 ns

    26

    52a

    26c

    Vsevolodov et al., 1973

    1060

    C2H5OH

    10-3

    ps

    3.6

    7.2a

    7.2

    Bradley et al., 1972

    1060

    C2H5OH

    10-6

    110 ns

    11 ± 5

    22 ± 10a

    11 ± 5c

    Li and She, 1982

    1064

    C2H5OH

    10-2

    cw

    12 ± 2

    48 ± 8d

    24 ± 4c

    Catalano and Cingolani, 1982

    1054

    CH3OH

    0.2

    5 ps

    10 ± 1

    -

    10 ± 1

    Penzkofer and Leupacher, 1987

    1064

    CH3OD

    2.2 10-6

    125 ns

    4.2 ± 0.8

    -

    4.2 ± 0.8

    Kaatz and Shelton, 1999

    1064

    C2H5OH

    10-4

    10 ns

    15 ± 1

    30 ± 2a

    15 ± 1c

    Rodriguez, et al., 2007

    1060

    CH3OH

    8 10-6

    100 fs

    10 ± 1

    -

    10 ± 2

    Makarov et al., 2008

    1060

    CH3OH

    n/a

    100 fs

    11 ± 1 

    -

    11 ± 1  

    de Reguardati et al., 2016

    Suggested conditions and cross section value

    1060

    CH3OH

    10-5

    100 fs

     

     

    10 ± 1

     


    Table 4. Other σ2 values of Rhodamine 6G at selected wavelengths. The σ2 values showing satisfactory matching between two or more measurements are italicized in column 7. Recommended values of σ2 are calculated as average of these numbers for each wavelength.

    Wavelength

    (nm)

    Solvent

    Conc.

    (M)

    Pulse

    Original

    σ2 (GM)

    Correction 1

    σ2 (GM)

    Correction 2

    σ2 (GM)

    (final)

    Ref.

    694

    C2H5OH

    n/a

    ns

    355 ± 170

    710 ± 355a

    277 ± 138b

    Hermann and Ducuing, 1972

    694

    CH3OH

    0.04

    30 ps

    180 ± 20

    -

    180 ± 20

    Sperber and Penzkofer, 1986

    690

    700

    CH3OH

    1.1 10-4

    100 fs

    136

    176

    -

    156e

    Albota et al., 1999

    694

    CH3OH

    8 10-6

    100 fs

    112 ± 22

    -

    112 ± 22

    Makarov et al., 2008

    692

    CH3OH

    n/a

    100 fs

    202 ± 16

    -

    202 ± 16

    de Reguardati et al., 2016

    Suggested conditions and cross section value

    694

    CH3OH

    10-5

    100 fs

     

     

    180 ± 23

     

     

    730

    CH3OH

    8 10-6

    100 fs

    50 ± 10

    -

    50 ± 10

    Makarov et al., 2008

    730

    CH3OH

    n/a

    100 fs

    50 ± 4

    -

    50 ± 4

    de Reguardati et al., 2016

    Suggested conditions and cross section value

    730

    CH3OH

    10-5

    100 fs

     

     

    50

     

     

    812

    CH3OH

    8 10-6

    100 fs

    71 ± 14

    -

    71 ± 14

    Makarov et al., 2008

    812

    CH3OH

    n/a

    100 fs

    79 ± 6

    -

    79 ± 6

    de Reguardati et al., 2016

    Suggested conditions and cross section value

    812

    CH3OH

    10-5

    100 fs

     

     

    75

     

     

    940

    CH3OH

    1.1 10-4

    100 fs

    7.7

    -

    7.7

    Albota et al., 1999

    940

    CH3OH

    8 10-6

    100 fs

    8.3 ± 1.7

    -

    8.3 ± 1.7

    Makarov et al., 2008

    940

    CH3OH

    n/a

    100 fs

    11.1 ± 0.9

    -

    11.1 ± 0.9

    de Reguardati et al., 2016

    Suggested conditions and cross section value

    940

    CH3OH

    10-5

    100 fs

     

     

    9.0 ± 1.0

     

     

    960

    CH3OH

    1.1 10-4

    100 fs

    12.7

    -

    12.7

    Albota et al., 1999

    960

    CH3OH

    8 10-6

    100 fs

    10.9 ± 2.2

    -

    10.9 ± 2.2

    Makarov et al., 2008

    960

    CH3OH

    n/a

    100 fs

    17 ± 1.4

    -

    17.0 ± 1.4

    de Reguardati et al., 2016

    Suggested conditions and cross section value

    960

    CH3OH

    10-5

    100 fs

     

     

    13 ± 2

     


  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. Table 5 summarizes the literature data for Fluorescein with recommended wavelengths and σ2 values.

    Table 5. Some suggested σ2 values of Fluorescein in alkaline water solution at different wavelengths. The σ2 values showing satisfactory matching between two or more measurements are italicized in column 7. Recommended values of σ2 are calculated as average of these numbers for each wavelength.

    Wavelength

    (nm)

    Solvent

    Conc.

    (M)

    Pulse

    Original

    σ2 (GM)

    Correction 1

    σ2 (GM)

    Correction 2

    σ2 (GM)

    (final)

    Ref.

    710

    H2O pH11

    8 10-5

    100 fs

    17.3 ± 4.4

     

    17.3 ± 4.4

    Xu and Webb, 1996

    710

    H2O pH11

    3 10-6

    100 fs

    16.5 ± 3.3

    -

    16.5 ± 3.3

    Makarov et al., 2008

    Suggested conditions and cross section value

    710

    H2O pH11

    10-5

    100 fs

     

     

    16.9 

     

     

    730

    H2O pH11

    8 10-5

    100 fs

    25.0 ± 6.3

     

    25.0 ± 6.3

    Xu and Webb, 1996

    730

    H2O pH11

    3 10-6

    100 fs

    24.2 ± 4.8

    -

    24.2 ± 4.8

    Makarov et al., 2008

    Suggested conditions and cross section value

    730

    H2O pH11

    10-5

    100 fs

     

     

    24.6 

     

     

    782

    H2O pH11

    n/a

    100 fs

    46 ± 10

     

    46 ± 10

    Xu et al., 1995

    782

    H2O pH11

    8 10-5

    cw (single mode)

    38 ± 10

     

    38.0 ± 10

    Xu and Webb, 1996

    782

    H2O pH11

    3 10-6

    100 fs

    46 ± 9

    -

    46 ± 9

    Makarov et al., 2008

    Suggested conditions and cross section value

    782

    H2O pH11

    10-5

    100 fs

     

     

    43 ± 3

     

     

    802

    H2O pH11

    8 10-5

    cw (single mode)

    36 ± 10

     

    36 ± 10

    Xu and Webb, 1996

    802

    H2O pH11

    3 10-6

    100 fs

    38 ± 8

    -

    38 ± 8

    Makarov et al., 2008

    Suggested conditions and cross section value

    802

    H2O pH11

    10-5

    100 fs

     

     

    37

     

     

    842

    H2O pH11

    8 10-5

    100 fs

    12 ± 3

     

    12 ± 3

    Xu and Webb, 1996

    842

    H2O pH11

    3 10-6

    100 fs

    13 ± 3

    -

    13 ± 3

    Makarov et al., 2008

    Suggested conditions and cross section value

    842

    H2O pH11

    10-5

    100 fs

     

     

    12.5

     

     

    900

    H2O pH11

    8 10-5

    100 fs

    16 ± 4

     

    16 ± 4

    Xu and Webb, 1996

    900

    H2O pH11

    3 10-6

    100 fs

    15 ± 3

    -

    15 ± 3

    Makarov et al., 2008

    Suggested conditions and cross section value

    900

    H2O pH11

    10-5

    100 fs

     

     

    15.5

     

     

    1000

    H2O pH11

    3 10-6

    100 fs

    3.1 ± 0.6

    -

    3.1 ± 0.6

    Makarov et al., 2008

    1000

    H2O pH11

    n/a

    100 fs

    3.3 ± 0.3

    -

    3.3 ± 0.3

    de Reguardati et al., 2016

    Suggested conditions and cross section value

    1000

    H2O pH11

    10-5

    100 fs

     

     

    3.2

     

     

    1060

    C2H5OH

    10-5

    60 ns

    0.18

    0.36a

    0.18c

    Bradley et al., 1972

    1060

    C2H5OH

     

    ps

    0.075

    0.15a

    0.15

    Bradley et al., 1972

    1050

    H2O pH11

    8 10-5

    100 fs

    0.23 ± 0.07

     

    0.23 ± 0.07

    Xu and Webb, 1996

    1050

    H2O pH11

    3 10-6

    100 fs

    0.17 ± 0.03

    -

    0.17 ± 0.03

    Makarov et al., 2008

    Suggested conditions and cross section value

    1050

    H2O pH11

    10-5

    100 fs

     

     

    0.2

     


    Footnotes for Tables 3-5:
    aMultiplied by factor 2 to conform with the more recent definition of σ2 (two photons are needed to excite one molecule), cf. Xu and Webb (1996); Kaatz and Shelton (1999); Makarov et al. (2008).
    bMultiplied by factor 0.39 to conform with the current reference value of quartz, d11 = 0.30 pm/V (versus 0.48 pm/V), see Kaatz and Shelton (1999).
    cRe-evaluated using the laser coherence function value g(2) = 2, see Weber (1971); Bradley et al. (1972); Kaatz and Shelton (1999). That factor was not included in original analysis.
    dMultiplied by factor 4 to conform with the more recent definition of σ2 (two photons are needed to excite one molecule), cf. Xu and Webb (1996); Kaatz and Shelton (1999); Makarov et al. (2008).
    eInterpolated between 690 and 700 nm measurements.

Recipes

  1. LDS 798 dye solution in CHCl3:CDCl3 (1:2) mixture
    1. Dissolve 0.1 mg of LDS 798 in 0.75 ml of CHCl3 in a 15 ml screw cap glass vial
    2. Add 1.5 ml of CDCl3. Stir overnight on magnetic stirrer. The optical density of the final solution at 594 nm (spectral maximum) in 3 mm cuvette should be OD ~1.2. This corresponds to a concentration of ~10-4 M
    3. It can be stored in closed vial in the dark at room temperature for at least 2 months
  2. Coumarin 540A dye solution in DMSO
    1. Dissolve 0.15 mg of Coumarin 540A in 3.3 ml of DMSO in a 15 ml screw cap glass vial
    2. The optical density of the final solution at 427 nm (spectral maximum) in 3 mm cuvette should be OD ~0.6. This corresponds to a concentration of ~10-4 M
    3. It can be stored in closed vial in the dark at room temperature for at least 2 months
  3. Rhodamine 590 dye solution in methanol
    1. Dissolve 0.05 mg of Rhodamine 6G in 10 ml of CH3OH in a 15 ml screw cap glass vial
    2. The optical density of the final solution at 528 nm (spectral maximum) in a 3 mm cuvette should be OD ~0.3. This corresponds to a concentration of ~10-5 M
    3. It can be stored in a closed vial in the dark at room temperature for at least 4 months

Acknowledgments

We thank Alexey Drobizhev for the custom LabView program. This work was supported by the NINDS grants U01 NS094246 and U24 NS109107 for MD, TEH, and RSM, and the Ruth L. Kirschtein National Research Service Award, number F31NS108593 for RSM.
  This protocol was derived from Molina, R.S., Qian, Y., Wu, J., Shen, Y., Campbell, R. E., Drobizhev, M. and Hughes, T. E. (2019) Understanding the fluorescence change in red genetically encoded calcium ion indicators. Biophys. J. 116: 1873-1886.

Competing interests

The authors declare no financial and non-financial competing interests.

References

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简介

[摘要] 两光子激光扫描显微镜(2PLSM)是用于组织内部深处的非侵入性成像的最新技术,具有高3D分辨率,最小的散焦光损伤和最小的自发荧光背景。为了在2PLSM中实现荧光探针的最佳应用,必须对以绝对截面表示的双光子吸收(2PA)光谱进行表征。以最佳波长进行激励将有可能降低激光功率,从而将光损伤降至最低。要获得2PA光谱和横截面,需要针对调谐范围内每个波长的激光特性的组合校正双光子激发的荧光信号,包括光束空间轮廓,脉冲持续时间和绝对功率。为了避免在绝对测量方法中进行日常繁琐的激光表征,通常使用基于独立表征的2PA参考标准的相对方法。通过仔细分析可用的文献数据,我们为2PA频谱形状和横截面测量选择了最可靠的标准。在这里,我们描述了一种使用这些参考标准物通过相对荧光法测量荧光蛋白和其他荧光团的2PA光谱形状和横截面的协议。我们的协议首先描述了如何构建光学系统,然后如何执行测量。在我们的协议中,我们使用在二甲亚砜中的香豆素540A和在氯仿中的LDS 798进行光谱形状测量,以覆盖680至1300 nm的范围,在甲醇中的若丹明590和在碱性水(pH 11)中的荧光素用于绝对的两个光子截面测量。
[背景技术两光子激光扫描显微镜(2PLSM)是一种先进的成像技术,用于神经科学,癌症生物学,免疫学以及其他生物科学领域(Denk 等,1990 ;Xu 等,1996; Zipfel 等, 2003;Helmchen 和Denk ,2005)。与基于单光子激发的荧光共聚焦显微镜相比,2PLSM提供了对组织的更深层询问,具有更少的离焦光斑和更少的自发荧光背景。对于2PLSM,约荧光探针的基于双光子激发在不同的激光波长的亮度信息的最佳应用(即,双光子吸收光谱和激发光谱)是必要的。该信息可帮助研究人员为特定应用选择合适的探针(例如,在近红外组织透明窗口内以最佳波长激发的最亮探针,或用于多色成像的探针组合)。有两种测量双光子吸收光谱和绝对截面值的方法(Rumi和Perry,2010),包括基于非线性透射(z扫描)的直接方法和诸如双光子激发的间接方法。荧光(2PEF)。在这里,我们选择2PEF方法是因为它更加灵敏,所需的荧光团浓度要低得多,并且2PLSM 中使用的所有探针本质上都是荧光的。与单光子(1P)激发相比,双光子(2P)激发效率在很大程度上取决于激光束的空间和(脉冲)时间分布。由于这些特征通常为波长在整个激光调谐范围波长而变化,这些变化小心的校正是必需的,用于提取纯的分子的双光子吸收(2PA)光谱形状和绝对横截面的值(σ 2 )。要获得这些属性,可以使用绝对方法或相对方法。在绝对测量方法中,必须在每个波长处表征激光属性,例如功率,脉冲持续时间和形状以及空间光束轮廓(即,强度对样品溶液中x,y和z坐标的依赖性)。然后,应该构造一个代表每个激光波长下这些参数的组合的函数,并将其应用于原始数据的校正。使用该绝对方法获得的光谱(徐和Webb,1996; Albota 等人,1998;马卡罗夫等人。,2008;马卡罗夫等人,2011;德Reguardati 等人。,2016)可以被认为是作为参考标准为在相关方法中的应用。

为了避免每天对激光特性进行繁琐的表征(绝对测量方法所必需),我们在协议中使用了一种基于先前表征的参考标准的相对方法。在该方法中,原料2PA光谱收集样品,˚F 2,S (λ ),以及用于参考,˚F 2,R (λ ),(指数S和R被分别用于样品和参考,这里和整个)在激发的相同条件下,然后将校正函数˚F ç (λ )使用参考的已知2PA光谱计算阿2PA,R (λ )和其原料光谱˚F 2,R (λ ):







然后将此校正函数应用于样品的原始光谱,以获得校正后的“真实”光谱(Makarov 等,2008),等式。(1):







类似的方法用于评估双光子横截面值。在这种情况下,样品的原始荧光信号,在特定的激发波长测量的λ EX 是通过使用在相同的激发和荧光注册条件测定的2PA剖面参考标准激光性能的组合进行校正。

假设˚F 2,S (λ EX ,λ REG )和˚F 2,R (λ EX ,λ REG )是记录在一窄小的双光子荧光信号周围登记波长,光谱范围λ REG ,具有相同的励磁条件样品和参考。为了计算2PA横截面,需要分别将这些信号归一化为荧光量子效率以及信号和参比物的浓度。让φ 小号(λ REG )和φ - [R (λ REG )是测量的差分的量子效率在同一波长具有相同的光谱带宽˚F 2个信号,但与单光子激发。在荧光收集中使用狭窄的光谱范围可避免对检测系统的光谱灵敏度进行校正。Ç 小号和Ç - [R 是相应的浓度,在双光子实验中使用,并使用比尔定律来计算:c ^ = OD 最大值/ ε 最大,其中OD 最大值为峰值光密度和ε 最大为峰值消光系数。然后读取样品的双光子截面(Makarov 等,2008),等式。(2):







其中σ 2,R (λ EX )是参考的横截面。

虽然积累了参考标准在过去几十年的文献资料开始收敛了一些人,显著偏差仍然存在于其他人。以前使用2PA表征的相对方法,但是经常缺少对测量细节的详细描述,标准的选择是任意的,从而导致不同实验室之间的结果差异很大。通过仔细分析可用的文献数据,我们为2PA频谱形状和横截面测量选择了最可靠的标准(请参见注释)。在我们的方案中,我们在DMSO中使用香豆素540A(de Reguardati 等人,2016),在氯仿中使用LDS 798(Makarov 等人,2011)进行光谱形状测量,以覆盖680至1300 nm的范围。我们在碱性水(pH 11)中使用荧光素(Xu 等人,1995 ; Xu和Webb,1996 ; Makarov 等人,2008 ; de Reguardati 等人,2016),在甲醇中使用若丹明6G(Hermann和Ducuing ,1972); 布拉德利等人,1972。; Penzkofer 和Leupacher ,1987 ; Albota 等人。,1998; 马卡罗夫等人。,2008 ; 德Reguardati 等人,2016)的绝对。σ 2点在选定的波长的测量。选择形状的标准品是因为它们报告的2PA和1PA光谱紧密重叠,这表明报告的2P光谱形状测量是可靠的。而且,光谱非常宽且无结构,因此由于有限的激光光谱形状和中心波长的较小偏移而引入了较少的潜在误差。该STANDAR 为DS σ 2 基于在所选波长两个至五个独立测量值之间的接近的匹配选择。使用该协议将使研究人员能够以标准化的方式并且具有高再现性来表征新型荧光探针和传感器的绝对2PA光谱。

我们的光学设置(图URE 1)由自动调谐(使用定制的LabView程序)飞秒激光(的的InSight DeepSee 加上光子计数,光谱物理)荧光分光光度计(PC1,ISS)。激光输出光束(100 - 120飞秒脉冲持续时间,680-1 ,300纳米的调谐范围,80MHz的重复率,0.6 - 1.3 W平均功率,水平偏振)的第一衰减到100-200 毫瓦和由垂直与偏振半波片和格兰激光偏振器的组合 然后将其过滤(使用6 45 长通滤光片)以除去所有残留的可见光,并用4个反射镜(M1 - M4)引导至分光荧光计的入射孔。我们在反射镜M3和M4之间使用连续可变的中性密度滤光轮,以将功率进一步衰减至实验所需的特定值。翻转镜(FM)用于将衰减的光束发送到功率计(Melles Griot),以在样品之前监测激光功率。

对分光荧光计光学器件进行了一些重新布置,以针对2PEF测量进行调整。从激发路径上卸下了激发灯源(未显示),激发单色仪(未显示),可选的分束器和两个准直透镜。添加了具有4个狭槽(ISS)的中性密度机动滤光轮,并将其连接到左荧光配准通道滤光轮的步进电机。将f = 45 mm的近红外消色差透镜(Edmund Optics)插入激发路径末端的光学管(ISS)中。该透镜将激光束聚焦到样品上,并置于3 x 3 mm的比色皿中(Starna Cells)。为避免溶剂吸收激光,从溶液的第一个0.7 mm层收集荧光(图1B)。为了最大程度地减少热透镜效应,在测量过程中搅拌所有染料溶液(图1B)。左荧光检测通道(没有单色)用于测量相对双光子激发光谱,因为在这种情况下,唯一的集成荧光信号需要,和右荧光通道(用单色仪)被用于测量双光子截面。



D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ 1.jpg

图1. 实验装置示意图。一。总体布局。飞秒InSight DeepSee (SpectraPhysics )激光束通过反射镜(M1-M4)系统导向光子计数光谱仪PC1(ISS)的入射孔。激光的自动波长到波长调谐是通过自定义LabView 程序控制的,荧光信号采集是通过ISS Vinci软件完成的。乙。顶部:3 x 3 mm比色皿与样品溶液的几何排列示意图(顶视图)。激光束(深红色)通过f = 45 mm透镜聚焦到溶液的正面部分。通过侧面收集荧光,该侧面通过遮罩指向左发射通道。面罩是固定在比色皿固定器内的黑纸窄矩形,仅允许从正面0.7毫米溶液中发出荧光。底部:从具有掩膜的支架侧面(左图)显示了在支架内部装有样品溶液的比色杯;从容器中取出的同一试管,其底部有一个微型搅拌棒(右图)。

关键字:双光子吸收, 双光子激光扫描显微镜, 双光子亮度, 双光子光谱, 截面, 荧光蛋白, 荧光基团, 双光子参考标准

材料和试剂


 


15 ml旋盖玻璃小瓶(Kesell ,目录号:BLP025,8包)
200 µl凝胶移液器吸头(Fisher Scientific,Basix TM ,目录号:13-611-116)
厚黑色建筑纸
香豆素540A染料(Exciton,目录号:05450)
LDS 798染料(Exciton,目录号:07980)
罗丹明590染料(激子,目录号:05901)
荧光素钠盐,一种分析标准品(Sigma-Aldrich,目录号:30181-100MG)
氯仿,CHCl 3 (Fisher Scientific,HPLC级,目录号:C607-1)
氘代氯仿CDCl 3 (Sigma-Aldrich,目录号:151858-10x0.75ML)
二甲基亚砜,C 2 H 6 OS(Fisher Chemical,ACS认证等级,目录号:151874-10x0.75)
甲醇,CH 3 OH(Fisher Chemical,HPLC级,目录号:A452-4)
LDS 798染料在CHCl 3 :CDCl 3 (1:2)混合物中的溶液(请参阅配方)
DMSO中的香豆素540A染料溶液(请参阅配方)
罗丹明590染料在甲醇中的溶液(请参见配方)
 


设备


 


光学平台:带可调阻尼的密封孔台式(Newport,型号:RS 2000 TM ),安装在高性能层流隔离器上(4件)(Newport,型号:S 2000)
飞秒脉冲激光(InSight DeepSee Dual,Newport Spectra-Physics )
光子计数荧光光谱仪(ISS,型号:PC1 TM )
镀银镜(纽波特,型号:New Focus 5103 (5片))
不锈钢光学接杆(Thorlabs,目录号:TR3(4个)和TR4(7个))
标准½ 英寸直径,3英寸长支柱(Thorlabs,目录号:PH3(11个))
标准½ 英寸直径1英寸长的接杆(Thorlabs,目录号:PH1(1个))
接杆支架底座(Thorlabs,目录号:BA1(11个)和BA1S(1个))
螺栓和螺母。¼” –20硬件套件(Thorlabs ,目录号:HW-KIT2 )
新焦点镜支架(新p ORT,目录号:9773 (5个。))
Flipper Optic Mount (新港,型号:New Focus 9891 )
安装的消色差半波片(690 - 1 ,200纳米)(Thorlabs公司,目录号:AHWP10M-980 )
Ø1英寸光学元件的旋转安装座(Thorlabs,目录号:RSP1(2个))
未安装的格兰激光偏振器,Ø10mm CA,未镀膜(Thorlabs,目录号:GL10P )
SM1透镜套管安装座,用于8毫米和10毫米已安装的偏振棱镜(Thorlabs,目录号:SM1PM10 )
安装无级变速ND过滤器,Ø50毫米,外径:0.04 - 4.0,ARC:650 - 1 ,050纳米(Thorlabs公司,目录号:MDC-50C-4M-B )
宽带能量/功率计(Melles -Griot ,型号13PEM001 )
直径25毫米 X45毫米FL,NIR II涂层,消色差透镜(埃德蒙光学,米Odel等:ACH-NIR 25x45 NIR-II,股票:45-802)
22毫米到25毫米内直径镜筒广告aptors,ISS配件,2块
ND New Focus反射滤镜(纽波特,目录号:5240(OD 1),5235(OD 0.5)和5233(OD 0.3))
带有步进马达的4槽滤光片轮,ISS附件
短通滤波器,633 / SP(Semrock ,目录号:BSP01-633R-25),680 / SP(Semrock ,目录号:FF01-680 / SP-25),694 / SP(Semrock ,目录号:FF02-694 / SP-25),745 / SP(Semrock ,目录号FF01-745 / SP-25 ),770 / SP(Semrock ,目录号FF01-770 / SP-25 )
长通滤波器645 / LP (Thorlabs,货号:FGL 645 )
RS 232串行电缆(15英尺)
微型,方形,塞子顶部专用光学玻璃比色杯,3 x 3毫米(Starna Cells ,目录号:23-3.45-SOG-3,数量6)
3毫米SOG比色皿适配器固定架(Starna Cells,目录号:FCA3,数量4)
1厘米标准GLAS 小号荧光计矩形单元(Starna 细胞,目录号:23-G-10 ,数量4)
微型旋转式磁力搅拌棒,5 x 1 毫米(Tho m as Scientific,目录号:1207Q08,数量6)
紫外/可见分光光度计(PerkinElmer,LAMBDA 950 )
发光光谱仪(PerkinElmer,LS55 )
200 μ 升移液管(吉尔森,米Odel等:移液器经典P200,目录号:F123601 )
 


软件


 


OriginPro 2017 (OriginLab ,https : //www.originlab.com )
Vinci 3 (ISS,http ://www.iss.com/ )
LabView2018(National Instruments,https: //www.ni.com )
可以通过向mikhail.drobijev@montana.edu发送电子邮件请求来获得用于扫描激光波长的定制Labview 程序。


 


程序


 


建立用于双光子吸收测量的光学装置
在光学平台上设置并固定飞秒激光和分光荧光计,相距约2 m 。
拆下激发灯光源(图2),从激发单色仪(图3)上取下带有两个反射镜的镜架,两个透镜(在管中)(图4和5),可选的分束器(图6)和可选的滤光片(图7)是PC1 荧光分光光度计的激发路径。还必须卸下装有第一个透镜并位于单色仪和可选分束器之间的灯管(图4)。进入样品室的第二根管(空)应保持在原位(图5)。
 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ 2.jpg


图2. 除去激发灯源后,PC1 ISS 荧光分光光度计的入口孔。翻转镜用于将激光束引导到功率计头中。红色虚线箭头示出了这里的激光束传播的方向和图URES 3 - 7。


 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ 3.jpg


图3.从激发单色仪隔室中取出带有两个镜子的镜子支架


 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ 4.jpg


图4.卸下带有准直透镜的灯管支架(放置在激发单色仪之后),并设置一个带有四个槽的ISS滤镜轮


 


 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ 5.jpg


图5.样品室的变化。从套筒透镜支架上取下一个指向样品的聚焦透镜。将3毫米比色皿适配器插入样品和参考比色皿支架中。在左侧的排放通道中,将一个带有短通滤光镜(770 / SP)的附加适配器镜筒支架(25 mm)滑到22毫米镜筒透镜支架上。


 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ 6.jpg


图6.卸下可选的分束器


 


 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ Figs jpg \ Figure7-MS.jpg


图7. 卸下可选的激励滤波器


 


断开“左发射波长”步进电机电缆与PC1内步进电机插孔的连接(图8)。
 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ 8.jpg


图8.将左发射波长滤光片轮与步进电机电气断开,并将激励路径中的另一个滤光片与左E 任务波长滤光片的步进电机连接


 


在具有4个插槽的备用电动滤镜轮(ISS配件)中,将OD = 1,OD = 0.5和OD = 0.3的New Focus ND反射滤镜分别插入插槽#1、2和3中。将插槽4留空。
在激发单色仪之后的PC1的激发光路中,将装有这些ND滤光片的电动滤光片安装到PC1的底座上(图4和8)。
将这个滤光片轮的步进电机连接到“左发射波长”轮的步进电机插孔(图8)。
将激发偏振片放入PC1的光路内。
打开PC1和控制它的计算机。
在Vinci中创建一个文件模板,以测量左或右发射通道中荧光的激发功率依赖性(功率依赖性文件),图9。为此,转到Vinci软件中的Experiment,然后单击Single Point,然后在药物中菜单中选择强度。右键单击名为“固定参数”的块,单击“删除”选项卡,然后选择“左发射波长”框,然后单击“确定”。右键单击名为“可变参数”的块,单击“添加”选项卡,然后选择“左发射波长”功能,然后单击“确定”。在“可变参数,左发射波长”框中,单击“ 编辑列表”,然后将过滤器#1-0.1T,#2-0.3T,#3-0.5T和#4- 清空。在“时基”窗口中,选择10 s,在“迭代次数”中,选择1。将此文件另存为“ Power Dependence”。
 


              D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ Figs jpg \ Figure9.jpg


图9.屏幕截图的左上部分显示了Vinci 3模板文件,用于在左右发射通道的“样本”位置中进行功率相关性测量


 


在Vinci中创建一个文件模板,以在使用左发射通道的一次激光扫描中,测量PC1的参考位置中两个样品的两个光子激发光谱(一个在样品中,另一个在r 中设置),如图2所示。10.为此,请在Vinci软件中进行实验,然后单击“慢动力学”,然后在药物菜单中选择“强度”。右键单击名为Variable Parameters的块,然后添加Sample函数。在“时基”窗口中,选择1.0 s,然后在“时间”窗口中的“变量参数”中,选择0 s(开始),6,500 s(停止)和6.00(步进)。选择“每__分钟测深200分钟” 。在“ 左侧发射波长” 下的“ 固定参数”块中,在“值” 框中选择空。将文件另存为“光谱扫描”。
 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ Figs jpg \ Figure10.jpg


图1 0.屏幕快照的左上部分显示了Vinci 3模板文件,用于一次扫描两个样品的光谱(在Sample和Reference位置设置)


 


在720 nm 下以对准模式(200 mW )打开激光器。使用护目镜。
在激光输出(主光束)之后,立即将组装在支架中的第一块反射镜M1 置于水平位置,平行于工作台将光束转向90 ° ,见图1。
在激光束中的M1之后,首先将一个半波片和第二个Glan激光偏振器安装在旋转安装座中。确保光束以其原始高度自由通过(当光束从激光器中出来时)。
将半波片快轴旋转大约45 度(将激光偏振从水平方向变为垂直方向)。
将格兰偏振镜的偏振轴转向接近垂直位置。
在其固定器组件中使用3个反射镜(M2 - M4),将激光束引导至分光荧光计的入射孔(见图1)。使用反射镜M2和M3使光束高度接近PC1的入射孔高度,并平行于工作台。使用M3和M4将光束引导到水平面中PC1入射孔的中心。
将无级变速中性密度滤镜放在M3镜后面的光束中。
将后视镜FM放在M4之后并将其向上翻转。
将功率计放在后视镜后面的激光路径中。
将无级变速的中性密度(ND)滤轮转到最大变速箱位置。
检查激光功率MEA 用功率计sured是〜200 毫瓦。
打开连续可变ND FIL 之三衰减的力量来〜20 毫瓦。
在VINCI实验软件(ISS)中,转到“仪器控制”选项卡并打开激发快门。
在同一选项卡中,将“左侧发射波长”设置为位置4(与激发ND滤光轮的空槽相对应)。
使用反射镜M3和M4,将光束沿连接PC1入射孔中心和样品架中窗口中心的轴对准。
插入45毫米的消色差透镜成22毫米- 至- 25mm直径的适配器管(ISS附件)和滑移吨与该透镜在22毫米ISS管帽适配器,具有朝向所述样本位置面向所述透镜(图11 )。
 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ Figs jpg \ Figure11-MS.jpg


图11. 将一个45 mm消色差透镜放入样品室内的激发光路中。左:一个f =45毫米交流hromatic透镜我š 插入并固定在一个22到25毫米直径的适配器管中。右:适配器管中的消色差透镜滑过样品室内的22毫米空适配器管。


 


确保激光束仍指向样品架开口的中心(现在已装有45毫米透镜)。如有必要,用反射镜M3和M4稍微重新对准光束。
关闭激光。
在右侧的排放通道中,将Semrock 745 / SP过滤器插入过滤器插槽。
使用InSight GUI软件从计算机上断开Laser的连接,并通过RS 232串行端口将其连接到具有自定义LabView程序的PC计算机,以调节激光波长。
使用LabView程序打开激光器(图1 2,补充文件1 Laser Scan)。
D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ Figs jpg \ Figure12-MS.jpg


图12 。定制LabView程序的前视图,以特定的步长和停留时间从初始波长扫描到最终波长


 


将激光调至850 nm。
翻转翻转镜。
使用LabView程序打开激光快门。
将激光功率调整为200 mW 。首先请确保将无级变速ND滤镜转到完全透射位置。然后,缓慢旋转半波片(紧接在激光之后坐下),直到激光功率读数为200 mW 为止。除非实验中需要更高的功率,否则请始终使用半波片的此位置。
将功率计的探头移至样品室中并进行固定(使用1英寸的立柱支架和短立柱支架底座BA1S ),以使其传感器位于激励路径的圆形开口内居中并面向激光束。使用PC1软件的仪器控制将激发偏振片设置在水平(90 °)位置。将外部格兰偏振镜的旋转偏振器旋转到最小,以调整其角度。用螺丝将其锁定在其旋转底座上。
将激发偏振器滑出激发光路。
在整个调谐范围内,在几个激光波长下校准ND滤镜。将第一激光波长设置为680 nm。通过使用Vinci中的“仪器控制”功能,将“左发射波长”滤镜的位置从1更改为4,并记录所有4个位置的功率。计算针对位置1-3测得的功率与针对位置4测得的功率之比。
重复步骤甲38,用于一组波长(的680,690,710,7 3 0,780,800,8 10 ,840,900,9 4 0,960,1 ,000,1 ,050,1060,1 ,100 ,1 ,200,1 ,在激光调谐范围为300nm)。
在“原始工作簿”中汇总数据,请参见图13 。
 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ Figs jpg \ Figure13-MS.jpg


图13 。原始工作簿包含一个查找校准表,用于校准在多个激光波长下在激发路径中的滤光片轮中的ND滤光片的透射特性


 


关闭激光器。
从样品室取下功率计头,然后将其放回翻转镜后放回其支架。
将770 / SP过滤器插入直径为22 mm至25 mm的适配器管(ISS附件)中,并将带有过滤器的适配器滑过22mm ISS管(其中装有PC1收集透镜),以用于排放物收集。左发射通道。
在该大号EFT ë 任务˚F ILTER W¯¯ 与4个时隙(从其电动千斤顶断开)插入脚跟Semrock 633 / SP,694 / SP,和在插槽1,2 680 / SP过滤器,和3。将插槽4留空。
卸下两个用于3 x 3毫米比色杯的适配器支架(松开4个固定支架一侧的微型螺钉),并在包含4个螺钉的一侧插入矩形的黑纸带(0.9 x 4厘米),这样只有一个狭窄的狭缝〜每个支架中都保留有0.7毫米的开口,请参见图1 B 右下方。当查看固定器时,该狭缝应位于小平面的左侧。
将支架装回,用4颗螺丝将其固定在其中。
 


使用左发射通道测量双光子激发光谱形状
准备用于双光子光谱测量的样品。
制备样品(多达两个新样本)和参考(LDS798和香豆素540A)的储备溶液,参见ř ecipes制备的参比溶液,并添加150μ升它们的进3×3mm的光学比色皿。
使用Lambda 950分光光度计,测量所有样品的吸收光谱。在置于3毫米适配器支架中的3 x 3毫米比色皿中进行测量时,所有溶液在最大光谱范围内的光密度应在0.2-1.5范围内。
将微型搅拌棒放入参考溶液中。
初始仪器设置preparatio ñ 。样品架中的香豆素540A解决方案。在左发射通道中过滤770 / SP和633 / SP。
在全功率模式下打开激光器。
从PC计算机启动激光控制LabView程序(补充文件3 LaserTestVI3.vi)。
打开PC1 荧光分光光度计。
将用于3 x 3比色杯的适配器支架插入PC1的样品和参考支架中,以便在将适配器适配器设置为测量值时,任一适配器支架的被掩盖的一面都指向左侧发射通道(可以将参考支架设置为通过选择在达芬奇的仪器控制转塔的参考位置)。在所有测量期间,将适配器固定器固定在适当的位置。
将“样品位置”设置为测量。
在仪器控制中,开始搅拌样品和参比样品。
将带有香豆素540A溶液的3 x 3毫米比色皿插入PC1的样品架中。
手动将左发射波长滤光轮转到位置1(对应于633 / SP滤光器)。
在发射路径中滑动左发射偏振器,并将其设置在“魔角”位置(具有仪器控制功能)。
将激光波长设置为800 nm。
使用连续ND滤光片将激光功率调节至〜20 mW 。
向下翻转反光镜,将激光束发送到PC1。
确保PC1中的激发偏振器移出激发光路。
确保使用Vinci的仪器控制将激励通道中的ND滤光轮(左发射波长)设置为空位置(插槽4)。
用仪器控制打开激励百叶窗。
在取下样品室盖的暗室中,通过将装有透镜的转接器滑过22毫米管(固定),并最大化左侧的荧光强度,来调节从45毫米透镜到样品的距离发射通道PMT。
用小螺钉固定适配器的位置(相对于22毫米管)。
调整样本#1和#2的功率范围。首先,在样品架中样品#1,然后样品#2 。在左发射通道中过滤770 / SP和633 / SP(或680 / SP,694 / SP或为空)。选择第二个短通滤光片,使其截止波长大于样品#1和#2的荧光峰值波长。
将装有样品#1溶液的比色杯放置在“样品”位置,而不是香豆素540A溶液。
用盖子盖上样品室。
在整个光谱2PA几个波长,测量最大信号例如,700,800,900,1 000,1100,1200纳米(使用左PMT)。
对2号样品重复相同的操作。
使用连续ND滤波器调整激光功率,以使两个样品在2PA频谱上的最大信号量约为1 x 10 6个计数。通常,此功率将在10 -100 mW 的范围内,具体取决于样品浓度和双光子亮度。
检查样品#1和#2在不同波长下荧光的功率依赖性。首先,在样品架中样品#1,然后样品#2。在左发射通道中过滤770 / SP和633 / SP(或680 / SP,694 / SP或为空)。
使用功率依赖性文件,检查样品#1荧光的功率依赖性在光谱的短波长区域内的波长处是否为二次光(功率指数在1.95和2.05之间),在该波长下单光子激发可以起作用。通过使用20 nm(即680、700、720 nm 等)的离散步长找到信号的二次波长,以找到最短的波长。
对样品2 重复步骤B 4a。
扫描样品1和2的2PA光谱。样品#1– i n样品位置,样品#2– 在参考位置。在左发射通道中过滤770 / SP和633 / SP(或680 / SP,694 / SP或为空)。
将比色皿放在样品仓转盘的“样品”位置的“样品1”和“样品”的“参考”位置。
在LabVIEW文件CONTRO的扫描部分灌装激光,扫描相等的开始波长设定为最大数目在步骤B4A和B发现4 湾
计算停止波长(λ停止)采取观察为单光子吸收光谱乘以2的长波长边缘的最长可能波长并取最接近的编号,以它在数字的序列开始于680。将此数字设置为停止波长。
设置步骤等于4纳米,每STE的时间p等于42秒。
打开“光谱扫描”文件,记下有关实验的注释(样品名称和位置,使用的过滤器等),然后开始扫描。
开始激光扫描。
扫描完成后,从适配器固定器中取出装有样品的比色杯。
扫描香豆素540A溶液的2PA光谱。一个装有香豆素540A溶液的比色皿– 处于“样品”位置,另一个带有香豆素540A溶液的比色皿– 处于“参考”位置。在左发射通道中过滤770 / SP和633 / SP。
将带有比色香豆素540A在DMSO中相同溶液的两个比色皿放入转盘的“样品”和“参考”位置。
重复步骤B5 b -B5 克与波长等于在步骤980处停止B5 c和所有其他设置保存在步骤相同的B5 b -B5 f和作出关于在步骤基准音符B5 即
扫描LDS 798溶液的2PA光谱。一个装有LDS 798溶液的比色皿– 在样品位置,另一个装有LDS 798溶液的比色皿– 在参考位置。在左发射通道中过滤770 / SP。
P UT斯达康2个比色皿与LDS 798的氯仿/氘代氯仿相同的溶液,1:2,到样品中,转台的基准位置。
手动将左发射波长滤光轮转到位置4(空)。
重复步骤B5B-B5 f控制波长等于940nm的步骤开始B5 B和所有其他设置保持相同的步骤B5B-B5 f和作出关于在步骤基准音符B5 即
用OriginLab 编写的程序计算样品#1和#2的校正后的双光子激发光谱。
对于包含双光子原始光谱数据(.ifx )的文件,其中在“样品”和“参考”支架中同时测量了两个样品:
在OriginLab中,创建并保存一个导入向导(“文件”>“导入”>“导入向导”),该向导会将文件打开到工作簿中,使其看起来类似于图14中显示的示例工作簿。Vinci程序保存的数据列为“时间”,“样本”,“强度”,“ IntensityStdError ”和“实时”。
将此导入向导过滤器命名为“ 2PA_sample_and_reference”。
 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ Figs jpg \ Figure14.jpg


图14 。创建“ 2PA_sample_and_reference”导入向导过滤器后,Origin工作簿的外观示例


 


将自动分析脚本添加到该导入向导过滤器。为此,首先导入用于上述步骤的文件,选择您刚刚创建的过滤器,然后单击“下一步”直到最后一个窗口。选中“保存过滤器”以及“指定高级过滤器选项”。将其另存为新的过滤器“ 2PA_sample_and_reference_autocalculation”。单击“下一步”,这将带您进入一个窗口,以在“每个文件导入后的脚本:” 窗口中复制和粘贴脚本1(请参阅补充文件2 Origin Script)。
在弹出的窗口中填写适当的值(第一个波长,最后一个波长,波长步长,每步秒数)后,生成的工作簿应类似于以下内容(图15 )。另外,将弹出一个图形(图16 ),显示脚本在激光稳定后自动删除点后原始数据的样子。它看起来像数据点聚集成两个不同的光谱。如果某些点的强度比其他点的强度低得多,则通常意味着脚本由于某种原因无法正常工作,因此应该手动对数据进行排序。此图(图16 )是脚本按预期工作的重要视觉检查。
 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ Figs jpg \ Figure15-MS.jpg


图15 。使用“ 2PA_sample_and_reference_autocalculation”导入向导过滤器打开文件后,最终工作簿的外观示例


 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ Figs jpg \ Figure16.jpg


图16 。该图的示例显示了脚本稳定删除激光点后脚本自动删除点后原始数据的外观。使用“ 2PA_sample_and_reference_autocalculation”“导入向导”过滤器打开文件后,将弹出该图。这是对脚本是否按预期工作的直观检查。


 


如果所测量的样品是用于测量设备校正功能的参考染料,则将“正确的”参考染料光谱复制并粘贴到其相应的列中(“ S 足够支架” 列为D,“ R ”参考支架为列E。)。确保复制的波长范围与测量的波长范围匹配。列F和G现在为“校正函数š 充足保持器”和' - [R 分别eference持有人。设置它们是为了将原始光谱除以正确的光谱。
合并分别与基准测得的短程校正函数中的染料香豆素540A在DMSO(λ ST 艺术- 980 nm),而LDS798在2:1 CDCL 3 :氯仿3 (940纳米- λ 停止)到全范围校正函数(λ ST 艺术- λ 停止)。(“样品架”和“参考架”的校正功能数据应分别处理。)
首先,通过将短距离校正函数中的每一个除以940-980 nm范围内的列值的平均值,对它们进行归一化。
现在,找到两个短程校正函数在940-980 nm范围内的行值的平均值,并将其作为该范围校正函数的一部分。
创建合并的校正函数。对于λ ST 技术-936纳米范围内,与粘贴香豆素540A在DMSO中测得的归一化的校正功能。对于940 nm - 980 nm 范围,粘贴上一步的平均值校正函数。对于980 nm - λ 终止范围,将用LDS798测量的归一化校正函数粘贴在2:1 CDCl 3 :CHCl 3中。
如果测得的样品具有未知2PA光谱,然后复制并粘贴您为样品保持器和所述基准保持器(列d为“先前测量的校正函数š 充足保持器”,E列的“ - [R eference保持器”)。确保复制的波长范围与测量的波长范围匹配。列F和G是现在经校正的2PA光谱形状在“测量样品š 充足保持器”和“ - [R eference保持器”,分别。设置它们是将原始光谱除以校正函数。
 


相对于若丹明590在甲醇中的红色荧光蛋白(例如,Zhao 等人,2011年描述的RGECO1 )在1,060 nm处的双光子吸收截面的测量使用了右发射通道,并在600 nm处记录了荧光。
准备若丹明6G在甲醇中的储备溶液。将3 x 3毫米比色杯中528 nm处的光密度调整为0.2到0.3之间的一个数字。记录OD 的确切数字。
调整在3个样品的光密度X 3毫米比色皿到0.2 - 0。3 。记录OD 的确切数字。
在1将激光波长,060纳米。
在右侧的发射通道中,将偏振器滑入并使用“仪器控制”功能将其设置为魔角。
将正确的发射单色仪设置为600 nm,然后将两个2 mm的狭缝手动插入单色仪中。
将装有参考罗丹明590溶液的比色杯放入转盘的样品架中。
调整使用ND滤光器连续激光功率使得右发光强度将是50000的数量级上- 100000当激励滤光轮被设置为空的位置(#4)。
记录此功率。
对样品溶液重复步骤C 7和C 8 。
在Rhodamine 590的右侧发射通道中运行并注册功率依赖文件。
对样品重复步骤C 10,而不改变条件。
使用OriginLab ,绘制荧光信号的功率依赖关系作为动力功能的平方和得到的比F 2,小号(λ EX ,λ REG )/ ˚F 2,- [R (λ EX ,λ REG )值的斜率的比率样品和参考标准品的线性拟合。
使用已知的消光系数,计算浓度比:
 






 


为了测量φ号码,稀释样品和参比溶液以具有光密度,在选定的波长(530纳米)和在0.05的范围内下降彼此相似- 在1-厘米比色皿的光0.07。记录它们各自在530 nm处的OD 值:OD S (530)和OD R (530)。
使用LS55 荧光分光光度计,在530 nm激发波长等于5 nm发射狭缝– 16 nm的激发下记录样品和参考的荧光光谱。
记录600 nm F 1,S (600)和F 1 ,R (600)的单光子信号并计算比率:
 






 


使用e q。(2),在所获得的所有值的比率的步骤C12-C16 ,和若丹明590的横截面σ 2 (1060)= 10 GM,计算样品的2PA横截面。
 


数据分析


 


的光谱曲线,所述变化也就是,当光谱在另一波长(归在一个波长的相对值的偏差例如,峰值)包含随机和系统的贡献。随机贡献来自同一样品连续扫描中激光参数的可重复性。我们已经观察到,在2-3次连续扫描中,光谱的偏差不大于4%。由于使用两次扫描(样品和参考标准品)来计算校正后的2PA光谱,因此导致6%的随机误差。系统的光谱偏差来自参考标准品2PA光谱的测量准确性。对于香豆素540A(de Reguardati 等人,2016 )估计约为5 %,对于LDS 798约为10%(Makarov 等人,2011)。这导致总的频谱形状误差8 %,在短波长范围内(680 - 980纳米)和12%的长波长范围(980-13 ,00纳米)。
2PA横截面测量的误差是各个参数的等效误差的组合。(2)。铒的F RORS 2 号码从荧光强度对功率的平方曲线的线性拟合估计(通常为1%为两个F 2,S 和F 2,R )。由一种蛋白质的12次独立测量确定了样品和参比物的差分量子产率φ 的误差,发现误差为7%。浓度的误差主要来自在样品的消光系数的测量结果的误差,等于5%(莫利纳等人。,2019)。罗丹明590的系统误差来自参考标准品2PA横截面的不确定性,为10%(请参阅“注释”部分)。因此,在正交计算出的总误差σ 2 在一个波长(1测量的样品,060 nm)的总计为15%。这转化为绝对误差σ 2 的值,在不同的测定波长小号的〜1 8 %(莫利纳等人。,2019)。
 


笔记


 


我们使用氯仿/混合物氘代氯仿(1:2)作为溶剂用于LDS 798摆脱氯仿吸收在1 ,150纳米。我们观察到几乎纯去时没有改变染料的光谱1PA 氯仿的米ixture。
在DMSO中香豆素540A的单光子和双光子吸收光谱的形状紧密地匹配彼此的745-1的区域,000纳米,如果1PA作图单光子波长加倍(德Reguarda TI 等人,2016 ; 图17 )。在较短的波长范围,680 - 7 45 纳米,相对2PA值中提出的(de Reguardati 等人。,2016)系统地超过1PA值。我们已经独立地测量香豆素540A在DMSO 2PA光谱,在DMSO中使用普罗丹作为参考标准(DE Reguardati 等人在680的区域中,2016。)- 800 nm,并且2PA和1PA光谱之间获得更好的相关性香豆素540A的形状,请参阅。红色和黑色的线图URE 1 7 。
 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ Figs jpg \ Figure17.jpg


图1 7 。DMSO中香豆素540A 1PA 和2PA光谱的光谱形状。所述1PA光谱测量在这项工作中,黑线,和2PA SPECTR 微米(德Reguardati 等人。,2016) ,蓝线,被在它们的峰值归一化。所述2PA光谱的短波长部分(680 - 800纳米),红色线,在这项工作中测量,使用P 在DMSO罗丹(德Reguardati 等为标准,2016。)。


 


要仔细检查香豆素540A(的频谱形状甲2PA,R (λ 在大范围从680至1)函数),000纳米,我们测量了2PA SPECTR 微米罗丹明590(=若丹明6G)在甲醇与香豆素540A在DMSO中,并将其与已知的文献数据(图URE 1 8 )。来自(Hermann and Ducuing ,1972; Makarov et al 。,2008; de Reguardati et al 。,2016)的数据以绝对横截面值的形式进行了测量(关于赫尔曼,请参见下表5的脚注a和b)和Ducuing 数据)。来自(Albota 等,1998 ; Rodriguez 等,2009 ; Rodriguez和Chiesa,2011 ; Rodriguez和Chiesa,2012)的数据被缩放为820 nm处的74 GM值,与(Makarov 等(2008 ;de Reguardati 等,2016)。的光谱由(Wakebe 和Van Keuren ,1999),最初呈现为在乙醇中的若丹明6G的相对光谱,被移位17纳米至红色(在2PA波长),并调整为在844 34 GM的值,匹配的测量(Makarov 等人,2008 ;de Reguardati 等人,2016)。在(690-960 nm)的香豆素540A吸收的几乎整个区域中,在(Albota 等,1998)和(de Reguardati 等,2016)中测量的光谱形状之间都有很好的相关性,因此我们得出结论:这些光谱代表了若丹明6G的2PA光谱的最佳近似值。这个结论是通过其他数据的到这些光谱在不同光谱区或在分离波长良好匹配的支持:赫尔曼和Ducuing 在694和1的数据,060纳米; Wakebe 和Van Keuren 数据- 在806 - 1 ,106纳米区域; 中号akarov 等。(2008 )d ata- 在720 - 900和1 ,060 - 1 ,100个纳米区域; Rodriguez 等。(2007)数据- 在720 - 860nm的区域。


 


 


表1.正确2PA香豆素540A在DMSO中的频谱形状,具有680 - 760nm的数据通过在普罗丹校正DMSO(德Reguardati 等人。,2016)


波长


相对值


波长


相对值


波长


相对值


波长


相对值


680


0.135


760


0.376


840


0.990


920


0.523


684


0.117


764


0.408


844


0.996


924


0.478


688


0.113


768


0.445


848


0.999


928


0.434


692


0.112


772


0.483


852


1.00


932


0.390


696


0.112


776


0.522


856


0.998


936


0.348


700


0.115


780


0.561


860


0.994


940


0.307


704


0.118


784


0.600


864


0.985


944


0.268


708


0.124


788


0.639


868


0.973


948


0.232


712


0.135


792


0.678


872


0.955


952


0.199


716


0.146


796


0.715


876


0.934


956


0.169


720


0.160


800


0.751


880


0.908


960


0.143


724


0.176


804


0.786


884


0.881


964


0.120


728


0.194


808


0.819


888


0.850


968


0.100


732


0.215


812


0.851


892


0.816


972


0.0832


736


0.234


816


0.881


896


0.780


976


0.0683


740


0.256


820


0.908


900


0.741


980


0.0556


744


0.279


824


0.931


904


0.700


984


0.0448


748


0.303


828


0.952


908


0.657


988


0.0355


752


0.325


832


0.968


912


0.613


992


0.0274


756


0.350


836


0.980


916


0.568


996


0.0203


 


当以DMSO中的香豆素540A的2PA光谱形状作为参考时,若丹明6G在甲醇中的2PA光谱在680-750区域内显示出强烈高估的吸收率(参见图18中的空心菱形和浅蓝色线)。这可能是由于香豆素540A的2PA的高估值,如图17所示。然而,当香豆素540A的2PA光谱在680校正- 740 nm范围内与普罗丹(见表1中的值)被取为甲2PA,R (λ )的功能,若丹明6G光谱(与图18中的三角形深绿线)相当不错的文献光谱(匹配Albota 等人。,1998)和(德Reguardati 等人。,2016)在的区域680 - 980纳米。因此,我们得出的结论是,当将DMSO中的香豆素540A的校正光谱用作680- 980 nm 范围内的参考时,在DMSO中的香豆素540A的校正后2PA光谱与A 2 PA,R (λ )函数很好地近似。。


 


 


表LDS 798的氯仿2.纠正2PA频谱形状从936 - 1 ,300纳米; 值来自将高斯拟合到Makarov 等人的LDS 798 z扫描数据中。,2011年。


波长


相对值


波长


相对值


波长


相对值


波长


相对值


936


0.116


1028


0.410


1120


0.847


1212


0.978


940


0.124


1032


0.428


1124


0.864


1216


0.970


944


0.132


1036


0.446


1128


0.879


1220


0.961


948


0.141


1040


0.465


1132


0.894


1224


0.950


952


0.150


1044


0.484


1136


0.907


1228


0.937


956


0.159


1048


0.503


1140


0.920


1232


0.923


960


0.169


1052


0.522


1144


0.933


1236


0.908


964


0.180


1056


0.542


1148


0.944


1240


0.892


968


0.191


1060


0.561


1152


0.954


1244


0.874


972


0.203


1064


0.581


1156


0.963


1248


0.855


976


0.215


1068


0.601


1160


0.972


1252


0.835


980


0.227


1072


0.621


1164


0.979


1256


0.813


984


0.240


1076


0.641


1168


0.985


1260


0.791


988


0.253


1080


0.661


1172


0.991


1264


0.767


992


0.267


1084


0.681


1176


0.994


1268


0.742


996


0.282


1088


0.701


1180


0.997


1272


0.717


1000


0.296


1092


0.720


1184


0.999


1276


0.692


1004


0.311


1096


0.740


1188


1.00


1280


0.665


1008


0.327


1100


0.759


1192


0.999


1284


0.638


1012


0.343


1104


0.777


1196


0.998


1288


0.611


1016


0.359


1108


0.795


1200


0.995


1292


0.584


1020


0.376


1112


0.813


1204


0.991


1296


0.556


1024


0.393


1116


0.831


1208


0.985


1300


0.529


 


 


 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ 18.jpg


图1 8 。罗丹明6G在甲醇或乙醇中的双光子吸收光谱(对数标度)。马卡罗夫(Makarov)等人的黑尔曼和杜翠(Hermann and Ducuing ,1972 )的数据。(2008 )– 蓝色圆圈,de Reguardati 等。(2016) - 淡蓝色线,以绝对值给出σ 2倍的值(见脚注a和b 表小号3-5 表5之后)。Albota 等人的数据。(1998年)– 红色圆圈,Rodriguez 等。(2009 )– ping直角三角形 罗德里格斯和基耶萨(2011 ) - 粉色左三角形,罗德里格斯和基耶萨(2012) - 粉红色的三角形向上缩放(见正文),并Wakebe 和Van Keuren (1999) - 橙色圆圈进行缩放和移动(见正文)。空心钻石显示了此处使用香豆素540A在DMSO 2PA 光谱(de Reguardati 等,2016)中得到的(标度)光谱。测得的光谱在这项工作中(缩放)相对于香豆素540A在DMSO中在740 - 1 ,000纳米区域和相对至P 罗丹在DMSO中在680 - 740nm的区域被示出暗绿色来与连续线连接的三角形。获得的光谱在这项工作中相对于LDS 798在氯仿/氘代氯仿中,通过紫色显示用实线连接的钻石。


 


在Makarov 等人。(2011年),使用荧光(相对于Styryl 9M [ Makarov 等,2008 ] )和z扫描(绝对测量)技术获得了LDS 798染料在氯仿中的2PA光谱。单光子和双光子吸收光谱的形状很广,无结构,并且紧密地匹配彼此在900的区域- 1 ,300纳米,如果1PA作图单光子波长加倍(马卡罗夫等人,2011 ; 图19 )。
 


D:\陈丹工作\ 1902694--1251 MikhailDrobizhev 793128 \ Figs jpg \ Figure19.jpg


图1 9 。LDS 798在氯仿中的1PA(紫色线)(此处测量)和2PA的光谱形状,绿色正方形(使用相对于苯乙烯9M的荧光技术,黑色正方形),使用z扫描和绝对方法(Makarov 等, 2011)。所有光谱均在其峰值处归一化。黑虚线表示高斯拟合到在该区域的z扫描数据从900至1 ,300纳米,其用作一个ñ 阿2 PA,R (λ 这里)函数。这个数字是改编自(Makarov et al 。,2011)。


 


由于z扫描数据是通过绝对方法获得的(LDS 798是主要标准),因此我们将相应的2PA形状(图1 9中的黑色正方形)用于A 2 PA,R (λ )函数。要将A 2 PA,R (λ )函数插值到中间波长,我们对900至1300 nm之间的实验数据使用了高斯拟合,如图1 9中的黑色虚线所示。该频谱被呈现于表2中要检查此功能的性能,若丹明590的在甲醇中的2PA光谱相对测量到LDS 798在CHCl 3 / CDCL 3 中940:(1:2)- 1140 MN 区域,请参见图1 8 。数据(紫色)与Albota 等人的光谱非常吻合。(1998年); Wakebe 和Van Keuren (1999),以及de Reguardati 等人。(2016),并且与使用香豆素540A(深绿线)在940-980 nm范围内测得的光谱重叠良好。这证实了我们选择LDS 798作为940-1,300 nm区域光谱形状的参考标准的事实。


 


在双光子截面的测量,我们使用的光密度OD <0.05 在荧光波长登记和OD <0.3的吸收峰(在3毫米比色皿)。这需要以排除可能的再吸收和再排放的影响。
我们建议使用甲醇中的若丹明590作为参考标准(有关文献数据,请参见表3和4),以测量选定波长下的2PA横截面。最一致的文献数据进行收集Ñ 耳1 ,060纳米(表3)。对于这个波长我们建议使用σ 2 = 10±1 GM。
 


表3.罗丹明6G在甲醇或乙醇中在1,054-1,064 nm区域的双光子截面的文献数据。大多数值(第7列)在10 GM附近。平均所有11个测量值的提供σ 2 = 12±2 GM(平均值±平均值的SE)。带下划线的值是通过锁模激光器或使用消除了激光强度g (2)的二次时间相关函数的不确定性的方法获得的。在这六次测量中,最低的(4.2 GM)(Kaatz and Shelton,1999)是通过使用Hyper Rayleigh散射技术校准包括g (2)的激光特性而获得的。作者发现,这种方法给了低估的值σ 2 ,相比于具有标准校准针对单光子荧光获得的那些。因此,所建议的值σ 2 通过排除该测量和平均在7列中斜体选择的数字计算。


波长


(纳米)


溶剂


浓缩


(男)


脉冲


原版的


σ 2 (GM)


校正离子1


σ 2 (GM)


校正离子2


σ 2 (GM)


(最后)


参考


1060


C 2 H 5 羟基


不适用


ns


12.9±6


25.8±12 一


10.1±4.7 磅


1972年,赫尔曼和杜庆


1060


C 2 H 5 羟基


10 -3


60纳秒


5.5


11 个


5.5 摄氏度


布拉德利(Bradley)等人,1972年


1060


C 2 H 5 羟基


10 -4


30纳秒


26


52 个


26 ℃


Vsevolodov 等。,197 3


1060


C 2 H 5 羟基


10 -3


ps


3.6


7.2 一


7.2


布拉德利(Bradley)等人,1972年


1060


C 2 H 5 羟基


10 -6


110毫微秒


11±5


22±10 a


11±5 摄氏度


李和社,1982


1064


C 2 H 5 羟基


10 -2


连续波


12±2


48±8 天


24±4 摄氏度


卡塔拉诺和辛戈拉尼(1982)


1054


CH 3 羟基


0.2


5 ps


10±1


--


10±1


彭兹科弗和勒皮查尔(1987)


1064


CH 3 外径


2.2 10 -6


125纳秒


4.2±0.8


--


4.2±0.8


Kaatz 和Shelton,1999年


1064


C 2 H 5 羟基


10 -4


10纳秒


15±1


30±2 a


15±1 摄氏度


Rodriguez 等。,2007


1060


CH 3 羟基


8 10 -6


100 fs


10±1


--


10±2


Makarov 等,2008


1060


CH 3 羟基


不适用


100 fs


11±1 


--


11±1 


de Reguardati 等,2016


建议条件和横截面值


1060


CH 3 羟基


10 -5


100 fs


 


 


10±1


 


 


 


表4.其它σ 2倍在选定的波长的值若丹明6G的。所述σ 2点的值表示两个或更多个测量之间满意的匹配列是斜体7的推荐值σ 2 作为平均这些数字对于每个波长的计算。


波长


(纳米)


溶剂


浓缩


(男)


脉冲


原版的


σ 2


(通用)


校正离子1


σ 2


(通用)


校正离子2


(最后)


σ 2


(通用)


参考


694


C 2 H 5 羟基


不适用


ns


355±170


710±355 a


277±138 羽


1972年,赫尔曼和杜庆


694


CH 3 羟基


0.04


30 ps


180±20


--


180±20


斯珀伯(Sperber)和彭斯科夫(Penzkofer),1986


690


700


CH 3 羟基


1.1 10 -4


100 fs


136


176


--


156 e


Albota 等,1999


694


CH 3 羟基


8 10 -6


100 fs


112±22


--


112±22


Makarov 等,2008


692


CH 3 羟基


不适用


100 fs


202±16


--


202±16


de Reguardati 等,2016


建议条件和横截面值


694


CH 3 羟基


10 -5


100 fs


 


 


180±23


 


 


730


CH 3 羟基


8 10 -6


100 fs


50±10


--


50±10


Makarov 等,2008


730


CH 3 羟基


不适用


100 fs


50±4


--


50±4


de Reguardati 等,2016


建议条件和横截面值


730


CH 3 羟基


10 -5


100 fs


 


 


50


 


 


812


CH 3 羟基


8 10 -6


100 fs


71±14


--


71±14


Makarov 等,2008


812


CH 3 羟基


不适用


100 fs


79±6


--


79±6


de Reguardati 等,2016


建议条件和横截面值


812


CH 3 羟基


10 -5


100 fs


 


 


75


 


 


940


CH 3 羟基


1.1 10 -4


100 fs


7.7


--


7.7


Albota 等,1999


940


CH 3 羟基


8 10 -6


100 fs


8.3±1.7


--


8.3±1.7


Makarov 等,2008


940


CH 3 羟基


不适用


100 fs


11.1±0.9


--


11.1±0.9


de Reguardati 等,2016


建议条件和横截面值


940


CH 3 羟基


10 -5


100 fs


 


 


9.0±1.0


 


 


960


CH 3 羟基


1.1 10 -4


100 fs


12.7


--


12.7


Albota 等,1999


960


CH 3 羟基


8 10 -6


100 fs


10.9±2.2


--


10.9±2.2


Makarov 等,2008


960


CH 3 羟基


不适用


100 fs


17±1.4


--


17.0±1.4


de Reguardati 等,2016


建议条件和横截面值


960


CH 3 羟基


10 -5


100 fs


 


 


13±2


 


 


我们建议在水中使用荧光素在pH 为分子在光谱的绿色部分的荧光参考标准11(500 - 550纳米)。然后应调整方案的C部分,以测量绿色区域的荧光。表5总结了文献中的数据进行荧光素与建议的波长和σ 2倍的值。
 


表5.有人建议σ 2个荧光素的值在不同的波长的碱性水溶液。所述σ 2点的值表示两个或更多个测量之间满意的匹配列是斜体7的推荐值σ 2 作为平均这些数字对于每个波长的计算。


波长


(纳米)


溶剂


浓缩


(男)


脉冲


原版的


σ 2


(通用)


正确离子1 σ 2 (GM)


校正离子2


(最后)


σ 2 (GM)


参考


710


H 2 O pH11


8 10 -5


100 fs


17.3±4.4


 


17.3±4.4


徐和韦伯(1996)


710


H 2 O pH11


3 10 -6


100 fs


16.5±3.3


--


16.5±3.3


Makarov 等,2008


建议条件和横截面值


710


H 2 O pH11


10 -5


100 fs


 


 


16.9 


 


 


730


H 2 O pH11


8 10 -5


100 fs


25.0±6.3


 


25.0±6.3


徐和韦伯(1996)


730


H 2 O pH11


3 10 -6


100 fs


24.2±4.8


--


24.2±4.8


Makarov 等,2008


建议条件和横截面值


730


H 2 O pH11


10 -5


100 fs


 


 


24.6 


 


 


782


H 2 O pH11


不适用


100 fs


46±10


 


46±10


徐等,1995


782


H 2 O pH11


8 10 -5


连续(单模式)


38±10


 


38.0±10


徐和韦伯(1996)


782


H 2 O pH11


3 10 -6


100 fs


46±9


--


46±9


Makarov 等,2008


建议条件和横截面值


782


H 2 O pH11


10 -5


100 fs


 


 


43±3


 


 


802


H 2 O pH11


8 10 -5


连续(单模式)


36±10


 


36±10


徐和韦伯(1996)


802


H 2 O pH11


3 10 -6


100 fs


38±8


--


38±8


Makarov 等,2008


建议条件和横截面值


802


H 2 O pH11


10 -5


100 fs


 


 


37


 


 


842


H 2 O pH11


8 10 -5


100 fs


12±3


 


12±3


徐和韦伯(1996)


842


H 2 O pH11


3 10 -6


100 fs


13±3


--


13±3


Makarov 等,2008


建议条件和横截面值


842


H 2 O pH11


10 -5


100 fs


 


 


12.5


 


 


900


H 2 O pH11


8 10 -5


100 fs


16±4


 


16±4


徐和韦伯(1996)


900


H 2 O pH11


3 10 -6


100 fs


15±3


--


15±3


Makarov 等,2008


建议条件和横截面值


900


H 2 O pH11


10 -5


100 fs


 


 


15.5


 


 


1000


H 2 O pH11


3 10 -6


100 fs


3.1±0.6


--


3.1±0.6


Makarov 等,2008


1000


H 2 O pH11


不适用


100 fs


3.3±0.3


--


3.3±0.3


de Reguardati 等,2016


建议条件和横截面值


1000


H 2 O pH11


10 -5


100 fs


 


 


3.2


 


 


1060


C 2 H 5 羟基


10 -5


60纳秒


0.18


0.36 一


0.18 ç


布拉德利(Bradley)等人,1972年


1060


C 2 H 5 羟基


 


ps


0.075


0.15 a


0.15


布拉德利(Bradley)等人,1972年


1050


H 2 O pH11


8 10 -5


100 fs


0.23±0.07


 


0.23±0.07


徐和韦伯(1996)


1050


H 2 O pH11


3 10 -6


100 fs


0.17±0.03


--


0.17±0.03


Makarov 等,2008


建议条件和横截面值


1050


H 2 O pH11


10 -5


100 fs


 


 


0.2


 


 


表3-5的脚注:


一个倍增通过因子2以更近的σ定义为符合2 (双光子小号是neede d激发一个分子),参见 徐和韦伯(1996 ); Kaatz 和Shelton (1999 ); Makarov 等。(2008年)。


b 乘以系数0.39以符合石英的当前参考值d 11 = 0.3 0 pm / V(相对于0.48 pm / V),请参阅Kaatz 和Shelton (1999)。


c 使用激光相干函数值g (2)= 2进行重新评估,参见Weber (1971 );布拉德利等。(1972年); Kaatz 和Shelton (1999)。该因素未包含在原始分析中。


d 倍增通过因子4更近的σ的定义符合2 (2 光子s为neede d激发一个分子),参见 Xu and Webb (1996 ); Kaatz 和Shelton (1999 ); Makarov 等。(2008年)。


e 在690和700 nm测量之间进行内插。


菜谱


 


LDS 798染料在CHCl 3 :CDCl 3 (1:2)混合物中的溶液
溶解0.1毫克的L的DS 798在0.75毫升三氯甲烷的3 在15毫升螺钉CA p 玻璃小瓶
加入1.5毫升的CDCl 3 。在磁力搅拌器上搅拌过夜。在3 mm比色皿中,最终溶液在594 nm(最大光谱)处的光密度应为OD〜1.2 。这对应于〜10 -4 M 的浓度
它可以存储在密封的小瓶中在黑暗中室温TEM perature为至少2个月
DMSO中的香豆素540A染料溶液
将0.15 mg香豆素540A溶于15 ml 旋盖玻璃瓶中的3 .3 ml DMSO中
在3 mm比色皿中,最终溶液在427 nm(最大光谱)处的光密度应为OD〜0.6 。这对应于〜10 -4 M 的浓度
可以在室温下在黑暗中密闭的小瓶中保存至少2个月
罗丹明590甲醇溶液
在15 ml 旋盖玻璃小瓶中的10 ml CH 3 OH中溶解0.05 mg罗丹明6G
在528处的最终溶液的光密度(SP ectral最大)1 N A3毫米试管应OD〜0.3。这对应于〜10 -5 M 的浓度
可以在室温下于黑暗中保存在密闭的小瓶中至少4个月
 


致谢


 


感谢Alexey Drobizhev 提供的自定义LabView程序。这项工作得到了NINDS授予MD,TEH和RSM的U01 NS0942 46和U24 NS109107的支持,以及Ruth L. Kirschtein 国家研究服务奖(RSM的编号为F31NS108593)的支持。


  该方案源自Molina,RS,Qian,Y.,Wu,J.,Shen,Y.,Campbell,RE,Drobizhev ,M.和Hughes,TE(2019)了解红色遗传编码的钙离子指示剂的荧光变化。生物物理。Ĵ 。116:1873-1886。


 


利益争夺


 


作者宣称没有任何金融和非金融竞争利益。


 


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引用:Drobizhev, M., Molina, R. S. and Hughes, T. E. (2020). Characterizing the Two-photon Absorption Properties of Fluorescent Molecules in the 680-1300 nm Spectral Range. Bio-protocol 10(2): e3498. DOI: 10.21769/BioProtoc.3498.
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